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Reactivation of TK-deficient Herpes simplex virus and homopolymer mutational hot spots Sasadeusz, Joseph John 1997

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R E A C T I V A T I O N OF TK-DEFICIENT HERPES S I M P L E X VIRUS A N D H O M O P O L Y M E R M U T A T I O N A L H O T SPOTS by JOSEPH J O H N S A S A D E U S Z M.B.B.S. Monash University, 1980 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y i n T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Experimental Medicine) We accept this tresis as conforming to the required standard T H E U N I V E R S I T Y OF BRITISH C O L U M B I A January 1997 © Joseph John Sasadeusz, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of (> * ^ f t ^ y vev^tr The University of British Columbia Vancouver, Canada Date ^ ~SftMW*t-f DE-6 (2/88) ABSTRACT The majority of herpes simplex virus (HSV) isolates resistant to acyclovir (ACV) have a deficiency of thymidine kinase ( T K D ) . Such mutants are able to establish latency i n animal models but are unable to reactivate w h i c h has contributed to the cl inical dogma that, fo l lowing effective eradication, such outbreaks are inevitably fol lowed by ACV-suscept ib le ( A C V s ) reactivations. In addi t ion , genotypic in format ion regarding mutations conferring the ACV-resistant ( A C V R ) phenotype onto H S V is very l imi ted . H S V type 2 strains 1737 and 89-063, A C V R and uniformly T K D by all conventional assays, clinically reactivated i n AIDS patients in the absence of antiviral drug pressure. Investigation of their neurovirulence and latency characteristics in mouse models, however, yie lded neurovirulent T K - w i l d type ( T K W T ) and T K - l o w producer ( T K L P ) populations. Two further A C V R reactivated isolates (89-353 and 90-150) contained mixed T K W T / T K D populations by plaque autoradiography while one (2370) l ikely exhibited a T K -altered phenotype. Mutations conferring the A C V R phenotype were assessed by plaque purification, amplification of the tk gene by polymerase chain reaction (PCR) and cycle sequencing. Three of 8 plaque isolates, 1737-14, 90-150-3 and 89-650-5 contained a guanosine insertion within a stretch of 7 guanosines. Correlation by immunoprecipitation and Western blot confirmed the predicted truncated 28 k d protein. Two further isolates, 89-063-1 and 89-353-1, produced truncated proteins 32 and 28 kd in size respectively while one isolate, 90-110-4, produced no detectable product. Reactivation is dependent on T K activity but may occur w i t h "Ultra-low" levels and may dissociate from the A C V R phenotype. The l i k e l y mechanism is in vivo complementa t ion between heterogeneous T K populations containing T K D or T K L P virus wh ich may be detectable by conventional means or masked. Eradication of T K D outbreaks does not ensure subsequent reactivations to be A C V S and alternating antivirals may be required for effective therapy. Mutations in the tk gene occur preferentially at homopolymer hot spots and the majority of mutants produce truncated products. Such homopolymer stretches may facilitate reactivation by high reversion frequencies or ribosomal frameshifting and may offer novel targets for detection and therapy of A C V R H S V isolates. i i i TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS x ACKNOWLEDGMENTS x i i DEDICATION x i i i CHAPTER 1. - INTRODUCTION 1 1.1. H E R P E S S I M P L E X VIRUS INFECTIONS 1 1.2. A N T I V I R A L A G E N T S A N D A C Y C L O V I R 5 1.3. A C Y C L O V I R RESISTANCE 9 1.4. T H Y M I D I N E K I N A S E 11 1.5. H E T E R O G E N E I T Y A N D C O M P L E M E N T A T I O N 13 CHAPTER 2. - MATERIALS AND METHODS 17 2.1. P A T I E N T S 17 2.1.1. Patient WJ 17 2.1.2. Cl inical isolates derived from A C T G 095 20 2.2. TISSUE C U L T U R E 21 2.2.1. Cel l Lines 21 2.2.2. Isolation and Growth of Cl in ica l Isolates of H S V 22 2.2.3. Susceptibility testing 24 2.2.4. Plaque isolation 25 2.2.5. Relative T K activities 25 2.2.6. Plaque autoradiography 26 2.3. A N I M A L STUDIES 27 i v 2.3.1. Mouse intranasal neurovirulence model 27 2.3.2. Mouse intracerebral neurovirulence model 29 2.4. M O L E C U L A R BIOLOGY 30 2.4.1. Preparation of Vi ra l D N A 30 2.4.2. Amplification of the T K gene by polymerase chain reaction 31 2.4.3. Cycle sequencing 33 2.4.4. Immunoprecipitation 35 2.4.5 Western blot analysis 36 CHAPTER 3. - RESULTS 38 3.1 M E C H A N I S M OF R E A C T I V A T I O N OF 1737 38 3.1.1. Characterization of clinical isolates from patient WJ 38 3.1.2. Plaque isolation of clinical isolates 1737 and 1773 38 3.1.3. Mouse neurovirulence studies of 1737-14 44 3.2 M E C H A N I S M OF R E A C T I V A T I O N OF A C T G 095 ISOLATES 46 3.2.1. Characterization of clinical isolates 46 3.2.2. Mouse intracerebral inoculation of 89-063 and 1737 51 3.3 M U T A T I O N C O N F E R R I N G T H E T K D P H E N O T Y P E O N T O 1737 61 3.3.1. D N A sequencing of isolates from patient WJ 61 3.3.2. Thymidine kinase protein studies of isolates from patient WJ 63 3.4 M U T A T I O N S C O N F E R R I N G T K D P H E N O T Y P E S O N T O A C T G 095 ISOLATES 67 3.4.1. D N A sequencing of A C T G 095-derived isolates 67 3.4.1.1. Spontaneously-reactivated isolates 67 3.4.1.2. Isolates followed by an A C V 3 reactivation 71 3.4.2. Protein studies of A C T G 095-derived isolates 71 3.4.2.1. Spontaneously-reactivated isolates 71 3.4.2.2. Isolates followed by an A C V S reactivation 75 v CHAPTER 4. - DISCUSSION 79 4.1 M E C H A N I S M S OF R E A C T I V A T I O N OF T K D ISOLATES 79 4.2 T K D M U T A T I O N S A N D R E A C T I V A T I O N 86 4.3 C L I N I C A L I M P L I C A T I O N S A N D A P P L I C A T I O N S 90 4.4 T H E F U T U R E 91 CHAPTER 5. - C O N C L U S I O N 93 B I B L I O G R A P H Y 95 v i LIST OF TABLES Table 1. A C V susceptibilities and thymidine kinase uptakes for all clinical isolates available for patient WJ 39 Table 2. A C V susceptibilities for plaque isolates derived from clinical isolates 1737 and 1773 41 Table 3. Mouse intranasal neurovirulence studies of WJ isolates 45 Table 4a. A C V susceptibilities and T K activities of A C T G 095 A C V R reactivated isolate pairs 49 Table 4b . A C V susceptibilities and T K activities of A C T G 095 A C V S reactivated isolate pairs 50 Table 5a. Percentage of T K W T plaques wi th in A C T G 095 isolates by 1 2 5 I d C plaque autoradiography 54 Table 5b. Percentage of T K W T plaques wi th in A C T G 095 isolates by 1 4 C TdR plaque autoradiography 55 Table 6. Intracerebral mouse neurovirulence studies of 1737 and 89-063 57 Table 7. A C V susceptibilities and T K uptakes of 1737 and 89-063 neurovirulent output strains 58 Table 8. A C V susceptibilities of ACTG-095 plaque isolates 70 LIST OF FIGURES Figure 1. Structures of acyclovir and foscarnet 6 Figure 2. Pathway of A C V activation and action 8 Figure 3. Timeline for clinical course of patient WJ 19 Figure 4a. 1 2 5 I d C plaque autoradiographs from WJ isolates 42 Figure 4b. 1 4 C TdR plaque autoradiographs from WJ isolates 43 Figure 5. Derivation of input and output WJ neurovirulence strains 47 Figure 6a. 1 2 5 I d C plaque autoradiography of A C T G 095 isolates 52 Figure 6b. 1 4 C TdR plaque autoradiography of A C T G 095 isolates 53 Figure 7a. 1 2 5 I d C plaque autoradiography of 89-063 and 1737 intracerebral output strains 59 Figure 7b. 1 4 C TdR plaque autoradiography of 89-063 and 1737 intracerebral output strains 60 Figure 8. 1 2 5 I d C plaque autoradiography of WJ-derived clinical and plaque isolates 62 Figure 9. T K mutations wi th in WJ isolates 64 Figure 10a. W i l d type T K 65 Figure 10b. Mutation wi th in 1737-14 66 Figure 11. Immunoprecipitation of W J isolates 68 v i i i Figure 12. Western blot analysis of WJ isolates 69 Figure 13. A C T G 095 A C V R mutational screen 72 Figure 14 . A C T G 095 A C V S mutational screen 73 Figure 15. Western blot analysis of A C T G 095 A C V R plaque isolates 74 Figure 16a. A C T G 095 plaque isolate 1 2 5 I d C plaque autoradiography 76 Figure 16b. A C T G 095 plaque isolate 1 4 C TdR plaque autoradiography 77 Figure 17. Western blot analysis of A C T G 095 A C V s plaque isolates 78 ix LIST OF ABBREVIATIONS H S V herpes simplex virus H I V human immunodeficiency virus A I D S acquired immunodeficiency syndrome D N A deoxyribonucleic acid R N A ribonucleic acid T K thymidine kinase L A T latency-associated transcript A C V acyclovir P F A phosphonoformate (foscarnet) A C V R acyclovir-resistant A C V S acyclovir-sensitive pol vira l D N A polymerase T K D TK-deficient T K A TK-altered X K W T TK-deficient T K L P TK- low producer ts temperature-sensitive PCP Pneumocystis carinii pneumonia A C T G AIDS Clinical Trials Group H F F human foreskin fibroblast M E M min imal essential med ium D M E M Dulbecco's minimal essential medium HEPES N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid FCS fetal calf serum E D T A ethylenediaminetetraacetic acid PBS phosphate buffered saline CPE cytopathic effect ID50 50% inhibitory concentration 1 2 5 I V a r a U l-fi-D-arabinofuranosyl-E-5-(2-[ 1 2 5I]-iodovinyl)uracil M O I multiplicity of infection C 0 2 carbon dioxide 125ldC 1 2 5 Iododeoxycytidine 1 4 C TdR 1 4 C thymidine N a C l sodium chloride NP-40 nonidet P-40 T A E tris-acetate, E D T A T B E tris, boric acid, E D T A D M S O dimethylsulphoxide T E M E D N,N,N' ,N' - te t ramethyle thylenediamine A P S ammonium persulfate SDS sodium dodecyl sulphate B U d R 5-bromo-2'-deoxyuridine Single letter code for nucleosides: A , adenine; C , cytosine; T, thymidine; G , guanosine xi ACKNOWLEDGMENTS I thank Dr. Sharon Safrin for her generous donation of the A C T G 095 clinical isolates and Dr. K e n Powel l for his donation of the ant i -HSV 2 T K monoclonal antibody. I thank Dr . Christopher H . Sherlock and The University of British Columbia Diagnostic Virology Laboratory for providing us wi th laboratory isolates of herpes simplex virus from patient WJ. I thank Kathryn Schubert and Mar ia Hubinette for their technical assistance i n the sequencing and protein studies. I thank the members of my committee: Drs. Frank Tufaro, Lome Kastrukoff, Grant Stiver and D ' A n n Rochon for their advice and guidance. I thank al l the individuals of the Sacks and Tufaro laboratories who taught me the basics of laboratory v i ro logy and helped troubleshoot the pitfalls along the way. I thank Dr. Frank Tufaro for allowing me to expand the project to include molecular studies by providing space in his laboratory and giving timely advice. Finally, I thank Professor Stephen Sacks, my supervisor, who init ial ly enticed me from Austral ia and then not only allowed me the latitude to pursue the avenues I wanted but paved the way and provided great encouragement. I was a recipient of the Burroughs Wellcome A n t i v i r a l Research Fellowship and the work was funded, i n part, by grants from the Medical Research Counci l of Canada and the Canadian Genetics Disease Network. x i i DEDICATION To my late father who was not able to see the efforts of his labors bear fruit i n his sons and my mother and brother from w h o m I am separated by thousands of miles. x i i i CHAPTER 1 INTRODUCTION 1.1. H E R P E S S I M P L E X VIRUS INFECTIONS Herpes simplex virus (HSV) infections occur worldwide and are one of the most common afflictions affecting mankind. Prevalence is proport ional to socioeconomic status, probably due to overcrowding. Recent seroprevalence studies from developed areas, such as Seattle, in the 1970's have demonstrated H S V antibodies in 40% of adults aged 25-29 years old w i t h a 1.5% increase per year thereafter up to age 50. Earlier studies from the 1940's and 1950's reported prevalence rates of up to 90% (1). Type-specific serologies to H S V 2, the most common causal agent of genital herpes, performed in the late 1970's demonstrated seropositivity in 16% of the United States population aged 15-74 years of age, a much larger percentage than generally appreciated (2). A follow-up study by the same group in 1990 demonstrated that H S V 2 seroprevalence had risen to 22% despite the intervening period encompassing the H I V pandemic which heightened awareness about safer sexual practices (3). This illustrates the scope of the growing epidemic. H S V 2 seroprevalence rates begin to rise coincident wi th the onset of sexual activity. Women, as is the case wi th other sexually transmitted diseases, are more commonly affected than men. Seroprevalence rates have also been shown to be influenced by socioeconomic status and race (2,4,5). H S V is a double stranded D N A virus approximately 150 nm i n diameter. It is surrounded by a l ip id envelope derived from modified cell membrane as it buds from the host cell and from which virus-specific 1 glycoproteins protrude, these being crucial in mediating vi ra l attachment and entry into the host cell. The membrane surrounds an icosahedral protein capsid composed of 162 capsomers, this in turn housing the genome. Between the capsid and envelope lies an amorphous material k n o w n as the tegument which contains a number of viral-encoded proteins. The genome is a linear double-stranded D N A molecule approximately 150 kb long which encodes over 70 protein products. A number of clinical, epidemiological and biological differences allow distinction between 2 H S V types, HSV-1 and HSV-2 , and there is a large degree of homology between the genomes of HSV-1 and H S V -2, about 50% of the sequences being highly conserved (4,6). Infection is initiated by inoculation of virus onto skin wi th an epithelial breach or mucosal surfaces. The new replication cycle of H S V commences wi th the attachment of membrane glycoproteins to heparan sulfate; a ubiquitous, normal component of host cells which acts as the cell surface receptor. After attachment, the viral envelope fuses wi th the host cell plasma membrane and nucleocapsids are liberated into the cytoplasm (7). Next the unenveloped virus migrates to the nucleus where it is uncoated at nuclear pores and the 'naked' D N A enters the nucleus and circularizes. Three sets of vira l genes are then expressed in an orderly sequence. The first set of genes, known as alpha or immediate-early, are expressed prior to protein synthesis and their transcription is initiated by a prepackaged transcriptional transactivator, VP16, a constituent of the tegument. A l p h a gene products are themselves transcriptional transactivators and induce expression of the beta (early) and gamma (late) genes while simultaneously downregulating cellular gene transcription. Beta genes are expressed prior to and are predominantly involved in D N A replication and include thymidine kinase 2 (TK). Late genes are expressed after D N A replication and are predominantly structural. The replicated D N A is then cleaved to an appropriate size and packaged. Finally, the v i r ion acquires an envelope as it buds from the inner nuclear membrane and, after glycoprotein modification in the endoplasmic reticulum and Golgi apparatus, leaves the cell as a mature v i r ion able to perpetuate infection in other cells by repeating the cycle of events detailed above. The generation of large numbers of virions i n permissive cells is associated wi th cell death and completes the lytic cycle of the virus (4,8,9). In a patient naive to H S V a primary infection ensues. This may be symptomatic or asymptomatic. In either case there may be sufficient v i r a l replication to result in infection of terminal branches of sensory endings i n which case the nucleocapsid is then transported centripetally inside axons to the cell body in sensory ganglia (10,11). A t this stage, a l imited degree of local replication occurs in ganglia which may extend to neighboring neuronal and nonneuronal cells, some of which may demonstrate cytopathic effect (12-14). The host immune system eventually controls the mucocutaneous v i r a l replication and epithelial damage, resulting in resolution of the primary infection. Simultaneously, the pattern of gene expression changes, such that all genes responsible for viral replication are shut off, leaving only one region of the genome transcriptionally active to produce a family of transcripts known as latency-associated transcripts (LAT's) (15-17). A t this time, infectious virus is undetectable except by ganglionic explantation, surface vi ra l proteins are undetectable and virus is latent, thereby evading both immune attack and currently available antiviral chemotherapies. This latent state underlies our current inability to "cure" patients of H S V infection. As a measure of extent of latent infection, in situ hybridization studies show that 3 0.1-3.0 percent of human neurons contain L A T ' s (16,17). H S V may be subsequently reactivated from its latent state via certain trigger factors, some defined, others not. Fol lowing reactivation, virus travels centrifugally to reinitiate productive mucocutaneous infection at or close to the site of in i t i a l infection. The recurrence is modified by the preexisting immune response and, in cases where the primary infection was clinically evident, recurrences are generally briefer in duration and/or less intense (18). Genital herpes can be caused by either HSV-1 or HSV-2 . HSV-1 may account for anywhere between 7-50% of first episode lesions, depending o n geographical variation. H S V 1, however, only causes 2% of recurrent genital herpes, suggesting that H S V 2 is better adapted to either exist wi th in and/or reactivate from sacral ganglia (18). Typical lesions of genital herpes may involve any region of the body supplied by the sacral plexus although the vulva and external genitalia are the sites most commonly involved. In male homosexuals and female recipients of anal intercourse, patients may sustain a proctitis and perianal ulceration. Herpetic lesions typically evolve through identifiable and distinct stages which, in temporal order, may include macules, papules, vesicles, ulcers and crusts before eventually healing. O n mucous membranes the early stages are not usually apparent and ulcers are often the sole manifestation. Patients who are immunocompromised , particularly those wi th cell mediated immuni ty defects such as those w i t h A I D S and transplant recipients, constitute a special subgroup as they may be susceptible to more frequent, more chronic and more severe recurrences and shed virus for more prolonged periods of time. In some such individuals lesions may persist for weeks to months and the prolonged associated v i r a l shedding predisposes to the development of antiviral drug resistance (19-22). 