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In vitro translation of cucumber necrosis virus RNA Johnston, Julie Catherine 1989

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IN VITRO TRANSLATION OF CUCUMBER NECROSIS VIRUS RNA By Julie Catherine Johnston B.Sc. (Agr.), The University of British Columbia, 1987 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES P L A N T SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A December 1989 © Julie Catherine Johnston, 1989 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 V^n.f Sd-Cnc-c The University of British Columbia Vancouver, Canada Date P'&-DE-6 (2/88) Abstract The in vitro translation products directed by cucumber necrosis virus (CNV) R N A were analyzed in both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of ca. 33 M r was produced. In wheat germ extracts, four proteins of ca. 41, 33, 21 and 20 M r were produced. Hybrid-arrested translation (HART) studies using synthetic C N V antisense R N A corresponding to the" entire C N V genome demonstrated that the four major proteins synthesized from C N V virion R N A in wheat germ extracts are virus-specific translation products. The genomic locations of the C N V in vitro translation products were determined using a number of experimental approaches including: (1) H A R T using antisense RNA corresponding to selected regions of the C N V genome; (2) in vitro translation of synthetic messenger-sense C N V transcripts; (3) immunoprecipitation of in vitro translation products with C N V polyclonal antisera and (4) in vitro translation of size-fractionated C N V virion R N A . Together, these experiments demonstrated that the ca. 33 M r protein is derived from the 5' proximal coding region, the ca. 41 M r protein is derived from an internal coding region, and that at least one but probably both of the ca. 20 and 21 M r proteins are derived from the 3' terminal coding region(s) of the C N V genome. In addition, immunoprecipitation experiments provided further evidence that the ca. 41 M r protein is the viral coat protein. The size, number, and genomic locations of the C N V in vitro translation products reported here are in agreement with those predicted from nucleotide sequence data (Rochon & Tremaine, 1989). The natural template for the expression of downstream cistrons in the C N V genome was investigated by in vitro translation of sucrose fractionated C N V virion RNA as well as in vitro translation of messenger-sense synthetic transcripts. These studies indicate that in vitro, both subgenomic and genomic-length C N V RNA molecules may act as templates for the synthesis of the ca. 41,21 and 20 M r proteins as well as the ca. 33 M r protein. i i Table of Contents Abstract ii List of Tables vii List of Figures viii List of Abbreviations x Acknowledgement xiii 1 Introduction 1 1.1 Plant viruses 1 1.1.1 General characteristics 1 1.1.2 Genome structure and organization 2 1.1.3 Translation strategies 2 1.2 Cucumber necrosis virus 6 1.2.1 General characteristics 6 1.2.2 Genome organization 7 1.2.3 Translation strategy 7 1.3 Thesis objectives 8 2 Materials and Methods 9 2.1 Virus propagation and isolation 9 2.1.1 Virus propagation 9 2.1.2 Virus purification 10 2.2 Antisera production and antibody purification 11 2.2.1 Production of polyclonal antisera 11 2.2.2 Purification of immunoglobulin (IgG) 12 i i i 2.3 Vir ion R N A extraction 13 2.3.1 R N A extraction 13 2.3.2 Denaturing agarose gel electrophoresis of R N A 14 2.4 Analysis of in vitro translation products 15 2.4.1 In vitro translation 15 2.4.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis...l6 2.4.3 Protein molecular weight estimates 16 2.4.4 Immunoprecipitation 17 2.5 C N V synthetic transcripts 18 2.5.1 C N V clones 18 2.5.2 Construction of subclones 18 2.5.3 Isolation of supercoiled plasmid D N A 20 2.5.4 In vitro transcription 21 2.5.5 Hybrid-arrested translation 22 2.6 Analysis of C N V subgenomic RNAs 23 2.6.1 Log-linear sucrose density gradients 23 2.6.2 Alkaline northern blot 23 2.6.3 Nick translated probes 25 2.6.4 R N A size estimates ...25 3 Results 2 6 3.1 Characteristics of C N V in vitro translation products 26 3.1.1 Comparison of C N V translation products in rabbit reticulocyte lysates and wheat germ extracts 26 3.1.2 Size estimates of C N V in vitro translation products 28 3.2 Optimal in vitro conditions for translation of C N V R N A 30 i v 3.2.1 Concentration of exogenous R N A 30 3.2.2 Time course of protein synthesis 30 3.2.3 R N A extraction from wheat germ extracts 33 3.2.4 Effect of magnesium and potassium 33 3.3 Hybrid-arrested translation 34 3.3.1 Use of synthetic negative sense R N A to identify CNV-specific in vitro translation products 34 3.3.2 Optimal molar amounts of C N V negative sense R N A 39 3.4 Genomic location and identification of C N V in vitro translation products39 3.4.1 Genomic location of the 34,600 M r protein coding region 41 3.4.2 Genomic location of the 41,600 M r protein coding region 45 3.4.3 Genomic location of the 20,000 and 24,000 M r protein coding regions 49 3.5 Natural template(s) for the expression of C N V proteins 52 3.5.1 Sucrose gradient fractionation of C N V virion R N A 52 3.5.2 In vitro translation of sucrose gradient fractionated C N V RNA