4 1.2. A N T I V I R A L A G E N T S A N D A C Y C L O V I R Ant iv i ra l therapy includes specific antiviral drugs as wel l as i m m u n e modulators, such as interferon, which augment the host immune system. The vira l replicative cycle offers mult iple potential sites of in tervent ion when considering the development of effective antiviral therapy. Stages i n this cycle include attachment to host cell receptors, penetration into the cell, uncoating of nucleocapsid, replication of genetic material, synthesis and processing of viral-encoded proteins, assembly of new virions and egress out of the cell. Due to their relative simplicity compared to other microorganisms, viruses need to "hijack" variable amounts of host cell machinery to replicate. Such a lack of distinct v i ra l targets has been a major stumbling-block in the development of antiviral agents as few agents have been found to be sufficiently selective in inhibit ing virus-specific functions while simultaneously avoiding unacceptable host cell toxicity. Agents directed at several sites of action have entered into clinical practice al though the main thrust of established antiviral therapy has been at the interrupt ion of D N A synthesis in herpesviruses and retroviruses. The main class of effective antiviral agents to enter clinical practice has been nucleoside analogues which are analogues of naturally occurring D N A bases and w h i c h block vira l D N A replication. The prototypic agent, acyclovir ( A C V ) was released for use in clinical practice in the late 1970's and quickly established a reputation as a safe and effective antiviral agent. A C V is an acyclic analogue of the naturally-occurring D N A base deoxyguanosine (Figure 1.) and is a selective inhibitor of H S V types 1 and 2 as we l l as varicella-zoster virus where in vitro assays show average median effective concentrations of 0.04, 0.10 and 0.50 | i g / m l respectively (23). Like all nucleosides, A C V needs to be 5 + re z CO 8 X X o O o — * — X o X _C '35 o c re 3 O) X o <D Q > O o >l o < re a '1 2 re"i-* r O O . c "> o § 1 .2 a u £ « 0) O re 0) •II o re _ E £ E re -I-. *•« •2 8 2 re ^ o) a> — i» +•* re S< a> O CL 0) c o _ a> c re re _a i - "O E o u re (A re re o o 0 •a 9 re re0) 3 U) o re c 3 re o -S 5 "35 m ° •g § c O "fe i to a) o 3 a> (/) o a> II re c re triphosphorylated to enable it to be incorporated into a growing D N A chain. In HSV-infected cells A C V is selectively phosphorylated to its monophosphate by a virus-encoded enzyme, thymidine kinase (TK), a reaction which does not occur in appreciable amounts in uninfected cells. Subsequent d i - and tri-phosphorylation is carried out by cellular kinases and ultimately results in the production of A C V triphosphate ( A C V - T P ) concentrations 40-100 times greater than in uninfected cells (24). V i r a l D N A polymerase also has a greater affinity for A C V - T P than does host D N A polymerase, resulting in little incorporation of A C V - T P into cellular D N A (25). A l l these properties confer selectivity, safety and potency on the drug. The mode of action of A C V - T P on vi ra l D N A is threefold. Firstly, it competes wi th deoxyguanosine triphosphate; secondly, the lack of a 3 'OH group results in an inability to form phosphodiester bonds wi th subsequent nucleotides, resulting in chain termination; thirdly, it acts as a "suicidal inactivator" of the polymerase by forming a tight association wi th v i ra l D N A polymerase and the terminated D N A to inhibit its function (Figure 2.) (26,27). Since its introduction A C V has established a track record of efficacy and become the standard of treatment and prophylaxis of herpes simplex virus infections i n both immunocompetent and immunocompromised hosts (23). Tr isodium phosphonoformate (foscarnet, PFA) , a pyrophosphate rather than nucleoside analogue (Figure 1.), also blocks vira l D N A replication but does not require vira l T K for activation and works by competit ively blocking the pyrophosphate binding site of the viral D N A polymerase (28). Its main clinical indication has been as alternative therapy for cytomegalovirus infections. Its independence of the requirement for T K activation, however , offers alternative therapy for patients wi th ACV-resistant ( A C V R ) outbreaks 7 V u "O 0) o 0) 3 « 0> c o y _c / / E / >» _ i (0 < z Q CO re -Q < 0> Z 8 4 2 3 Q. O h S i 4 « O (Q ** £ T3 Q. - 8 o a > |< >».*; re tn > O < o > c o o tn o tn re c o .> u re c re (0 re E >» o a> Z O Q III re <D f_ > -° o t) « c ra 8 . 2 > c £ 0 3 5 < rope -C -C c ra ~ .2 °- 1! re j "o .F o c CM ° P TO " E il § 3 and controlled trials have demonstrated its efficacy in this setting (29). This less selective mode of action is associated wi th increased host cell toxicity including renal dysfunction, nausea and vomit ing, disturbances in ca lc ium and phosphate homeostasis, anemia, penile ulceration and local thrombophlebitis. In addition, P F A can only be administered intravenously. These drawbacks preclude its use as a first line agent in the treatment of H S V infections (30,31). 1.3. A C Y C L O V I R RESISTANCE A s wi th all antimicrobial agents, reports of resistance to A C V began to emerge soon after the release of the drug. In vitro resistance of herpes simplex to A C V was first reported in 1980 when it was shown that less sensitive herpes simplex viruses could be selected during exposure to A C V in tissue culture (32). The init ial clinical A C V R isolates were isolated from immunodeficient patients in 1982 (33,34). Mutat ion at two sites in the H S V genome have been shown to confer resistance; the T K and polymerase (pol) genes. Mutations in the T K gene may result in two different phenotypes. They may result i n deficiency of enzyme activity to all substrates and these are termed TK-deficient ( T K D ) . Alternatively, the mutation may cause the enzyme to narrow its normally promiscuous spectrum of nucleoside activity to exclude A C V but still be able to phosphorylate its naturally occurring substrate, thymidine; the latter are termed TK-altered ( T K A ) . The overwhelming proportion of isolates exhibiting the A C V R phenotype are due to T K deficiency, T K A or pol mutants being rarely reported (32,35-37). Al though occasional studies have demonstrated A C V R isolates in the n o r m a l host prior to and developing during treatment, sensitivity patterns bear no relation to subsequent clinical course and such isolates were rapidly cleared 9 (38-40). Presumably, in such cases a normal immune system clears v i rus despite in vitro resistance. There has only been one clinically-significant A C V R isolate reported in an immunocompetent host. This was a T K A isolate which was unresponsive to A C V and which clearly retained T K activity for thymidine (41). The population almost exclusively subjected to clinically significant A C V resistance has been the immunocompromised host where this has been estimated to occur in 4.7% of such patients (42). Immunocompromised states associated wi th clinical resistance include organ (particularly bone marrow) transplantation (43,44), AIDS (45-47), hematological malignancy (48,49) and congenital immunodeficiency states (50-52). Such patients suffer chronic mucocutaneous lesions unresponsive to and often progressive on A C V despite high doses. The overwhe lming majority of clinically resistant isolates have been of the T K D phenotype although pol and T K A mutants have occasionally been described (42, 53-57). The biological behaviour of T K D isolates in vitro and in an imal models differs from T K w i l d type ( T K W T ) strains. Such mutants are capable of efficient replication in actively d iv id ing cells but not in serum starved or non-dividing cells. They can also replicate efficiently at peripheral sites of inoculation but, although they are able to establish latency, they are less efficient, resulting in a lower incidence of infection and yielding a lower titer of virus. These mutants are also at least one hundred times less neurovirulent in a mouse model and this may reflect a need for T K activity i n non-dividing cells where such endogenous activity may be lacking (58-64). Finally, and especially germane to the clinical situation, despite having the ability to establish and maintain latency, these mutants have been repeatedly demonstrated to be unable to reactivate in this animal model (65-70). This 10 concept has been further reinforced by the demonstrated ability of T K inhibitors to reduce reactivation (71). These findings, together wi th the dearth of reports of reactivation ability in patients, have contributed to the cl inical dogma that T K D reactivations, once effectively sterilized and in the absence of any ongoing antiviral pressure, w i l l be followed by a ACV-susceptible ( A C V S ) reactivation. It has also been shown that the degree of T K activity correlates wi th the neuropathogenicity, impacting on both virus ganglion titer and encephalitis (63,64). In addition, despite a requirement for T K activity, even low levels of T K activity below 10% are sufficient to allow reactivation i n mouse models (72). Finally, it needs to be appreciated that the level of T K expressed by different viruses seems to be a spectrum and strains can be identified which exhibit the A C V R phenotype during in vitro susceptibility testing but demonstrate low but detectable levels of T K , so called T K low producer ( T K L P ) strains. Such isolates have been arbitrarily defined as hav ing <15% activity by enzyme kinetic experiments. The mechanistic basis underlying these strains is unknown at present (56,63). 1.4. T H Y M I D I N E K I N A S E The T K of H S V was first identified i n 1963 by K i t and Dubbs (73). Al though initially it was thought to exclusively catalyze the phosphorylation of thymidine the enzyme was later subsequently shown to be able to phosphorylate a number of other pyrimidine deoxyribonucleosides. It may thus be more accurately termed a deoxypyrimidine kinase (74-76). In its denatured form, T K has a molecular weight of 40,000-45,000 daltons o n polyacrylamide gels (77,78) while in its native form it has a molecular weight of about 82,000 daltons (79). The enzyme exists as a homodimer of identical subunits and one putative mechanism of complementation proposed the 11 formation of a heterodimer, such that one w i l d type polypeptide compensates for the lack of activity of its mutated partner (80,81). For some nucleoside analogue antiviral agents (e.g. A C V ) the init ial phosphorylation in the sequence of triphosphorylation to the active compound is selectively catalyzed by a viral-encoded T K rather than a host-specified kinase, conferring specificity for virus-infected cells. A s described above, resistance to pyrimidine analogues, including A C V , is most commonly mediated by alterations in the function of T K and by far the most common such alteration is a deficiency of enzyme activity. Mutations in the gene coding for T K which result i n resistance to A C V are usually caused by the alteration i n a single base (82). Two types of T K D mutants have been described. The gene may harbor a base pair substitution, resulting in a missense mutation and producing an intact peptide wi th negligible enzymatic activity. Alternatively, there may be a base pair insertion or deletion to produce a nonsense mutation in which there is a frameshift resulting in the coding of a premature stop codon to produce a truncated peptide wi th absent activity. Some mutants do not exhibit a detectable peptide; this may be the result of rapid degradation of the peptide, there may be a very proximal nonsense mutation or there may be mutations i n the promoter and /or enhancer elements (83). H S V 1 and 2 T K genes have been sequenced for strains CI 101 and 333 respectively. They are located at a collinear position on the H S V genome around map position 0.30. The complete gene, including the coding region and its flanking sequences, is 1656 base pairs in length. There is extensive (80%) homology between the coding regions of H S V 1 and 2, suggesting a common ancestry, the basis of variations between the 2 types being a series of single base changes. The site for initiation of translation is located at 12 nucleotide 333 which signals the start of an open reading frame of 1128 nucleotides followed by an T A G termination codon, beyond which is a polyadenylation signal. The open reading frame codes for a polypeptide of 376 amino acids (84,85). Three conserved regions have been identified on the basis of altered substrate binding properties i n mutants as wel l as homology studies and these constitute the putative "active centre" of the enzyme. The three sites consist of the binding site for A T P , the phosphate donor, located between amino acids 51 to 63 near the N-terminus of the protein, the nucleoside binding site between amino acids 168-176 located in the middle of the protein and amino acid 336 near the C-terminal end of the protein w h i c h participates in both A T P and nucleoside binding (86). 1.5. H E T E R O G E N E I T Y A N D C O M P L E M E N T A T I O N Naturally occurring A C V R mutants can be detected wi th in c l inical isolates in normal hosts in the absence of drug pressure even i n patients naive to A C V . They occur at a relatively high frequency of 1 i n 10 4 pfu, although the clinical isolate as a whole population tests as A C V S by susceptibility assays. Such subpopulations can be demonstrated in diverse clinical settings including cases of gingivostomatitis, genital herpes, encephalitis and systemic infection. These isolates have been shown to consist of heterogeneous mixtures of A C V S , T K D , T K A and pot mutants and the existence of such subpopulations illustrates how selection pressure may ultimately select out a clinically significant A C V R population (54,87,88). This is supported by animal models which have shown the progressive emergence of A C V R isolates coincident wi th a reduction in T K activity using serial passages of a H S V 1 strain in mice treated wi th suboptimal doses of A C V . The same work also demonstrated the re-emergence of an A C V S popula t ion 13 when antiviral pressure was removed (89). The relative contributions of passive selection as opposed to increased mutational rates due to such pressure is currently unknown. H S V has been shown to be capable of complementation, such that a mutant virus can have its defective gene function compensated for by another virus wi th intact gene function. This phenomenon has been demonstrated between intertypic temperature sensitive (fs) H S V mutant pairs, for neuroinvasiveness and for T K activity. Genetic recombination has been demonstrated to occur between some but not all such complement ing pairs and likely underlies compensation of function in some cases. In fact, recombinant frequencies of up to 2% have been reported between pairs of T K mutants. Some investigators draw a distinction between recombination and situations where complementation occurs in the absence of demonstrable recombinant events. The relative contribution of recombination and n o n recombinant complementation in the above settings is unclear. In practice genetic recombinants are often generated when complementation studies are conducted, making the separation of underlying mechanisms difficult. For the purposes of this paper, such a distinction w i l l not be made and the te rm complementation w i l l include all potential underlying mechanisms including recombination. (81,90-95). Several lines of evidence support the concept of complementat ion wi th in A C V R isolates. In vitro work wi th cells infected wi th such mutant pairs can be shown to increase T K activity beyond levels of cells infected w i t h each mutant alone while, conversely, the addition of T K D virus to a T K W T population can reduce T K activity wi th in that population (81). In an ima l models, studies have demonstrated complementation can occur between 14 T K D / T K D and T K D / T K W T pairs to produce increased T K D titers of v i rus from mouse trigeminal ganglia. Such experiments showed a correlation between trigeminal ganglion virus titer and percent T K activity and showed that as little as 20% activity could yield virus titers near T K W T levels (63,67). The most striking demonstration of such complementation is shown by the reactivation of a genetically-engineered T K D virus, having established latency in mouse cervical ganglia, only if ganglia were superinfected wi th a T K W T strain; so called "superinfection rescue" (65). In the clinical setting complementation has been documented for both T K D and pol mutants. In one report of an isolate from a case of esophagitis from a bone mar row transplant recipient, plaque purification of 10 plaques from the clinical isolate showed that 2 T K D plaque isolates and 2 pol plaque isolates imparted clinically-significant A C V resistance onto the larger 6 A C V S plaque isolates (54). In another report of a clinically A C V R throat isolate from an A I D S patient, a subpopulation of 3 of 17 T K D plaque isolates allowed for cl inical resistance i n the face of an A C V S phenotype by susceptibility testing, suggesting that such testing could not always reliably predict clinical outcome (96). In all cases, it seems that the nature of the complementation functions to enhance virus survival . In v iew of the principle that T K D isolates are not able to reactivate, the work described wi th in this thesis pr imari ly set out to determine why a T K D H S V 2 clinical isolate in an AIDS patient described below exhibited apparently paradoxical behaviour by reactivating in the absence of antiviral drug pressure. The init ial hypothesis was that the isolate was inherently different from other T K D isolates and the work was originally directed at proving this. The first phase of the work involved tissue culture characterization to 15 exclude T K heterogeneity by currently-available methods followed by a n i m a l model studies to determine whether reactivation ability was associated w i t h increased neurovirulence. In the next phase, molecular characterization of this isolate was performed in an attempt to correlate reactivation ability to T K genotype. Finally, the above work was extended to characterize other A C V R clinical isolates which had also reactivated. 16 C H A P T E R 2 M A T E R I A L S A N D M E T H O D S 2.1. P A T I E N T S 2.1.1. Patient WJ WJ was a 45 year old mother of two who sustained a probable pr imary episode of genital herpes in A p r i l 1984. She presented wi th a 3 week history of genital ulceration and a necrotic cervical lesion associated wi th discharge, both of which yielded positive cultures for H S V 2. The first week of her illness was characterized by marked systemic symptoms and dysuria. Her partner had likely suffered intermittent genital herpes lesions for several years which had never been diagnosed. Her past history included gonorrhea, trichomonas and yeast infections, the removal of a benign cervical polyp, a dilatation and curettage following a threatened abortion, 2 episodes of shingles and a tonsillectomy. In addition, in February 1984 she suffered an undiagnosed illness lasting 1 week and characterized by fever, night sweats and rash. Her partner suffered a similar illness lasting 2 weeks in September 1983. Both these are l ikely to have been a human immunodeficiency v i ru s (HIV) seroconversion illnesses. She experienced regular (approximately 3 monthly) recurrences of genital herpes which she self medicated wi th short courses of A C V . In 1985 she was found to be H I V antibody positive fo l lowing the H I V diagnosis of her bisexual partner. In March 1986 she was admitted following a curtailed vacation to South America where she had been diagnosed wi th cervical carcinoma and treated by cone biopsy but continued to suffer persistent cervical discharge, lower abdominal tenderness and night sweats. She clinically improved on clindamycin and gentamicin and her CD4 17 count at this time was 990 /mm 3 . In M a y 1988 she suffered a bout of Pneumocystis carinii pneumonia (PCP) which was successfully treated but by this time her CD4 count had dropped to 3 0 / m m 3 and she was diagnosed as suffering from the acquired immune deficiency syndrome (AIDS). A further episode of P C P followed in M a y 1989 and was successfully treated. In September 1989, she was admitted to hospital wi th a 6 week history of an H S V recurrence unresponsive to 4 weeks of oral A C V at doses of up to 800 mg qid. She was initially treated wi th intravenous A C V at doses of up to 10 m g / k g 8 hourly for 3 weeks without effect unt i l A C V susceptibility testing demonstrated the clinical isolates from this outbreak ( 1090,1091,1104,1105 and 1106) to be A C V R . She was treated wi th foscarnet (PFA) 60 m g / k g intravenously 8 hourly for 14 days wi th subsequent rapid clinical and virological resolution of her lesion. She sustained a further brief recurrence in November 1989 which responded to oral A C V (isolate 1201). Further recurrences clinically resistant to A C V soon followed, however, i n January 1990 (isolates 1280,1286,1288,1307), A p r i l 1990 (isolates 1420,1423,1424) and M a y 1990 (isolates 1563,1564,1566). The first of these was after attempted self medication wi th A C V and the other two in the setting of A C V prophylaxis. Each of these episodes also responded rapidly to intravenous P F A as above. Due to the frequency of recurrences, after the fourth episode she was placed on P F A prophylaxis 60 m g / k g twice daily. This was successful unt i l , after weaning her prophylaxis down to every third day, she suffered a further recurrence in July 1990 (no cultures obtained). She self medicated wi th three doses of 60 m g / k g P F A 8 hourly to good clinical effect as documented at two office visits and decided to cease all antiviral treatment. In August 1990, some 18 re CD Q o z o n ^ 3 3 3 O t D O O S 00 CO 00 o CM CM Ol CO o CM o T - f m io fflfflOOO O O T - T - T ~ re E > C CO 0. X Q T> 8- °> o en to " D) on I ? o — o> 3 CO a S o CD o C CO re <» > s IS CD CO CO r-00 0. co u m + I I > < o o > 0. > 5> </> C a, — * J 73 flj 1 1 ° CJ) > w > * C/5 ^ ° I (/> u, O) l i J3 II f o o z to o a CO c — CD O £ </> ~ •~ > I | O = 0 ra 1 ° iE o * 2 -o 0) c CO 2 a> o -o ^  a> -r o> i l l _ 73 CO CO CD O CO N ° ^ 2 * W S o co co o S2 o ^ ^ > c E c C 3 CD co -2 g CO w TOO "9 five weeks after cessation of antiviral therapy, she was readmitted w i t h neurological complications of H I V infection and was noted to have a further H S V recurrence which was later shown to be A C V R (isolate 1737). She was treated for only 3 days due to PFA-induced symptomatic hypocalcemia but had a satisfactory clinical and virological response and was discharged on n o antiviral treatment. In September 1990 she sustained another recurrence which was found to be A C V S (isolates 1773,1774,1775) and for which she was discharged home for terminal care on P F A 60 m g / k g q8h. She died at home in December 1990 of causes unrelated to her H S V infection. Post mortem was not performed. A time line depicting these events is outlined in Figure 3. 2.1.2. Clinical isolates derived from A C T G 095 The remaining clinical isolates were graciously provided by Dr. Sharon Safrin from The University of California, San Francisco. They were sourced from a randomized clinical trial comparing P F A wi th vidarabine (ara-A), another nucleoside analogue but one which does not require activation by T K , in the treatment of resistant H S V in fourteen AIDS patients. This tr ial was sponsored by the AIDS Cl in ica l Trial Group (ACTG) and was ascribed study number 095 (29). Inclusion criteria required patients to be H I V positive and have a mucocutaneous H S V lesion unresponsive to 2 weeks or more of oral A C V therapy or 10 days or more of intravenous therapy and that cl inical isolates from these outbreaks have in vitro demonstration of resistance to A C V and susceptibility to P F A and ara-A. After random assignment, patients were administered either P F A 40 m g / k g 8 hourly or ara-A 15 mg per day intravenously for a min imum of 10 days. Patients who had completely healed by day 10 ceased therapy while those wi th a partial response continued for up to 42 days. Patients who failed treatment or were intolerant were allowed to 20 cross over to the alternative drug. Fol lowing completion of therapy, patients who completely healed were monitored for 3 months for recurrence of H S V lesions during which time the use of antiviral agents was not permitted. The study was terminated after the enrollment of 14 patients due to toxicity and lack of efficacy of ara-A. We initially received 4 pairs of isolates. These consisted of all first clinical reactivations which tested A C V R and their respective A C V R clinical isolates which qualified the patients for study entry. These pairs were: clinical isolates 89061/89063, 89353/89350, 90150/90030 and 2370/89660. We later received a further 3 pairs of clinical isolates w h i c h consisted of A C V R isolates qualifying for study entry but in whom first reactivations were A C V s . These isolates were clinical isolates 89390/89391, 89650/89641 and 90110/3187. These clinical isolates were used for further evaluation to extend the ini t ial findings from isolates originating f rom patient WJ. 2.2. TISSUE C U L T U R E 2.2.1. Cel l lines A l l tissue culture flasks, tubes and dishes were Falcon brand and purchased from Baxter Diagnostics Corporation, Canlab D i v i s i o n , Mississauga, Ontario, Canada. Unless otherwise stated, all media and supplements were purchased from Canadian Life Technologies Inc., (Burlington, Ontario, Canada). African green monkey kidney cells ( V E R O , Flow Laboratories, Inc., McLean, Va.), and human foreskin fibroblasts (HFF, Clonetics Corp. San Diego, Ca.) were purchased commercially and passaged i n 5% M E M [minimum essential medium containing Earle's Salts ( M E M ) , supplemented wi th 5% inactivated fetal calf serum (FCS), penici l l in (100 21 IU /ml ) , streptomycin (100 ug/ml) , amphotericin B (2.5 ug/ml) , glutamine (2uM), buffered to a p H of 7.4 wi th 10 m M N-2-hydroxyethylpiperazine-N'-2-ethansulphonic acid (HEPES), and sodium bicarbonate (0.15%)]. Ce l l line 143B (HOSFTK-; [human osteosarcoma, thymidine kinase-deficient]), was provided by Dr. Si lvia Barchetti, McMaster Universi ty (Hamilton, Ontario, Canada). 143B cells were maintained in M E M as above except that this was supplemented wi th 10% FCS (10% M E M ) , and 50 u g / m l bromodeoxyuridine (Sigma Chemical Company, St. Louis Missouri). Cells were split by trypsinizing wi th 0.25% trypsin in versene [0.02% E D T A (ethylenediamine-tetraacetic acid) i n PBS] and maintained at 37° C in 5% CO2. 2.2.2. Isolation and Growth of Clinical Isolates of H S V Clinical isolates were obtained by swabbing lesions wi th D a c r o n ™ swabs (American Scientific Products, M c G r a w Park, IL.) by hospital personnel and inserted into vials containing v i ra l transport medium [Medium 199 w i t h Hank's salts and L-glutamine supplemented wi th 1% FCS, penici l l in (100 I U / m l ) , streptomycin (100 jxg/ml) and amphotericin B (2.5 ixg/ml) and buffered to a p H of 7.4 wi th 10 m M N-2-hydroxyethylpiperazine-N'-2-ethansulphonic acid (HEPES), and sodium bicarbonate (0.075%)]. They were then inoculated into duplicate monolayers of human foreskin fibroblasts (HFF's) (passage <20) in duplicate 16 x 125 mm polystyrene tissue culture tubes seeded wi th 4 X 10 5 cells containing 5% M E M . After one day growth at 37° in 5% CO2, they were inoculated wi th 100 ul of virus media. Cells were then incubated at 37°C in 5% CO2 and observed daily by microscopy for cytopathic effect (CPE). Infected cells from positive cultures were confirmed and typed by direct immunofluorescence. They were suspended in 200 ul of a 1:1 solution of ethanol and PBS, p H 7.2 and the suspensions were air dried o n 22 microscope slides, fixed with acetone, incubated for 30 m i n at 37° C w i t h fluorescein isothiocyanate-labelled monoclonal antibodies directed against type-specific antigens of HSV-1 and HSV-2 (Syva, Palo Al to , California) and examined under a mercury vapor fluorescence microscope (Nikon, Canada, Inc., Vancouver, British Columbia, Canada). V i r a l stocks were grown i n confluent V E R O cell monolayers in 175 c m 2 flasks at 37° C i n 5% carbon dioxide. The monolayer was inoculated wi th approximately 200 pll of v i r a l transport med ium containing the swab and incubated unt i l approximately >70% CPE was seen. The infected monolayer was removed wi th glass beads, centrifuged at 1000 rpm for 5 min (Beckman J-6B, Beckman Instruments, Palo Al to , Ca, U S A ) to pellet cells, media removed to leave a residual volume of 4 ml , cells resuspended by vortexing (Vortex-Genie, Scientific Industries, Bohemia, N Y , U S A ) , frozen at -70° C and thawed. They were then sonicated (Braunsonic 1510 sonicator, Baxter Diagnostics Corporation, Canlab D i v i s i o n , Mississauga, Ontario, Canada) for 15 s at 150 watts, the cell debris was removed by centrifugation at 1200 rpm for 5 min, the supernatant transferred to a new tube and aliquots removed into 1.8 m l cryotubes (Nunc, Canadian Life Technologies Inc., Burlington, Ontario, Canada). V i ra l stocks were titered after adsorbing onto confluent V E R O cell monolayers in 24 we l l plates. 200 (xl of duplicate serial fivefold dilutions of vira l stock in 2% Hanks media [Medium 199 wi th Hank's salts and L-glutamine, supplemented w i t h 2% FCS, penici l l in (100 I U / m l ) , streptomycin (100 p:g/ml) and amphotericin B (2.5 pig/ml) and buffered to a p H of 7.4 w i th 10 m M N-2-hydroxyethylpiperazine-N'-2-ethansulphonic acid (HEPES), and sodium bicarbonate (0.15%)] were adsorbed onto the monolayer for 1 h and then overlaid wi th 2% Hanks media containing a 1:750 dilution of anti-HSV-1 rabbit polyclonal antibody (Dakopatts a/s, Dimension Laboratories Inc., Mississauga, Ontario, Canada) to 23 prevent secondary infection and incubated for 48 h. Media was then removed and cells fixed wi th 0.5 m l 10% formalin for 10 min , stained wi th 0.5 m l 0.5% crystal violet for 5 min, washed 3 times wi th water and air dried. The number of plaques was counted and expressed as plaque forming units (pfu's) per ml . 2.2.3. Susceptibility testing Plaque reduction assays (50% reduction; ID50) were performed by adsorbing approximately 50 pfu of titered virus stock in 200 ul 2% Hanks media onto confluent V E R O cell monolayers in 24 wel l plates for 1 h as described above. Wells were then overlaid in duplicate wi th 0.5 m l of 6 serial dilutions of A C V (Burroughs Wellcome, Research Triangle Park, Nor th Carolina) or P F A (Astra Arcus A B , Sodertalje, Sweden) in 2% Hanks media containing a 1:750 dilut ion of polyclonal anti HSV-1 antibody arid incubated for 48 h. Posi t ive virus control wells without drug and negative virus control wells wi thout drug or virus were included. Monolayers were fixed, stained, washed and dried as described above. Plaques in duplicated wells were counted manual ly , a mean of the two derived and the subsequent values used to generate a dose-response curve. Statview 512 software (Abacus Concepts Inc., Berkeley, Ca, U S A ) was then used to derive a linear regression equation from the linear part of the curve. The concentration of A C V or P F A required to reduce plaque formation by 50% compared to drug-free inoculated control wells was calculated from this equation and nominated as the ID50. Concentrations greater than 2 u g / m l and 100 f ig /ml were taken as the cutoff for resistance to A C V and P F A respectively. 24 2.2.4. Plaque isolation Duplicated dilutions of approximately 100, 10 and 1 pfu in 0.5 m l 5% M E M media from titered vi ra l isolates were adsorbed onto confluent V E R O cell monolayers in 6 wel l plates for 1 h. Wells were then overlaid wi th a mixture of 1.5 m l of 1% agarose and 1 m l double strength 5% M E M at 46° C which was allowed to set and incubated for 48 h. Individual well-separated plaques were picked by drawing up 1 fil using a P20 Gilson P i p e t m a n ™ (Mandel Scientific, Guelph, Ontario, Canada) and inoculated into a confluent V E R O cell monolayer in 25 c m 2 flasks . When flasks showed >70% CPE, infected monolayers were harvested as described above. 2.2.5. Relative TK activities This was done by comparing the uptake of l-B-D-arabinofuranosyl-E-5-(2-[125]-iodovinyl)uracil ( l ^ IVaraU) (2200 C i / m m o l ) by HFF's infected w i t h the strain of interest compared to H S V 2 strain G (American Type Tissue Collection, Rockville, Maryland). Polystyrene tissue culture tubes (16 x 125 mm) were seeded wi th approximately 3 x l 0 5 H F F cells in 2.0 mis 5% M E M and incubated overnight. Media was removed and replaced wi th 1 m l of 2% Hanks containing virus at a mult iplici ty of infection (MOI) of 0.1 and 2 x l 0 6 counts per minute (cpm) of 1 2 5 I V a r a U and incubated for a further 18 h. After inspection of the tubes to confirm the presence of early CPE, the media was then removed, the tubes were washed four times wi th 4 m l phosphate buffered saline (PBS), p H 7.4 at 37°C, and drained on a paper towel. Cells were trypsinized with 1 m l trypsin in versene as described above and the contents transferred to 12 X 75 m m polystyrene tubes and counted in a gamma counter (LKB-Wallac 1282 Compugamma, Fisher Scientific, Ottawa, Canada). 25 Uninfected cell background was subtracted and activity expressed as % T K activity relative to strain G . 2.2.6. Plaque autoradiography The method used for plaque autoradiography was a modification of that described by Mart in et. al. (97). A l l incubations were performed in 5% CO2 and 37°C. Tissue culture dishes (60 X 15 mm) were seeded wi th 4 m l 5% M E M containing 5 X 10 5 V E R O cells or 10 X 10 5 143B cells per m l and incubated overnight to achieve a confluent monolayer. Approximately 100 pfu of titered virus stocks in 1 m l of 5% M E M were inoculated, adsorbed for 1 h, over la id wi th a mixture of 2 m l of 1% methyl cellulose and 1 m l double strength M E M and incubated until adequate sized plaques formed as determined by daily microscopic evaluation. The overlay was then aspirated, 0.5 | i C i 1 2 5 I iododeoxycytidine ( 1 2 5 IdC) (2200 C i per mil l imole) added to V E R O cells and 1 4 C deoxythymidine ( 1 4 C TdR) (62.8 m C i / m i l l i m o l e ) added to 143B cells respectively and incubated for 4 h. Isotopes were purchased from Dupont N E N , Boston M A , U S A . Cells were then fixed wi th 4 m l 10% formal in , stained wi th 4 m l 0.5% crystal violet as described above, washed twice wi th 4 m l PBS and air dried. Circumferential rims were then removed, taped onto a paper template and placed in a F B X C 810 autoradiography cassette and exposed to Kodak X-Omat X A R X-ray film for 5 days (Fisher Scientific, Ottawa, Canada). The fi lm was then developed in a Kodak M 3 5 A X-Omat processor (Eastman Kodak, Rochester, N Y , U S A ) . Plaques on the monolayer were then assessed as to whether they incorporated radiolabelled nucleoside on the basis of the darkness of plaque rims on their corresponding autoradiographs. Dark rims indicated incorporation and demonstrated a TKWT phenotype w h i l e plaques lacking corresponding dark rims did not incorporate label and 26 indicated a T K D phenotype. Plaque numbers on monolayers and dark rims on autoradiographs were then counted and compared and a % of T K W T plaques generated. Finally, some isolates were seen to have very faint but discernible corresponding rims but tested as A C V R on susceptibility testing. These were only visible by direct inspection of autoradiographs against a light box and it was not possible to demonstrate these light rims on photographs of the autoradiographs such as those in the text below. These plaques were designated as expressing the TK- low producer ( T K L P ) phenotype. 2.3. A N I M A L STUDIES 2.3.1. Mouse intranasal neurovirulence model. The init ial animal model studies were designed to characterize the neurovirulence of isolates from patient WJ compared to controls. Therefore, in order to generate a dose-response curve for neurovirulence, the in i t i a l mouse studies were conducted using a modified intranasal model described elsewhere (98). Four week old female B A L B / c mice (Animal Care Center, University of British Columbia, Vancouver) were lightly anesthetized w i t h methoxyflurane (Pitman-Moore Ltd., Mississauga, Ontario, Canada) us ing drop anesthesia in a 600 m l beaker wi th a P e r s p e x ™ l id following which the right nostril was inoculated wi th titered virus stock diluted in 20ul of 5% M E M via a P 20 P i p e t m a n ™ fitted wi th a 0.5 m m diameter 200 m l microflex pipette tip (Island Scientific, Bainbridge, W A , USA) . Six mice were inoculated wi th each virus strain at varying inocula, ranging between 10 1 and 10 6 pfu. Isolates tested were T K D plaque isolate 1737-14 and T K W T plaque isolate 1773-5 which originated from the same patient and served as an internal control. Other controls included sham-infected mice (inoculated wi th 5% M E M 27 alone), strain G (a T K W T , H S V 2 reference strain) and strain A C G r 4 , a T K D , H S V 1 reference strain (courtesy of Dr. Don Coen, Harvard Medical School, Boston, Massachusetts). Mice were then observed daily for evidence of encephalitis for 21 days. The brains of any TK D - inocula ted mice which died were surgically removed. Surgery was performed in sterile 100 X 20 m m tissue culture dishes in a biohazard hood (Nuaire model N u 425FM-600, Plymouth, M N , USA) . Mice were sprayed wi th ethanol and allowed to dry, the skin was then cut and the skul l opened with sterile dissecting scissors and forceps. A l l surgical instruments were purchased from Baxter Diagnostics Corporation, Canlab Divis ion, Mississauga, Ontario, Canada. The brain was transected at the brainstem, shelled out wi th a sterile nickel spatula, homogenized in sterile Wheaton 15 m l tissue grinders (Fisher Scientific, Ottawa, Ontario, Canada), and the supernatant inoculated onto confluent V E R O cell monolayers in 25 c m 2 flasks. The media was changed after 24 h and, following the development of greater than 70% CPE, infected cells were harvested and stocks prepared as previously described. A t 30 days, a l l surviving mice were sacrificed by administering CO2 into a mouse cage wi th a P e r s p e x ™ l id (Lab Products Inc., Maywood , NJ , USA) . Both trigeminal ganglia of 10 mice inoculated wi th the highest inocula of 1737-14 ( the T K D plaque isolate) were then removed to assess the latency characteristics of this isolate. The brain was removed as described above, the ganglia located and removed wi th sterile dissecting scissors and forceps. Ganglia were then minced wi th a 40 m m scalpel blade (Fisher Scientific, Ottawa, Ontario, Canada) and cocultivated on confluent V E R O cell monolayers in 25 c m 2 flasks. After the development of >70% CPE, infected cells were harvested and vira l stocks were prepared as described above. Isolates obtained from the animal experiments 28 were recharacterized by susceptibility testing, T K uptake and plaque autoradiography as described above. 2.3.2. Mouse intracerebral neurovirulence model The aim of subsequent animal model studies was to tease out T K D subpopulations. Therefore, as opposed to the above intranasal m o d e l designed to assess neurovirulence, these were designed using a modificat ion of an intracerebral mouse model previously described to enhance this possibility (99). Animals were also 4 week old female B A L B / c mice but, due to lack of availability, were purchased from Charles River (Montreal, Canada) and allowed to settle at the A n i m a l Care Center, Universi ty of Br i t i sh Columbia for 48 h after delivery and prior to use. Isolates to be inoculated were diluted and prepared as described above. Isolates to be tested included clinical isolates 1737 and 89-063. Both of these were spontaneously-reactivated T K D and were shown to be homogeneous by plaque autoradiography, thereby testing as pure T K D by all conventional criteria Control isolates included strains G, A C G r 4 and sham-inoculated animals as described above. Six animals were inoculated via a 100 ul Hamil ton syringe (Mandel Scientific, Guelph, Ontario, Canada) wi th 50 | i l of 5% M E M containing 10 4 pfu of each isolate into the right cerebral hemisphere and observed for 21 days as above. Dying or sick animals had brains surgically removed and vira l stocks were harvested and prepared as described above. Successfully recovered isolates were characterized by susceptibility testing, T K uptake and plaque autoradiography as described above. The purpose of using 1737 was that, using the parental clinical isolate of 1737-14 combined wi th the increased sensitivity of an intracerebral model, the observations made originally o n 1737-14 could be verified and reproduced wi th an increased sensitivity. 29 2.4. M O L E C U L A R B I O L O G Y 2.4.1. Preparation of vira l D N A A l l reagents and gel apparatus, unless otherwise specified, were purchased from Canadian Life Technologies Inc., Burlington, Ontario, Canada. D N A purification was performed after inoculation of confluent V E R O cell monolayers in 175 c m 3 flasks wi th an M O I of 0.1 and incubated unti l greater than 70% CPE was seen. The cell monolayer was then washed wi th 5 m l cold PBS and incubated on ice for 5 m in wi th 1 m l cold lysis buffer ( lOmM Tr i s -HCl p H 7.6, 150 m M N a C l , 1% Nonidet P-40 (NP-40), 1% N a -deoxycholate). Cells were harvested wi th a 25 cm cell scraper (Falcon, Baxter Diagnostics Corporation, Canlab Divis ion , Mississauga, Ontario, Canada) and resuspended in a 15 m l polyethylene snap cap. A further 1 m l of lysis buffer was added to the flask, the flask was scraped again the solution aspirated and this volume was added to the tube which then was topped up to 4 m l w i t h lysis buffer. Nucle i were removed by centrifugation at 3000 rpm for 10 min at 4°C (Beckman J-6B, Beckman Instruments, Palo Al to , Ca, USA) and the supernatant transferred to a new tube and incubated for 2 h at 37°C wi th 50 ( ig /ml proteinase K , 5 m M E D T A and 12.5 | i g / m l of R N A s e A . The solu t ion was deproteinized by 3 rounds of the addition of an equal volume of a 1:1 ratio of phenol and chloroform, mixing by inversion, centrifugation at 5,000 rpm for 5 m in at room temperature (Beckman J2-21, JA-2 rotor, Beckman Instruments, Palo Al to , Ca, USA) and transfer of the aqueous supernatant into a fresh tube. Residual phenol was removed by the addition of an equal volume of chloroform, inversion, centrifugation and transfer of the aqueous phase into a fresh tube. D N A was precipitated by the addition of 2.5 vo lumes of 100% cold ethanol together wi th 0.3 M N a acetate. The tube was inverted 30 several times, incubated at -20° C for a min imum of 1 h and centrifuged again at 10,000 rpm for 30 min. The ethanol was removed and the pellet washed wi th 0.5 m l cold 70% ethanol, air dried for 5 min and resuspended in 100 ul of distilled water. The D N A concentration of the resulting product was estimated by diluting 1 ul in 1 m l distilled water and measuring in a spectrophotometer (Ultrospec II, L K B Biochrom, Cambridge England). D N A yields were consistently approximately 1000 u g / m l . Presence and purity of H S V D N A was confirmed by restriction enzyme digest wi th Eco R l . Reaction conditions included 5 ul D N A , 2 ul (20 units) of enzyme, 10 ul XI0 buffer and distilled water to a total volume of 100 ul in a 1.7 m l eppendorf tube. After incubation for 2 h at 37°C, the digested D N A was ethanol precipitated as described above, resuspended in 10 ul of distilled water, 2 ul of running buffer was added and the total volume run in a 0.8% agarose gel in a Hor izon 58 gel apparatus using T A E buffer (0.04 M Tris-acetate, 0.001 M E D T A ) at 100 volts for 1 h. The gel was removed and bathed in 100 m l distilled water to which 5 ul of a 10 m g / m l solution of ethidium bromide was added. After staining for 15 min, the gel was washed three times and destained for 5 m in wi th disti l led water. The gel was then examined for D N A by visualization under ultraviolet light using an ultraviolet transilluminator (Fotodyne Inc., N e w Berlin, WI, USA) and photographed using a Polaroid M P 4 Land camera (Polaroid Corporation, Cambridge, M A , USA) . 2.4.2. Amplification of the TK gene by polymerase chain reaction. Polymerase chain reaction (PCR) was performed using 2 primers designed to lie external to the T K open reading frame (JS1: C T G A T C A G C G T C A G A G C G T T and JS8: C G C T T A T G G A C A C A C C A C A C ) (Oligos Etc. Inc., Wi l sonvi l l e , OR, USA) . Ampl i f ica t ion was performed in a 31 Techne PHC-3 thermocycler (Mandel Scientific, Guelph, Ontario, Canada) and conditions included init ial denaturation at 100°C for 5 m in followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 2 m in and extension at 72°C for 3 m in which was followed by a final extension step at 72° C for 10 min. Reactions were carried out in 0.5 m l eppendorf tubes (National Scientific Supply Co. Inc., San Rafael, C A , U S A ) and included 2 f i l (2 ug) template, 1 ul (50 pmoles) of each primer, 8 ul of 1.25 m M dNTP's , 0.5ul (2.5 Units) of Taq polymerase, 5 ul X10 buffer, 2 ul (4%) dimethylsulphoxide (DMSO) and distilled water to a final volume of 50 ul. T K D N A was separated electrophoretically in 0.8% agarose using a Hor i zon 11.14 gel apparatus and the band visualized by staining wi th ethidium bromide as described above. The T K band was cut out with a razor blade and the gel block was placed into a 1.7 m l eppendorf tube. The TK product was then purified using a Sephaglas D N A purification kit (Boerhinger Mannhe im Biochemica, Laval , Quebec). After weighing the gel fragment, 1 ul of gel solubilizer was added per mg of agarose, the sample was vortexed and incubated at 60°C for 5 m in to dissolve the gel after which 5 ul of sephaglass beads per tube was added. The mixture was vortexed, incubated at room temperature for 5 min, centrifuged at 12,000 rpm for 1 min in a microcentrifuge (Microspin 12, Sorval Instruments, Wi lmington , DE, U S A ) and the supernatant removed to a new tube. The pellet was washed and resuspended in 8 times the volume of wash buffer, centrifuged as above and the supernatant removed. This wash was repeated twice and the resultant pellet was air dried for 10 m i n and resuspended in 10 ul of elution buffer. After further centrifugation as above the supernatant was removed to a new tube. Finally, 2 ul of the resulting product was electrophoresed in a Hor izon 58 gel apparatus together wi th a Mass ladder, stained wi th ethidium bromide and visualized under U V light as described 32 above. This was used to estimate the concentration of D N A template to be used for cycle sequencing; results consistently yielded approximately 30 n g / u l . 2.4.3. Cycle sequencing. Cycle sequencing was performed by designing 14 overlapping internal primers oriented in both coding and noncoding directions to cover the entire T K open reading frame. Internal primer sequences were as follows; (JS2: C T C A T C A G C G T C A G A G C G T T , JS4: T C A T T G T T A T C T G G G C G C T G , JS5: A A T G G C G G A C A G C A T G G C C A , JS6: T G T C T A C G A T C T A C T C G C C A A , JS7: A A T C C A G G A C A A A T A G A T G C , JS9: T A C C T C A T G G G A A G C A T G A C , JS10: C T G C T G C G G G T T T A T A T A G A , JS11: G T A A G T C A T C G G C T C G G G G A , JS12: G G G G A G G C G G C G G T G G T A A T , JS13: G G G T A G C A C A G C A G G G A G G C , JS14: G G A A C A G G G C A A A C A G C G T G , JS15: C A C A T T T T T G C C T G G G T C T T , JS17: G T T C G G T C A G G C T G C T C G T G , JS18: C A A A C G T G C G C G C C A G G T C G , JS19: G T G G G G T C C G T C T A T A T A A A ) . Sequencing was also performed using external primer JS8. Reactions were carried out using a d s D N A Cycle Sequencing System (Canadian Life Technologies Inc., Burlington, Ontario, Canada). Sequencing primers were end-labeled wi th 3 2 P d A T P (6000 C i / m m o l , Amersham Life Sciences, Oakville, Ontario). Labeling reaction mixtures included 1 pmol (2 ul) of primer, 2 pmol (1 ul) y ^ P d A T P , 1 unit (1 ul) T4 polynucleotide kinase and 1 ul X5 kinase buffer, incubated at 37°C for 10 min after which the reaction was terminated by incubation at 55°C for 5 min. Sequencing reactions consisted of assembling a pre-reaction mixture in a 0.5 m l eppendorf tube which consisted of 1 pmol (5 ul) of labeled primer, 50 pmol (2 ul) template D N A , 1.25 units (0.5 ul) of 2.5 un i t s /u l Taq polymerase, 4.5 ul of X10 sequencing buffer, 1 ul D M S O and distilled water to a total of 36 ul. For the reaction mixture, 8 u l of the pre-reaction mixture was then added 33 to each of 4 x 0.5 ul eppendorf tubes containing 2 uT of termination mixes, each containing one dideoxynucleotide as wel l as a mixture of the four deoxynucleotides. Reactions were then placed in the thermocycler for the sequencing reactions. Reaction conditions included denaturation at 95°C for 3 min, 20 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 70°C for 60 s followed by 10 cycles of denaturation at 95°C for 30 s and extension at 70°C for 60 s. The reactions were then terminated wi th 5 | i l of stop solution. Products were then denatured by heating at 100°C for 5 m i n and run on a 6% polyacrylamide gel [40.4 g urea, 12.6 m l of 40% acrylamide (19:1 ratio of acrylamide to N,N'-methylenebisacrylamide) 16.0 m l X5 TBE (54 g Tris base, 27.5 g boric acid, 20 m l 0.5 M EDTA) and 25.2 m l distilled water and dissolved at 37°C]. The gel was polymerized by the addition of 700 ul of 10% ammonium persulfate (APS) and 40 ul T E M E D ( N , N , N ' , N ' -tetramethylethylenediamine) and injected using a 60 m l syringe and 16 gauge needle between 2 clean glass plates separated by 0.4 m m spacers and a comb and sealed wi th bulldog clips and electrical tape. Gels were allowed to polymerize for 1 h, the tape and clips were removed and the gel clipped into a model S2 sequencing gel apparatus wi th I X TBE buffer added to the upper and lower reservoirs. The gel was prerun for 1 h at 60 Watts and then 3 ul aliquots of sample were electrophoresed for 1.5 and 3 h at 65 Watts using a Pharmacia ECPS 3000/150 power supply (Pharmacia, Uppsala, Sweden). The gel was then removed onto a double layer of Grade No . 3 m m Chr. W h a t m a n chromatography paper (Fisher Scientific, Ottawa, Ontario, Canada), covered wi th plastic cling wrap and dried at 80°C for 1 h in model SE 1160 Drygel Sr slab gel dryer (Hoefer Scientific Instruments, San Francisco, C A , U S A ) After removal of plastic wrap and the outer layer of Whatman paper, baby powder was sprinkled over the gel and it was exposed to Kodak X-Omat X-ray f i lm i n 34 a FB-XC-1417 autoradiography cassette wi th image intensification (Fisher Scientific, Ottawa, Ontario, Canada) at -70°C. The fi lm was then developed as previously described and the sequence read manually using a light box. 2.4.4. Immunoprecipitation Identification of T K protein was performed by immunoprecip i ta t ion and Western blot using an ant i-HSV 2 T K mouse monoclonal antibody, T K 30/4 (kindly provided by Dr. Ken Powell , Burroughs Wellcome, Beckenham, United Kingdom). For immunoprecipitation, 60 x 15 m m tissue culture dishes were inoculated wi th an M O I of 0.1 and adsorbed for 1 h. The infected monolayer was washed three times wi th 1 m l of methionine /cysteine-free D M E M without L-glutamine ( ICN Biomedicals Canada Ltd, St. Laurent, Quebec, Canada), overlaid wi th a 5 m l mixture containing a 9:1 ratio of methionine/cysteine-free to regular D M E M , 4% dialyzed fetal calf serum and 50 u C i / m l Trans 3 5 S label (1101 C i / m m o l I C N Biomedicals, St. Laurent, Quebec, Canada) and incubated overnight. The monolayer was then washed wi th 2 m l ice cold PBS, the cells lysed wi th 1 m l cold lysis buffer for 15 m i n on ice, removed wi th a cell scraper, aspirated and transferred to a 1.7 m l eppendorf tube. The nuclei were removed by centrifugation at 15,000 rpm for 5 min at 4°C. The supernatant was incubated overnight on ice wi th 100 ul of a mixture of 10% NP-40, 10% Na-deoxycholate and 1% sodium dodecyl sulfate (SDS) together with a 1/100 dilution of T K antibody. Immune complexes were collected by adding 100 ul of a 10% suspension of Staphylococcus aureus cells (Sigma Chemical Company, St. Louis, M O , U S A ) , rocking at 4°C for 2 h and centrifugation at 12,000 rpm for 1 min . The pellet was washed sequentially wi th 0.5 m l of the following solutions; wash buffer #1 (20 m M Tr is -HCl p H 7.5, 150 m M N a C l , 1% NP-40), wash buffer #2 (20 m M Tr i s -HCl p H 8.8, 150 35 m M N a C l , 1% NP-40, 0.2% SDS) and wash buffer #3 (20 m M Tr i s -HCl p H 6.8, 150 m M N a C l , 1% NP-40, 0.2% SDS). The pellet was then resuspended in 50 ul of SDS sample buffer (2% SDS, 5% 2-mercaptoethanol, 62.5 m M T r i s - H C l p H 6.8, 10% glycerol and 0.001% bromophenol blue), denatured at 100°C for 5 min and electrophoresed. Electrophoresis was carried out in a model SE 400 vertical slab gel unit (Hoefer Scientific Instruments, San Francisco, C A , U S A ) wi th 1.5 m m wide spacers. After clamping, a 10% resolving gel was poured [10 m l of 30% acrylamide, 7.5 m l of X4 resolving buffer (1.5 M Tris base, 0.4% SDS, p H 8.8) 12.5 m l distilled water, 200 ul 10% A P S and 40 ul T E M E D ] , covered wi th a layer of distilled water and allowed to polymerize for 1 h, after w h i c h the water was poured off and a 6% stacking gel was poured over the resolving gel [3 m l 30% acrylamide, 3.8 m l of X4 stacking buffer (0.5 m Tris base, 0.4% SDS, p H 6.8), 8.2 m l distilled water, 100 ul 10% A P S and 20 m l TEMED] , a comb inserted and the gel allowed to polymerize for 20 min . The comb was then removed and wells flushed wi th running buffer (X10 = 0.25 M Tris base, 1.92 M glycine and 1% SDS). Samples were loaded into wells, running buffer added to upper and lower chambers and samples were electrophoresed at 25 m A for 4 h. Gels were removed and then fixed in 40% methanol wi th 5% acetic acid for 1 h. The gel was then dried in a gel dryer, exposed to X-ray f i l m and developed as described above. 2.4.5 Western blot analysis Inoculation and adsorption of isolates for Western blot analysis was performed as described for immunoprecipitation except that an M O I of 3 was inoculated and the infected monolayers were subsequently incubated overnight wi th regular D M E M . Cells were washed and lysed and nucle i removed as described above. Protein was then precipitated from the 36 supernatant by adding 4 volumes of 100% acetone, incubated at -20°C for a m i n i m u m of 1 h, centrifuged at 15,000 rpm for 10 min at 4°C, washed w i t h 80% acetone. The resulting pellet was dried and resuspended in 50 ul SDS sample buffer. The samples were boiled and electrophoresed as described above. The gel was then removed and incubated in transfer buffer (56.5 g glycine, 12.0 g Tris base and 800 m l methanol made up to 4000 m l w i t h distilled water) for 20 min. The gel was then transferred onto a BA-S N C nitrocellulose transfer and immobi l iza t ion membrane (Schleicher and Schuell, Keene, N H , USA) using an electroblotting transfer apparatus at 1 A m p for 1 h filled wi th transfer buffer and stirred wi th a magnetic stir bar (Bio-Rad Laboratories, Mississauga, Ontario). The membrane was removed and transfer of protein confirmed by staining for 5 m in wi th Ponceau S [X10 =2% Ponceau S (Sigma Chemical Company, St. Louis, M O , USA) , 30% trichloroacetic acid, 30% sulfosalicylic acid) followed by 3 washes in distilled water. Antibody incubations and detection was performed using a L u m i G L O H R P Western blot kit (Kirkgaard and Perry Laboratories, Gaithersberg, Maryland, USA) . The membrane was blocked with 0.5% mi lk block for 1 h at room temperature, incubated wi th a 1/5000 dilut ion of anti- H S V 2 T K monoclonal antibody (primary antibody) in 0.1% milk block for 1 h at r o o m temperature, washed 3 times in wash buffer, incubated wi th 60 u / m l secondary antibody (goat anti-mouse, Boehringer Mannhe im Biochemica, Laval , Quebec, Canada) in 0.1 % mi lk block and washed 3 more times. Chemiluminescent substrates were then applied to the membrane for one minute which was then covered wi th plastic cling wrap. The membrane was then exposed to X-ray film in an autoradiograph cassette, removed and developed as described above. 37 C H A P T E R 3 R E S U L T S 3.1 T H E M E C H A N I S M OF R E A C T I V A T I O N OF 1737. 3.1.1. Characterization of clinical isolates from patient WJ A l l available clinical isolates were characterized. Patient WJ sustained 7 clinical episodes of genital herpes between September 1989 and September 1990. Twenty clinical isolates were obtained from these episode and all were shown to be H S V 2. Characterization of these isolates by A C V susceptibility testing and TK uptake studies demonstrated 5 of these outbreaks to be T K " deficient and ACV-resistant while 2 were ACV-susceptible. A l l isolates sourced from the same clinical outbreak were consistent in their phenotype (Table 1). The T K D clinical isolate which reactivated spontaneously, 1737, was further characterized together wi th a clinical isolate from the next outbreak which was ACV-susceptible, 1773, and which served as an internal control. 3.1.2. Plaque isolation of clinical isolates 1737 and 1773. Prior cases have been reported where A C V R subpopulations w i t h i n heterogenous mixtures of vira l phenotypes were able to impart properties, i n this case antiviral resistance, to the population as a whole (54,96). Our in i t i a l aim was to determine whether heterogeneous vira l populations containing TKWT subpopulations may have explained the apparently paradoxical activity of isolate 1737 by providing sufficient T K activity to the population as a whole to enable reactivation while simultaneously maintaining the A C V R phenotype. Initial investigation of heterogeneity was performed by plaque 38 Table 1. A C V susceptibilities and thymidine kinase uptakes for al l cl inical isolates available for patient WJ. Isolates are grouped according to the outbreak they were sourced from.*Denotes spontaneously-reactivated A C V R isolate of interest (1737) and w i l d type internal control (1773). Date Clinical Isolate I D 5 0 (ug/ml) T K Act iv i ty (%) September 1989 1091 5.4 11.8 1090 7.4 10.5 1104 7.8 6.4 1105 6.5 8.6 1106 8.4 0.6 November 1989 1201 0.08 55.8 January 1990 1280 6.0 0 1286 6.5 0 1288 5.7 0 1307 5.6 6.8 A p r i l 1990 1420 13.4 6.1 1423 6.8 3.4 1424 7.4 0 M a y 1990 1563 8.1 10.1 1564 5.1 3.8 1566 7.6 2.8 Aueust 1990 *1737 6.6 9.7 September 1990 *1773 0.11 198.6 1774 0.08 137.4 1775 0.09 68.6 39 purification of individual plaque isolates from clinical isolates and recharacterization of these to identify T K W T plaques. Nineteen plaque isolations were successfully regrown from 1737 and twenty from 1773. A l l plaque isolates derived from 1737 was A C V R w i th IDso's to A C V ranging from 2.1 to 17.1 u g / m l and all plaque isolates derived from 1773 were A C V S wi th IDso's ranging between 0.06 and 0.56 u g / m l (Table 2.), suggesting homogeneity wi th in both clinical isolates. Because of this apparent homogeneity, an individual plaque isolate sourced from clinical isolate 1737, 1737-14 ( A C V ID50 11.8 ug /ml ; T K activity 5.4%), was selected for further neurovirulence studies. Another plaque isolate, 1773-5 ( A C V ID50 0.14 u g / m l ; thymidine kinase activity 134.7%), sourced from clinical isolate 1773 (obtained from the reactivation following successful sterilization of 1737 wi th P F A and which was A C V S ) , was chosen as an internal control. Both plaque isolates underwent P F A susceptibility testing and 1737 was thus proven not to harbor a pol mutant (PFA ID 5 0 ' s : 1737-14= 30.9 and 1773-5= 34.0 u g / m l respectively). In v iew of the poor sensitivity of T K uptake studies in discr iminat ing between low and absent TK levels, subsequent assessment of T K phenotype was performed by plaque autoradiography of v i ra l populations and demonstrated both 1737 and 1737-14 to be homogeneous T K D whi le 1773 and 1773-5 were homogenous T K W T - This also excluded the possibility of a T K L P phenotype. In addition, similar results using 1 4 C TdR, the natural substrate for TK, excluded the possibility of a T K A phenotype. This technique also enabled the sampling of a larger number of approximately 100 plaques to more comprehensively exclude T K heterogeneity (Figures 4a. and 4b.). 40 Table 2. A C V susceptibilities for plaque isolates derived from clinical isolates 1737 and 1773. * Denotes plaque isolates selected for further characterization. D N G = d id not grow. 1737 A C V I D 5 0 1773 A C V I D 5 0 fue/ml) (ue/mD 1 3.7 1 0.08 2 17.1 2 0.23 3 2.1 3 0.27 4 13.5 4 0.39 5 6.2 5* 0.14 6 13.3 6 0.30 7 4.1 7 0.19 8 9.3 8 0.27 9 5.1 9 0.49 10 3.2 10 0.06 11 12.6 11 .0.54 12 D N G 12 0.55 13 13.7 13 0.14 14* 11.8 14 0.17 15 13.4 15 0.17 16 11.7 16 0.19 17 8.5 17 0.16 18 7.9 18 0.24 19 13.6 19 0.23 20 2.7 20 0.12 41 Figure 4a. 1 2 5 I d C plaque autoradiographs from WJ isolates. Strain G is a T K w i l d type control and strain A C G r 4 is a TK-deficient control. The upper panels demonstrate plaques grown and the lower segments demonstrate corresponding plaques either exhibiting dark-rims and thereby expressing T K activity or lacking dark rims due to T K deficiency. This figure shows results using 0.5 |xCi [ 1 2 5I]-iododeoxycytidine ( 1 2 5 IdC) as the thymidine kinase probe in V E R O cells and demonstrates uniformity of the T K D plaques w i t h i n clinical isolate 1737 and plaque isolate 1737-14. w.j. REACTIVATED TK", HSV-2 '"IdC PLAQUE AUTORADIOGRAPHY Patient Isolates 1737 1773 1737-14 1773-5 Encephalitic Isolate 1737-14/ME 1737-M/lCA 1737-14/105B Patient Isolates 1737 1773 1737-14 1773-5 Encephalitic Isolate Figure 4b. 1 4 C TdR plaque autoradiographs from WJ isolates. Plaque autoradiography are in similar arrangement to Figure. 4a. and are interpreted in a similar fashion. Results using 0.5 | i C i 1 4 C thymidine ( 1 4 C TdR) i n a thymidine kinase-deficient cell line (143B) again confirm the homogeneity of T K D plaques wi th in clinical isolate 1737 and plaque isolate 1737-14. 1737-14/ME 1737-14/10'A 1737-14/105B 1737-14/ME 1737-14/106A J737-14/10SB 43 3.1.3. Mouse neurovirulence studies of 1737-14 Because clinical isolate 1737 d id not harbor a subpopulation of T K W T virus when this was sought by al l conventional methods, the neurovirulence of the isolate was assessed to determine whether the unusual reactivation ability of the isolate was associated wi th other phenotypic markers w h i c h were also atypical for T K D isolates and which might offer further clues to reactivation mechanisms. As already alluded to, T K D isolates typically have reduced neurovirulence. Accordingly, plaque isolate 1737-14 was assessed for neurovirulence and latency characteristics in a mouse model. Reference T K W T strain G and internal T K W T control, 1773-5, both demonstrated marked neurovirulence as predicted by their phenotype wi th no mice s u r v i v i n g beyond an inoculum of 10 2 pfu. In contrast, reference T K D strain A C G r 4 was demonstrated to be non-neurovirulent, also as predicted, wi th no resultant mouse deaths. Animals inoculated wi th plaque isolate 1737-14, however, yielded an interesting and unexpected result. The isolate overall showed reduced neurovirulence as predicted from its phenotype but surprisingly, 1 of 6 mice at the highest inoculum (10 6 pfu) died from encephalitis (Table 3.). Two explanations were possible for this finding; either the in vivo assay was demonstrating the lower threshold of the reduced neurovirulence of the isolate or it was detecting unexpected neurovirulence. In view of this finding, the isolate resulting in neurovirulence was grown from the brain of the dying animal. The output encephalitic isolate, 1737-14ME, was characterized and surprisingly was shown to be fully A C V S (ID50 0.12 u g / m l ; T K activity 154.9%), demonstrating a complete change in phenotype from the input strain. Simultaneously, ganglia were extracted from surviv ing mice inoculated wi th 1737-14 and attempts to cocultivate virus were successful in 44 Table 3. Mouse intranasal neurovirulence studies of WJ isolates. The table shows the number of surviving mice out of a total of 6 inoculated at each inoculum of each strain. (* Only 5 mice were inoculated wi th 1737-14 at this inoculum). Inoculum (pfu) G A C G r 4 1773-5 1737-14 S h a m 10 6 0 6 0 5 10 5 0 6 0 5* 10 4 0 6 0 6 103 0 6 0 6 10 2 1 6 4 6 10 1 6 6 5 6 0 6 45 2 mice. Characterization of these 2 ganglionic strains demonstrated one, 1737-14 /10 6 A, to be a similar A C V R , T K D phenotype to the input strain ( A C V ID50 9.0 ug/ml; T K activity 8.9%; P F A I D 5 0 11.8 ug/ml) . In contrast, the other isolate, 1737-14/106B, despite being A C V R , demonstrated a surprisingly h igh level of T K activity ( A C V I D 5 0 15.2 u g / m l ; T K activity 53.5%; P F A I D 5 0 9.7 ug/mi) . In view of these apparently ambiguous results, plaque autoradiography was performed on these isolates. This demonstrated 1737-14ME, despite the input strain being homogenous T K D , to consist of a homogeneous T K W T population. Strain 1737-14/10 6A, as expected, was a homogeneous T K D population similar to the input strain. Strain 1737-14/10 5 B, however, was a heterogeneous mixture of almost equal populations of T K D and T K W T plaques (Figures 4a. and 4b.), thus explaining its unexpectedly high T K activity. P F A susceptibility excluded a v i ra l D N A pol mutant phenotype. The derivation of the abovementioned isolates is depicted in Figure 5. 3.2 M E C H A N I S M OF R E A C T I V A T I O N OF A C T G 095 ISOLATES 3.2.1. Characterization of clinical isolates. In view of the initial findings seen in patient WJ and to determine whether other spontaneously-reactivated T K D H S V 2 isolates also harbored T K W T subpopulations, we sought to detect T K heterogeneity wi th in cl inical isolates sourced from other patients which had exhibited similar behaviour. We were fortunate to receive a gracious donation of 4 isolate pairs w h i c h were part of a clinical trial. The trial, as already described, was A C T G protocol number 095 and compared the efficacy of foscarnet to vidarabine for the treatment of A C V R genital H S V infections in AIDS patients. Inclusion criteria 46 Q. CO o T3 CO i_ o < CD 3 CT ro Q . <D o co CO 3 o CD C CD C CD O) O E o X IU CO I-co 3 o CD c CD c CD O) O E o X o co Q t; CO 3 O CD C CD C CD D) O CD 4-1 CD X m m o CO 1^  a o co (•>» s : 8 CO 3 O CD CD s> cn £ o E o X .2 "5> c CO o> 15 c I CD cn E o co CD 3 O CD > co 5 CO o Q. cn c "•5 CO J C Q. 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Fol lowing eradication of the outbreak by therapy, the patients were prohibited from further antiviral therapy until a subsequent reactivation. Four post-treatment reactivated isolates which were A C V R (clinical isolates 89-063, 89-353, 90-150 and 2370) were sent to us for further characterization together wi th the corresponding initial A C V R isolates which qualified these 4 patients for enrollment into the trial (clinical isolates 89-061, 89-350, 90-030 and 89-660). A l l isolate pairs underwent confirmatory A C V susceptibility testing and T K uptake studies. A l l isolates were confirmed to be A C V R w h i l e T K uptake studies demonstrated pretreatment clinical isolates to have negligible activity while reactivated isolates, although having low activity, showed a trend to increased activity. In addition, 3 post-treatment reactivated isolates which were A C V S (clinical isolates 89-391, 89-641 and 3187) and the respective pre-treatment isolates (clinical isolates 89-390, 89-650 and 90-110) also underwent A C V susceptibility testing and were found to be A C V S and A C V R respectively (Tables 4a. and 4b). A s an initial assessment of TK heterogeneity, all A C V R reactivated clinical isolates underwent plaque autoradiography to determine whether this, on its own, might demonstrate TK heterogeneity without the need for neurovirulence studies. Three of the 4 reactivated isolates (clinical isolates 89-353, 90-150 and 2370) demonstrated heterogeneity on autoradiography. The fourth (clinical isolate 89-063) was homogeneously T K D as originally described for 1737 from patient WJ. A s a result 89-063 was selected for assessment in a mouse neurovirulence model to detect "masked heterogeneity" as performed for 1737. 48 Table 4a. A C V susceptibilities and T K activities of A C T G 095 A C V R reactivated isolate pairs. The table depicts characteristics of A C V R c l inical isolates which qualified for enrollment to A C T G 095 and their corresponding spontaneously-reactivated, A C V R first-reactivation clinical isolates. Enrollment Isolates Reactivation Isolates Isolate A C V I D 5 0 T K (%) (Ug/ml) 89-061 9.5 89- 350 10.5 90- 030 10.2 89-660 11.1 Isolate A C V I D 5 0 T K (%) (Kg/ml) 1.3 89-063 12.8 4.2 0 89-353 6.2 11.2 0 90-150 3.7 23.6 0 2370 2.1 20.1 49 Table 4b. A C V susceptibilities and TK activities of A C T G 095 A C V S reactivated isolate pairs. The table depicts A C V characteristics of cl inical isolates which qualified for enrollment to A C T G 095 and corresponding first reactivations which were A C V S . Enrollment Isolates Reactivation Isolates Isolate A C V I D 5 0 ( H g M l ) Isolate A C V I D 5 0 (Ug/ml) 89-390 6.0 89-391 0.23 89-650 10.0 89-641 0.15 90-110 8.7 3187 0.30 50 The 3 reactivated clinical isolates which demonstrated T K heterogeneity on plaque autoradiography were then assessed to determine whether there was an increase in T K W T plaques on plaque autoradiography from the enrollment A C V R isolates to the reactivated A C V R isolates. The plaque autoradiographs on these isolate pairs demonstrated an increase in the proportion of T K W T plaques between enrollment and reactivation when both 1 2 5 I d C and 1 4 C TdR were used as the probe. As the percentage of T K W T plaques was at all times greater by 1 2 5 I d C than by 1 4 T d R (the natural substrate) this excluded the emergence of a T K A phenotype (Figures 6a. and 6b. and Tables 5a. and 5b.). Isolate 2370 is of particular interest because by 1 2 5 I d C it already shows the majority of plaques demonstrating the T K W T phenotype despite it being A C V R . This suggests the presence of either a T K A phenotype or a mutation in the v i ra l D N A polymerase locus. 3.2.2. Mouse intracerebral inoculation of 89-063 and 1737. Due to the detection of a T K W T subpopulation wi th in clinical isolate 1737 despite an apparent homogeneous T K D population by plaque purification and plaque autoradiography we attempted to confirm the in i t i a l findings. O n this occasion the primary focus of the work was not to assess neurovirulence but rather to specifically use this animal model to tease out such a TKWT subpopulation. Accordingly, an intracerebral inoculation m o d e l was used and inoculated at a high M O I of 1 x 10 4 pfu. In addition, as the previous assessment of clinical isolate 1737 was via its plaque isolate, 1737-14, we inoculated the parent clinical isolate. Both these strategies were aimed at increasing the sensitivity of the assay. In addition, because clinical isolate 89-063 was phenotypically identical, we also inoculated it in an attempt to detect TKWT strains. Input strains ,1737 and 89-063 were shown to be uniformly T K D 51 Figure 6a. 1 2 5 I d C plaque autoradiography of A C T G 095 isolates. The upper panel demonstrates the total number of inoculated plaques after fixing and staining of monolayers while the lower panel demonstrates the corresponding autoradiograph after exposure to X ray fi lm, showing T K W T plaques which are identified by those which produce dark rims. There is an increase in TK w i l d type plaques from the time at which isolates which A C V R isolates qualified patients for inclusion into A C T G 095 to the time of first-reactivation isolates which were A C V R , indicating an increase in TK activity coincident wi th reactivation. ACTG 095 Reactivated TK-, HSV-2 125idC Plaque Autoradiography Pre Foscarnet First Reactivation 89-063 89-353 90-150 2370 Figure 6b. 1 4 C TdR plaque autoradiography of A C T G 095. The figure again demonstrates an increase in T K wi ld type plaques between isolates w h i c h developed A C V R to qualify for inclusion into A C T G 095 and first-reactivation isolates which were A C V R . ACTG 095 Reactivated TK-, HSV-2 1 4 C TdR Plaque Autoradiography Pre Foscarnet First Reactivation 89-063 89-353 90-150 2370 53 Table 5a. Percentage of T K W T plaques wi th in A C T G 095 isolates by 1 2 5 I d C plaque autoradiography The table compares the percentages of T K W T plaques wi th in A C V R clinical isolates which qualified for inclusion into A C T G 095 and corresponding first reactivations which were A C V R . A n increase in the proportion of w i l d type (dark rim) plaques is noted in reactivated isolates for all pairs of isolates except for 89-061/89-063 which were homogeneously T K D . Pre-Foscarnet First reactivation Isolate % T K W i l d Type Isolate % T K Deficient 89-061 0 89-063 0 89- 350 0 89-353 48.0 90- 030 0 90-150 58.5 89-660 84.2 2370 100.0 54 Table 5b. Percentage of T K W T plaques wi th in A C T G 095 isolates by 1 4 C T d R plaque autoradiography The table lists percentages of T K W T plaques as depicted in Table 5 a. but using 1 4 C TdR plaque autoradiography. This also demonstrates an increase in the proportion of TK w i l d type plaques between original A C V R isolates and first reactivations which were A C V R for all pairs of isolates except for 89-061/89-063. Pre-Foscarnet First reactivation Isolate % T K W i l d Type Isolate % T K W i l d Type 89-061 0 89-063 0 89- 350 0 89-353 16.3 90- 030 0 90-150 18.3 89-660 . 0 2370 51.5 55 by plaque autoradiography (Figures 4a. and 4b. and Figures 6a. and 6b.). A t the end of the experiment on day 21, all 6 mice inoculated wi th 1737 and 5 of 6 mice inoculated wi th 89-063 had died. A l l mice inoculated wi th T K W T reference strain G and no sham-infected mice or mice inoculated wi th T K D reference strain A C G r 4 had died as expected (Table 6.). Virus was recovered from all dying mice inoculated wi th 89-063 or 1737. Characterization of these output strains demonstrated them to be all A C V R w i th negligible T K levels except for 1737 F which exhibited 23.7% T K activity (Table 7.). Plaque autoradiography of 1737 output strains using 1 2 5 I d C demonstrated a very small subpopulation of dark-rimmed T K W T plaques in addition to a population of plaques wi th faint rims consistent wi th T K L P phenotype. Output strains derived from 89-063-inoculated animals, in contrast, demonstrated the presence of a uniform population of T K D plaques. Plaque autoradiography using 1 4 C TdR demonstrated that 1737 output strains harbored a similar subpopulation of T K W T plaques as seen by 1 2 5 I d C but faint rims suggestive of the T K L P phenotype were not seen. Output strains derived from 89-063, however, demonstrated a significant population of faint r i m plaques suggestive of the T K L P phenotype (Figures 7a. and 7b.), suggesting that the selection of this phenotype was the major mechanism responsible for the contribution of T K activity and reactivation ability of this isolate. Attempts to recover virus from the brain of the single surv iv ing mouse inoculated w i t h 89-063 was unsuccessful. 56 Table 6. Intracerebral mouse neurovirulence studies of 1737 and 89-063. The table depicts the number of surviving mice at 21 days. Six mice were inoculated wi th each strain at an inoculum of 10 4 pfu. Isolate N u m b e r S u r v i v i n g G 0 A C G r 4 6 1737 0 063 1 S h a m 6 57 Table 7. A C V susceptibilities and T K uptakes of 1737 and 89-063 neurovirulent output strains. The table depicts characterization of strains derived from the brains of mice dying after intracerebral inoculation w i t h clinical isolates 1737 and 89-063. Isolate A C V ID 5 o (Ug/ml) % T K 1737 A 5.3 0 1737 B 11.3 0 1737 C 9.5 3.3 1737 D 11.4 2.5 1737 E 8.2 2.7 1737 F 9.4 23.7 063 A 14.1 2.7 063 B 12.5 0 063 C 12.0 1.3 063 D 10.1 0 063 E 14.9 0 58 Figure 7a. 1 2 5 I d C plaque autoradiography of 89-063 and 1737 intracerebral output strains. The figure depicts isolates recovered from mouse brains after intracerebral inoculation wi th strains 89-063 and 1737. This demonstrates the recovery of a subpopulation of T K W T plaques wi th dark rims in 1737-derived output isolates. These isolates also contain a population of faint-r immed plaques suggestive of a T K L P phenotype which is not evident on the photographs below but is only visible on direct autograph v isua l iza t ion against a light box. Output strains derived from 89-063-inoculated animals demonstrate a lack of rims by any method of inspection, suggesting a u n i f o r m T K D population. 1 2 5 Iododeoxycytidine Plaque Autoradiography Strain G ACGr4 Input Output 1737 1737-A 1737-B 1737-C 1737-D 1737-E 1737-F 89-063 Figure 7b. 1 4 C TdR plaque autoradiography of 89-063 and 1737 intracerebral output strains The figure is similar in outlay to figure 6a and depicts plaque autoradiography using 1 4 C TdR. This again demonstrates the recovery of a subpopulation of T K W T wi th dark rimmed plaques in 1737 although the faint rims suggestive of a T K L P seen by 1 2 5 I d C are not visible even wi th direct visualization against a light box. In contrast, 89-063-derived plaques do demonstrate faint rims by light box examination although this is not evident on the photograph below, again suggesting a T K L P population. 14 C Thymidine Plaque Autoradiography Controls Strain G Input Output 1737 1737-A 1737-C 1737-D 1737-E Input Output 89-063 89-063-A ACGr4 1737-B 1737-F 89-063-B 89-063-C 89-063-D 89-063-E 60 3.3. M U T A T I O N C O N F E R R I N G T H E T K D P H E N O T Y P E O N T O 1737 3.3.1. D N A sequencing of isolates from patient WJ. To determine the genetic lesion conferring the T K D phenotype onto clinical isolate 1737 and to determine whether this may be mechanistically implicated in the ability of 1737 to reactivate, D N A sequencing of the T K gene was performed. Specifically, sequencing was performed on plaque isolate 1737-14, the plaque isolate originating from 1737, as this was the strain w h i c h resulted in mouse neurovirulence and yielded output strain 1737 lO^ME as previously described. The homogeneity of the mutation wi th in the v i r a l population was suggested by the uniform plaque autoradiography of 1737 (Figures 4a. and 4b.), the uniform A C V R phenotype of the 20 plaque isolations derived from 1737 (Table 2.) and the detection of the same mutation i n another plaque isolate (1106-2) originating from a different T K D c l inical isolate from the same patient (1106) which was also uniformly T K D by plaque autoradiography (Figure 8.). A T K W T plaque isolate (1773-5) originating from clinical isolate 1773 also obtained from patient WJ was used as an internal control wi th the expectation that the difference in TK D N A sequence between the two strains should only differ from 1737-14 by the mutation conferring the T K D phenotype. The sequence of 1773-5 was compared to published H S V -2 T K W T sequence of strain 333 and demonstrated 4 base pair differences (85). These were; adenosine for guanosine at position 79 of the open reading frame, guanosine for thymidine at position 85, guanosine for adenosine at position 232 and thymidine for cytosine at position 476. A l l four base pair changes were non-conservative but none of the amino acid substitutions occurred in the three regions which comprise the putative active center as 61 Figure 8. 1 2 5 I d C plaque autoradiography of WJ-derived clinical and plaque isolates. The figure depicts all plaque isolates derived from WJ w h i c h underwent D N A sequencing of the TK gene. Isolates include internal control clinical and plaque isolates, 1773 and 1773-5 respectively, to be un i formly T K W T wi th dark-rimed plaques while clinical isolates, 1737 and 1106, and their respective plaque isolates, 1737-14 and 1106-2, demonstrate plaques without rims consistent wi th uniform T K D populations. 62 previously described (86). The T K D N A sequence of 1737-14 differed from strain 333 by the same 4 base pair substitutions as 1773-5 but differed from 1773-5 by a single G addition in a homopolymer string of 7 G's residing between positions 433 and 439 of the open reading frame (Figure 9.), suggesting both to have a common origin. Primer JS 12, which spanned the region containing the string of 7 guanosines, was then used to sequence the same region of 1106-2. This was demonstrated to have the same guanosine addition as 1737-14 (Figure 9.). Plaque isolate 1106-2 was sourced from clinical isolate 1106 which also exhibited the T K D phenotype but, unlike 1737, it was followed by the expected T K W T reactivation. This demonstrated that, even wi th in the same patient, this mutation could spontaneously reactivate as a T K D isolate or be followed by a TKWT reactivation, suggesting that such a mutation does not confine the isolate to one mode of reactivation behaviour. The predicted consequences of this guanosine insertion were to produce a frameshift which wou ld result in a premature stop codon at position 684 of the open reading frame and which would result in a truncated protein of 228 amino acids of approximately 28 kd molecular weight compared to the w i l d type 376 amino acids of 40 kd molecular weight. It wou ld also alter every amino acid in the putative nucleoside-binding site and eliminate the combined nucleoside and ATP-binding site. Such a protein should theoretically be devoid of T K activity and confer the T K D phenotype (Figures 10a. and 10b.). 3.3.2. Thymidine kinase protein studies of isolates from patient WJ. Hav ing predicted the translational consequences of the guanosine addition which conferred the T K D phenotype onto isolates from patient W J , 63 Figure 9. TK mutations wi th in WJ isolates. The figure depicts D N A sequencing gels of the T K genes of T K ^ T control plaque isolate 1773-5 and T K D plaque isolates 1737-14 and 1106-2 originating from patient WJ. These show 1773-5 to have a stretch of 7 G's while both 1737-14 and 1106-2 have an identical G insertion mutation to produce a stretch of 8 G's. G A T C G A T C G A T C 1773-5 1737-14 1106-2 64 I CO JZ t-o S a (/) O) c H5 c !5 • 10 3 C • M E CD I— • o o o o o c o a! d < > < 2 c o CO (A CP CO CO a> CO ll#llslll 3 3 0) _ l O >» 2 a. < < o» co ** "w C 73 C 15 cu w o U 3 < U) 3 C a> •5 o O CD H " o • 4-1 *— u ro CO £ » ."2 o ro a) IE CD i_ ro ro co CO CD co »-o c 'a> 4-» o CD O CD > o o _ ro °-Sa> * CD I- £ W £ O % o J2 c £ « « o).S2 _ c » 3 '55 o - u a cu a to •C C 4-i2 8-= .9 <fl-co a t j c •g « .2 E o 2 CO C 3 co E E CO CO CO T3 CD </) a, £ ro r r «j c o ^ ro ^ iZ I s- O I s- c CD T - 3 a 0 *-o ^ I- m 33 CD 3 CO CT CD ro c o. '35 o > E C O ro o CD 3 CO CD CD •*-> "<7> c "O c • a. </> > » 0 1 6 u OL a m < 5 C O O O O O O o o 3 (!) - J 6 Q. JO < « d) 00 CO CO • CD T3 "55 O _Q) O 3 CO a) o o 2 o) -a 3 .E a> -a -a a. 2 .E </> a - ? "D o> a> o •- "O « w w o o> o n c a> c ^ o C 3 S-E N !E < o o ~a c « » "55 ° •= 2 c E Q. T3 O (0 •— CO 0 w w 4—' * L ^ a> * "I 13 «re 3 0) (0 Q> 0) 4-> c w _ =5 » o c .E CO i o •D Q. = c i - 5 re o) ™ o c re ro a> ._ ^ - «- g> •a .E 0) m £ £ ^ S5f * r*. ^ a> T - o>.c = = = £ re > c+ o v c ".3 O re co *<P K / t. 3 £ ® 5 » ^ 0) — • o «> 2 3 o » s s UL Q. D) 66 the presence of the predicted truncated T K protein product was sought by protein studies. Initial studies by immunoprecipitation using 3 5 S meth ion ine metabolic labeling and an anti-HSV-2 monoclonal antibody demonstrated 1773-5 to produce the expected full length 40 kd protein while both 1737-14 and 1106-2 produced the predicted truncated 28 kd protein product (Figure 11.). The technique of Western blot analysis using the same antibody was then perfected and, due to its superior specificity and the lack of a requirement for radioisotopes, was adopted in favor of immunoprecipitation. Western blot analysis also confirmed 1773-5 to produce a full length 40 kd protein and 1737-14 and 1106-2 to produce a 28 kd truncated protein product as predicted from the amino acid sequence resulting from the mutation and seen by immunoprecipitation (Figure 12.) 3.4 M U T A T I O N S C O N F E R R I N G T K D P H E N O T Y P E S O N T O A C T G 095 ISOLATES. • 3.4.1. D N A sequencing of A C T G 095-derived isolates 3.4.1.1. Spontaneously-reactivated isolates. In view of the genetic lesion detected in isolates 1737-14 and 1106-2, the genetic lesions in other T K D spontaneously-reactivated clinical isolates were evaluated. Accordingly, A C V R isolates derived from clinical trial A C T G 095 which exhibited such behaviour were sequenced to determine whether there was a common genetic basis for this ability. In an attempt to improve isolate purity, 5 plaque isolates were picked from these clinical isolates and retested for A C V resistance (Table 8.). These plaque isolates were screened for the guanosine addition identified in 1737-14 and 1106-2 by sequencing the region covering this mutation using the primer spanning this region (JS 12). 67 Figure 11. Immunoprecipitation of WJ isolates. The figure depicts immunoprecipitat ion using 3 5 S methionine labeling and ant i -HSV 2 T K monoclonal antibody. The left hand lane is a V E R O cell extract which was mock infected and shows labelled cellular protein. G strain is a T K W T control while 1773-5 is a T K W T plaque isolate from patient WJ; both demonstrate a full length 40 kd protein. KpnA333 is a control T K D deletion mutant and demonstrates a truncated T K protein at approximately 35 kd. Plaque isolates 1737-14 and 1106-2 are T K D and originate from patient WJ; both demonstrate an identical truncated product at 28 kd. kDa 68 Figure 12. Western blot analysis of WJ isolates. The figure depicts plaque isolates derived from patient WJ clinical isolates. The left lane is mock infected and shows no signal. Reference T K W T strain G and plaque isolate 1773-5 demonstrate full length 40 kd proteins. Plaque isolates 1737-14 and 1106-2 show a truncated 28 kd product. k D a 97-68-69 Table 8. A C V susceptibilities of ACTG-095 plaque isolates. The table depicts characterization of plaque isolates originating from ACTG-095 clinical isolates and demonstrates all to be A C V R . Reactivated A C V R Reactivated A C V S Plaque A C V I D 5 0 Plaque A C V I D 5 0 Isolate p ig /ml ) Isolate p ig /ml ) 89-063-1 19.2 390-4 9.5 89- 353-1 21.8 650-5 10.1 90- 150-3 23.7 110-4 9.5 2370-2 19.5 70 Sequence screening demonstrated 1 of 4 of these A C T G 095-derived plaque isolates (90-150-3) to have the identical G insertion as 1737-14 and 1106-2 (Figure 13.). 3.4.1.2. Isolates followed by an A C V S reactivation To see whether the G insertion only occurred in A C V R isolates wh ich were able to spontaneously reactivate or whether they also occurred in isolates which were followed by the expected A C V S reactivation, we also screened for the mutation in 3 such isolates sourced from A C T G 095. Of these, one plaque isolate, (89-650-5), also demonstrated the identical guanosine insertion as seen in 1737-14 and 1106-2 (Figure 14.), suggesting that such mutations can result in reactivations which could either spontaneously reactivate as a T K D phenotype or be followed by the expected A C V S phenotype. 3.4.2. Protein studies of A C T G 095-derived isolates. 3.4.2.1. Spontaneously-reactivated isolates. Western blot analyses of A C T G 095-derived plaque isolates also confirmed a 28 kd truncated T K product for both 90-150-3 and 89-650-5 w h i c h corroborated the predictions of the sequencing data. Of other A C T G 095-derived plaque isolates, 2370-2 produced a full length 40 kd protein, 89-353-1 demonstrated a truncated product identical in size to the 28 kd product associated wi th the above reported guanosine insertion while 89-063-1 demonstrated a slightly larger truncated product of approximately 32 kd (Figure 15.). Plaque autoradiography using 1 2 5 I d C performed on these plaque isolates demonstrated 90-150-3 to consist of 89% T K D plaques. Of particular interest was plaque isolate 2370-2 which produced a full length product. It, as 71 Figure 13. A C T G 095 A C V R mutational screen. The figure depicts D N A sequence screening of the gene region covering the stretch of 7 guanosines i n plaque isolates derived from A C T G 095 spontaneously-reactivated A C V R clinical isolates. Of 4 isolates screened, plaque isolate 89-150-3 has the identical guanosine insertion wi th in the stretch of 7 guanosines as was demonstrated in 1737-14 and 1106-2. G A T C G A T C G A T C G A T C 063-1 353-1 150-3 2370-2 72 Figure 14. A C T G 095 A C V S mutational screen, the figure depicts D N A sequences screening of the gene region of the 7 guanosines in plaque isolates derived from A C T G 095 which were followed by the expected A C V S reactivations. Of 3 isolates screened, plaque isolate 89-650-5 also has the identical guanosine insertion wi th in that stretch as was demonstrated i n 1737-14 and 1106-2. G A T C G A T C G A T C 110-4 390-4 650-5 73 Figure 15. Western blot analysis of A C T G 095 A C V R plaque isolates. The figure depicts proteins produced by plaque isolates derived from cl in ical isolates which were A C V R and spontaneously reactivated. Reference T K W T strain G and plaque isolate 2370-2 demonstrate full length 40 kd proteins. Plaque isolate 90-150-3 produces a truncated 28 kd product identical to 1737-14 and 1106-2. Reference T K deletion mutant KpnA333 demonstrates a truncated product of approximately 35 kd. Plaque isolate 89-353-1 demonstrates a truncated product identical in size to the 28 kd product associated wi th the guanosine insertion described while plaque isolate 89-063-1 demonstrates a slightly larger truncated product of approximately 32 kd. kOa 9 7 -6 8 -4 3 -2 9 -1 8 -1 4 -u o CO CO 3 c t T T 7 CO CO o co in in O CO CM • o CO CM 74 wel l as it parent clinical isolate 2370, despite testing A C V R by susceptibility testing and having low T K uptake by 1 2 5 IVaraU, demonstrated most plaques to have an almost uniform population of plaques exhibiting a high level of T K activity by both 1 2 5 IdC and 1 4 C TdR (Figures 16a. and 16b.). This suggested that the basis of its A C V resistance was due to mechanisms other than T K deficiency. 3.4.2.2. Isolates followed by an A C V S reactivation. Western blot analysis of plaque isolates from A C V R clinical isolates sourced from A C T G 095 which were followed by the expected A C V $ reactivation also confirmed a 28 kd truncated T K product identical to 1737-14 and 1106-2 for 89-650-5. Of other isolates, 89-390-4 produced a full length 40 kd protein while 90-110-4 d id not produce a detectable product (Figure 17.). Plaque autoradiography using 1 2 5 I d C performed on these plaque isolates demonstrated 89-650-5 to be a homogeneous T K D population. Also of interest was plaque isolate 89-390-4 wh ich also produced a full length 40 kd product and which, by plaque autoradiography was shown to consist of plaques wi th a faint r im, suggestive of a T K L R phenotype (Figures. 16a and 16b.). Further characterization of both 2370-2 and 89-390-4 is ongoing. 75 Figure 16a. A C T G 095 plaque isolate 1 2 5 I d C plaque autoradiography. The figure depicts 1 2 5 I d C plaque autoradiography of plaque isolates sourced from clinical isolates from A C T G 095. Plaque isolate 89-650-5 is uniformly T K D while 90-150-3 is 89% T K D . Also of note is the almost uniform marked T K activity of plaques wi th in 2370 and 2370-2. The barely perceptible faint r ims produced by 89-390-4 are not evident on the photograph below but are vis ible by direct inspection against a light box Clinical Isolates 89-063 89-353 90-150 2370 Plaque Isolates 89-063-1 89-353-1 90-150-3 2370-2 Clinical Isolates 89-390 89-650 90-110 Plaque Isolates 89-390-4 89-650-5 90-110-4 76 Figure 16b. A C T G 095 plaque isolate 1 4 C TdR plaque autoradiography. The figure depicts 1 4 C TdR plaque autoradiography of plaque isolates sourced from clinical isolates from A C T G 095. Plaque isolate 89-650-5 again is un i fo rmly T K D while 90-150-3 is mostly T K D . Again the marked T K activity of plaques within 2370 and 2370-2 is apparent. 89-063 89-353 90-150 2370 Plaque Isolates Clinical Isolates 89-390 89-650 90-110 Plaque Isolates 89-390-4 89-650-5 90-110-4 77 Figure 17. Western blot analysis of A C T G 095 A C V s plaque isolates. The figure depicts proteins produced by plaque isolates derived from clinical isolates which were A C V R and which were followed by the expected A C V S reactivations. Plaque isolate 89-390-4 produces a full length 40 kd protein identical to reference T K W T strain G. Plaque isolate 89-650-5 produces an identical 28 kd protein as was demonstrated in 1737-14 and 1106-2 whi le plaque isolate 90-110-4 d id not produce a detectable protein product. kDa 9 7 -6 8 -4 3 -2 9 -1 8 -1 4 -"3-6 o CO • o in CO 78 C H A P T E R 4 D I S C U S S I O N 4.1 M E C H A N I S M S OF R E A C T I V A T I O N OF T K D ISOLATES . The original aim of this work was to define the mechanisms underlying the apparent paradoxical behaviour of a clinical isolate which was T K D but reactivated. A t the outset our working hypothesis was that the reactivation potential of such isolates was due to a fundamental phenotypic difference from other T K D isolates. Subsequent work, as detailed above, disproved this. Al though reactivation by T K D isolates had been previously noted (98), the work wi th isolate 1737, originating from patient WJ, is the first report which documents, fully characterizes, and suggests a mechanistic basis for the spontaneous clinical reactivation of a truly T K D H S V isolate. Resistance was acquired during the treatment of previous episodes and then the T K D isolate reactivated without selection pressure by continuing ant iv i ra l administration. Complete eradication of H S V from the affected area between episodes was well-documented, demonstrating that latency of the T K D strain had been established. This was further supported by experimental data demonstrating the ability of the isolate to establish latency wi th in mouse trigeminal ganglia. The reactivation of this isolate in the patient appeared paradoxical in the face of current dogma suggesting that T K D H S V should not be able to reactivate. This isolate was homogeneously T K D by a l l conventional tests including plaque isolation and plaque autoradiography. Al though T K uptake studies may have suggested a low level of activity, this assay has proved insensitive in discriminating between low and absent activity and its true phenotype is much more accurately defined by the 79 homogeneously T K D plaques shown by plaque autoradiography as wel l as the failure to produce full length product by protein studies. Despite this apparent absence of TK activity, inoculation of the T K D isolate into a mouse model by either intranasal or intracerebral routes was able to both result in encephalitis wi th a pure T K W T population as wel l as establish trigeminal ganglia latency wi th a mixed populations of both T K D and T K W T strains. In addition, the intracerebral neurovirulence model was able to demonstrate 1737 to harbor an additional population suggestive of a T K L P phenotype, suggesting yet another possible mechanism of furnishing T K activity. Further work characterizing clinical isolate 89-063 also identified it as a reactivated truly T K D isolate by all conventional means. Mouse intracerebral inoculation of this isolate also extracted virus bearing a T K L P phenotype but, on this occasion, without a demonstrable T K W T population. The nature of the T K L P phenotype is very poorly understood at present; such isolates may constitutively express low levels of T K or, alternatively, r ibosomal frameshifting may play a role in their generation. Whether only the intracerebral model is capable of teasing out T K L P subpopulations is uncertain at present. Regardless of the underlying mechanism, it is very likely that the T K activity demonstrated in the neurovirulence studies also enabled these isolates to reactivate in the clinical setting and further enhances the principle that T K activity is required for reactivation of H S V . The intracerebral mode l , given its ability to detect T K activity in nearly all inoculated mice, clearly proved itself to be a more sensitive assay in the detection of low levels of T K activity than the intranasal model. It has been previously documented that the T K activity of a c l in ical isolate correlates wel l wi th the proportion of T K W T virus wi th in a mixed 80 population (97) and that mutants wi th low levels of T K activity can reactivate from latency (72). The above work suggests that very sensitive assays challenge old phenotypes and demonstrate that the levels of T K required for reactivation are quantitatively much lower than previously appreciated. Indeed, these data demonstrate that T K activity may fall below the threshold of conventional assays and, in this case, T K activity was amplified using passage in the animal model. Herein we have designated such strains as "ultra-low" producers. This is the first clinical demonstration that 'ultra l o w ' level thymidine kinase expression, predicated on masked heterogeneity, is sufficient for reactivation of clinically-significant A C V R disease. The distinction between isolates wi th very low levels of T K as opposed to absolute deficiency remains confused and we define the latter as being a pure T K D population. Our data suggest that the T K activity of isolates represent a continuum, based on the proportion of T K W T virus, wi th the threshold for reactivation yet to be determined and hence, such pure T K D populations may, indeed, be unachievable from clinical isolates. There are two possible explanations for the emergence of T K W T v i rus wi th in apparently pure populations; either a T K W T population, present i n such low numbers as to be undetectable by conventional means, became unmasked due to the exquisite sensitivity of the animal model in amplifying such small numbers of preexisting T K W T virus or, alternatively, in vivo reversion to T K W T took place from a pure T K D population during passage i n the animal model. In either case, the resulting T K W T virus wou ld have a virulence advantage and would explain the recrudescence of the neurovirulent phenotype. The frequency of reversion of the thymidine kinase gene has been estimated at 10~4 to 10~6, (87) although mutational hot 81 spots wi th much higher frequencies may exist wi th in the viral genome and a later discussion regarding the genetic lesion in this isolate w i l l further address this issue. W h i c h of the two mechanisms operated in these cases is s t i l l unresolved and it wou ld require further plaque purifications followed by reintroduction into an animal model to determine whether T K W T emergence could be reduced, thereby suggesting the washout of a preexisting populat ion. In addition, at present the relative contributions of T K W T and T K L P subpopulations to T K activity and therefore reactivation is also unresolved. Regardless of the underlying mechanisms, if one extrapolates from the experience of the animal model, the demonstrated T K heterogeneity suggests that reactivation of both 1737 and 89-063 was due to small subpopulations wi th intrinsic T K activity wi th in a larger T K D population and that such subpopulations were able to impart their phenotypic T K expression onto the larger T K D population and enable it to reactivate; this may be another example of in vivo complementation. This is yet another example whereby a larger virus population can widen the repertoire of its phenotypic expression by util izing those of a much smaller subpopulation. The virus is thus able to retain A C V resistance while extending its phenotypic expression to include reactivation potential, such a strategy offering a survival advantage i n the face of antiviral pressure. Further work on clinical isolates sourced from A C T G 095 which also reactivated as A C V R showed that 3 of 4 reactivated isolates, (89-353, 90-150 and 2370) harbored subpopulations of virus wi th the T K W T phenotype demonstrated simply by performing plaque autoradiography and without the need to resort to very sensitive mouse neurovirulence studies as was done for 1737 and 89-063. Characterization of the pre-reactivation A C V R c l in ical 82 isolates from these 3 patients demonstrated an increase in the number of TKWT plaques from the init ial pretreatment A C V R isolate to the corresponding A C V R isolate wh ich reactivated, showing that, in cases where T K activity is demonstrable wi th in reactivated isolates by convent ional means, the ability to reactivate is associated wi th an emergence of the T K W T population and a corresponding increase in TK activity. In any case, these experiments conclusively demonstrate that supposed T K D clinical isolates which reactivate retain some T K activity, further enhancing the principle that T K activity is essential for reactivation. It also demonstrates that c l inical isolates can selectively retain some phenotypic expression of thymidine kinase activity, thereby retaining the characteristics of reactivation and neurovirulence, while still testing as A C V R in susceptibility assays, . This is the first demonstration that there can be a dissociation between these characteristics. The ability of a T K W T subpopulation to impart its characteristics onto a larger T K D population to enable it a wider phenotypic expression by reactivating suggests in vivo complementation. There have been mul t ip le prior reported examples of such interactions wi th in H S V viral populations. V i r a l heterogeneity wi th possible complementation has been previously demonstrated to be an important component of clinical disease progression wi th in A C V R clinical isolates bearing both the T K D and pol phenotypes (54,96). In addition, evidence for in vivo complementation has been documented in pairs of temperature sensitive mutants (90,91), neuro invas ive mutants (93) and between pairs of thymidine kinase strains, both T K W T / T K D and T K D / T K D (63,67,81). There are three possible mechanisms by w h i c h complementation may occur. First, there may be genetic recombination and at 83 least some of the above mentioned complementations were shown to be due to this mechanism, although there were many complementation pairs where no such evidence could be detected, suggesting the possibility of other mechanisms (90,91,93). Secondly, as the thymidine kinase gene exists as a homodimer, it has been proposed that a heterodimer may form where a TKWT polypeptide may in some way combine wi th a T K peptide lacking activity, thereby compensating for its deficient activity (81). Finally, local exchange of phosphorylated nucleotide pools may occur, such that T K W T virions may phosphorylate nucleosides which, due to their small size may enter cells infected wi th T K D virions, bypassing the need for T K activity. The contribution of theses 3 putative mechanisms is at present unclear. Cl in ica l isolate 89-063 was uniquely interesting in that, even using an animal model, a phenotypically T K W T subpopulation could not be extracted. Rather, it yielded a population of viruses consistent wi th a T K L P phenotype on plaque autoradiography This phenotype would also test as A C V R o n susceptibility assays and produce min imal T K activity. Arbitrarily, such isolates have been defined as expressing 1-15% T K activity based on their thymidine phosphorylating ability (56). Again this data strengthens the principle that T K activity, even if i n only small amounts, is essential for reactivation but suggest a second mechanism by which the virus could generate it. The issue of whether thymidine kinase activity can be dissociated from neurovirulence has also been recently debated. There have been 2 reports of neurovirulent T K D H S V isolates which have supposedly retained neurovirulence. Chatis and Crumpacker reported a plaque-purified T K D H S V isolate from an AIDS patient which , due to a single base substitution, 84 produced a full length protein and retained full neurovirulence (98,100). Plaque autoradiography in those studies, however, was conducted in a T K -producing cell line using a 1 4 C-thymid ine overlay, potentially reducing assay sensitivity in demonstrating heterogeneity or a T K - l o w producer phenotype. In addition, the combination of a full length T K polypeptide and a base substitution which is not in any of the three described conserved binding domains, calls into question the significance of this mutation. The second report by Tanaka et al. (101) performed only a single plaque purification and omitted plaque autoradiography altogether. In addition, although the identified T K mutation was close to the putative nucleoside binding site, again it was not within it and this was not corroborated wi th protein studies. These discrepancies raise the possibility that masked heterogeneity, as described in our report, or a low T K producer phenotype may have provided sufficient T K activity for neurovirulence while testing as T K D . Contrary to this, the isolates from the above work demonstrated neurovirulence to be directly linked to T K activity, a finding which supports conventional wisdom. It thus seems likely that T K activity in the above isolates may have been left sufficiently intact to allow for neurovirulence and the proof of a dissociation between neurovirulence and T K activity has not yet been convincingly proven. The clinician w i l l need to appreciate that effective clinical and virological eradication of mucocutaneous viral shedding through foscarnet (or alternative) therapy, followed by no antiviral treatment whatsoever, may stil l eventuate in a subsequent T K D H S V reactivation episode. Accordingly, the use of A C V may, in some cases, fail even for treatment of a new and unchallenged recurrence. Despite the documentation of T K D recurrences, 85 they are rare and T K W T activity prevailed in the vast majority of untreated reactivations. One possible explanation for this is that clones expressing T K are more efficient reactivators. That withdrawal of A C V treatment pressure in this patient eventually led to an T K W T reactivation further supports this hypothesis. Alternatively, T K W T reactivations may have originated from different latently-infected ganglionic sites altogether which had not been subjected to the development of A C V resistance. Regardless of the under lying mechanism, for patients wi th refractory disease, this reversion of herpes simplex virus over time to an A C V S population may provide hope for a useful clinical alternative, such as alternating antiviral interventions. 4.2 T K D M U T A T I O N S A N D R E A C T I V A T I O N . The second aim of this work was to identify whether specific genotypic lesions were associated wi th reactivation potential. Initially, work was directed at identification of the mutation in the T K gene of the original reactivated T K D H S V 2 isolate, 1737, and identification of its T K product. The mutation in 1737 was identified as a guanosine insertion in a stretch of 7 guanosine bases. The effects of this mutation would be to alter every amino acid in the nucleoside-binding site, and to introduce a premature stop codon which wou ld eliminate the combined ATP/nucleoside-binding site (Figs. 10a and 10b). Such a protein should theoretically be devoid of T K activity. G iven that this was the longest homopolymer stretch of bases in the gene and that the gene is particularly G-C rich, it is likely purely by chance, that such a long stretch would be composed of G / C pairs. Alternatively, it would be tempting to speculate that A C V , by being a guanosine analogue, may cause the viral D N A polymerase to selectively slip or stutter in a region 86 containing a stretch of guanosines and induce such a mutation. A C V is an obligate chain terminator of vira l D N A , hence any such mutational influence it exerted wou ld have to influence vi ra l D N A polymerase wi thout incorporation. Such mechanisms remain intellectually attractive but speculative at this stage. Al though only a single plaque isolate from 1737 was sequenced, the clinical isolate was uniformly T K D by plaque autoradiography and another plaque isolate originating from an independent prior outbreak in the same patient whose parent clinical isolate was also uniformly T K D by plaque autoradiography, contained the same mutation. This suggests that the guanosine insertion was a homogeneous mutation. Reports of mutations i n the T K gene of other T K D H S V isolates is very limited. It has been w e l l documented that the majority of T K D H S V 1 mutants produced truncated T K proteins, suggesting that frameshift mutations are common in this setting, although this early work was not correlated wi th D N A sequencing of the T K gene (83). Of available genotypic data, K i t et al. (102) has previously described a deletion mutation in the same string of guanosines in a laboratory-derived 5'-bromodeoxyuridine (BUdR)-resistant H S V 2 isolate and Hwang et al. (103) have described the same G addition as 1737-14 in a plaque-purified T K D H S V 1 clinical isolate. In addition, screening of A C T G 095-derived plaque isolates has identified the same G insertion i n 1 of 4 other reactivated A C V R H S V 2 plaque isolates and 1 of 3 A C V R H S V 2 clinical isolates which were followed by an A C V S reactivation. These data identify this stretch of 7 G's as a mutational hot spot for the H S V T K gene and are in marked distinction to the varicella-zoster tk gene where mutations are widely distributed throughout the gene (104) 87 Homopolymer stretches have been shown to be particularly susceptible to frameshift mutation in other biological systems. Consecutive runs of single nucleotides have been shown to also be mutational hotspots for the lysozyme and r l l genes in T4 bacteriophage (105,106), the T antigen gene in polyoma virus (107), as wel l as for eucaryotic cells such as mouse i m m u n o g l o b u l i n heavy chain locus (108). This suggests homopolymer nucleotide stretches as general hot spot mutational mechanisms distributed throughout diverse biological systems. Such hot spots can be susceptible to both spontaneous and mutagen-induced mutations and the l ikel ihood of mutation is directly related to the number of bases within the homopolymer stretch. The putative mechanistic model is of local misalignment of base pairs w i t h i n homopolymer stretches which provide frequent sites for misaligned but complementary base pairing (105). Such hot spots resulting in frameshift mutations may not only occur in reiterations of single base pairs but also wi th in more complex repeats, raising the possibility of such sequences also operating within H S V T K (109,110). As wel l as forward mutation, homopolymer base stretches have also been shown to have an increased reversion frequency to w i l d type (111). This may offer a putative explanation for the emergence of heterogeneity wi th in a seemingly pure population as a high rate of reversion at such hot spots may potentially generate T K W T subpopulations producing sufficient T K activity to allow reactivation in animal models and in the clinical situation as described i n the initial characterization of 1737. A n alternative potential explanation has been reported by H w a n g et al. (103). They have demonstrated an in vitro net +1 translational frameshifting by an H S V isolate harboring the same guanosine insertion described above; previously a phenomenon only 88 reported in R N A viruses. Whereas such strains produce a truncated product in the majority of translations, occasional frameshifts may produce fu l l length protein in 2% of translations. We have already demonstrated that T K activity required for reactivation is very small and such mechanisms may allow production of a full length T K protein in sufficient quantity to a l low reactivation. The frequency of such frameshifts are homopolymer length-dependent (Dr. D o n Coen, personal communication), again suggesting that the string of 7 guanosines is the most l ikely site of such events. Such low level T K production may explain the neuropathogenic faint-rimmed plaques seen in output strains from mice inoculated wi th 89-063 and may play a role i n the genesis of the T K L P phenotype in at least some isolates. The relative contributions of the 2 genetic mechanisms is unclear. The demonstration of TKWT heterogeneity wi th in the majority of T K D isolates suggests that such subpopulations are important in most instances. In these cases a h i g h reversion frequency at such homopolymer sites may be crucial to generate such subpopulations. In the case of 89-063, however, where no such subpopulations can be demonstrated but reactivated faint plaques are identified, frameshifting may wel l be the dominant mechanism. Frameshifting may also contribute to T K activity wi th in homopolymer T K D isolates already harboring T K W T subpopulations, as suggested by 1737 where both T K W T and T K L P subpopulations can be identified. Only two reports of T K D H S V T K gene sequences have documented single base substitutions (100,101). Interestingly, these isolates were the isolates described above w i t h retained neurovirulence. By contrast, the Western blot results from the above work indicated truncated products in 5 of 8 isolates (1737-14, 90-150-3, 89-650-5, 89-063-1 and 89-353-1). Two isolates (2370-2 and 89-390-4) produced full length proteins while the remaining isolate (90-110-4) failed to produce a detectable 89 product. This, together wi th the literature cited above, suggests that frameshift mutations constitute the majority of T K D isolates. F u l l sequencing of isolates not containing the G insertion is in progress to identify any other potential hot spots; specifically at other homopolymer sites. 4.3 C L I N I C A L I M P L I C A T I O N S A N D A P P L I C A T I O N S These data have multiple implications for the management of patients. Firstly they reinforce the observation that, as wel l as wi th in the context of selection of resistant subpopulations during antiviral therapy, that T K D reactivations can occur in the absence of any therapy. Hence, the long held belief that, following successful virological sterilization of a T K D lesion, the next reactivation would be A C V s no longer applies and the clinician can no longer invariably rely on the efficacy of A C V . This may mandate the use of different management strategies such as alternating a TK-dependent antiviral, such as A C V , wi th one independent of TK, such as P F A . In addition, the identification of a limited number of hot spots allows for the design of specific probes to detect common mutations, thereby allowing for easier detection of resistant isolates. Protein probes to detect truncated proteins are feasible and may be used to screen large populations of viruses wi th in clinical specimens and are currently under development. The current proposed strategy is to produce one polyclonal antibody directed at the N-terminus of the T K protein which wou ld detect all truncated proteins while another antibody directed at the C-terminus would only detect fu l l length T K proteins. If the cell monolayer was grown and inoculated wi th the clinical isolate on a membrane and the proteins detected using a Western blot system, a subtraction fi lm analogous to plaque autoradiography wou ld a l low 90 the detection of such truncated proteins wi th in a large heterogeneous cl inical population. This work also has therapeutic implications. Homopolymer hot spots may also offer a novel mechanism of antiviral attack, perhaps by permitt ing targeting of mutational sites either to stabilize such homopolymer stretches or inactivate established mutations. One way this could conceivably be implemented would be via antisense strategies. These goals are further away from realization but may be attainable in the future. Further work to better define the frequency and distribution of such mutations is needed. Finally, the strengthening of the principle that T K is required for reactivation increases the l ikelihood that strategies to neutralize T K may be useful in preventing reactivation altogether. Pharmacological T K inhibitors exist and are currently undergoing development. They w i l l l ikely enter clinical trials in the not too distant future. Alternatively, T K inact ivat ion could be achieved through a different mechanism; i.e. immunological ly v i a an anti-TK vaccine which offer the prospect of long-lasting inhibition. 4.4 T H E F U T U R E This work begun here w i l l be extended. Firstly, the mutations conferring resistance on the remaining clinical isolates need to be identified to determine whether there are any other hot spots wi th in the gene. The development of a protein probe to detect truncated proteins wi th in entire populations of clinical isolates has already been discussed above. In addition, the detected mutations need to be repaired using site-directed mutagenesis and the resultant virus recharacterized to prove that the identified muta t ion is indeed responsible for the observed mutant phenotype. This work is 91 currently ongoing. The mechanism of the low T K producer needs to be identified; specifically the promoter region needs to be sequenced and m R N A detected by Northern blot to determine whether mutations wi th in control elements outside the coding sequence are responsible for lower expression of TK. In addition, many more resistant isolates need to be sequenced to strengthen the data presented here and more fully describe the proportions of different type of mutations conferring the A C V R phenotype. Integral to this wou ld be a commitment to sequence a large bank of T K W T isolates to help define whether base substitutions seen may be a phenomenon of strain variation or whether they may confer the A C V R phenotype, given that such mutations have not yet been convincingly proven to do so. Finally, further plaque purifications of isolates 1737 and 89-063 need to be performed and reintroduced into animal neurovirulence models to discriminate whether the appearance of T K W T output strains is due to small preexisting subpopulation or in vivo genetic reversion. 92 C H A P T E R 5 C O N C L U S I O N This thesis set out to prove the hypothesis that a truly T K D virus can reactivate and disproved it. The present work elucidated the mechanisms whereby a clinical isolate which tests as A C V R by susceptibility testing and can even appear as a uniform T K D population by conventional assays can defy existing dogma and reactivate. The work still supports the principle that T K activity is necessary for reactivation but defines ways in which H S V can circumvent such restrictions by genetic reversions at hot spots in the T K gene or ribosomal frameshifting such that T K activity is present. By doing so, the work also demonstrates that the level of T K activity required to reactivate is far lower than previously appreciated and defines the mouse neurovirulence model as a more sensitive assay for teasing out such low levels of T K activity in virus populations where conventional techniques fail to identify it and defines such isolates as having "ultra low" T K activity. Furthermore, the work demonstrates for the first time that the A C V R phenotype can be dissociated from the ability to reactivate. The other major finding of this work was to identify a homopolymer string of guanosines within the T K gene as a mutational hot spot. 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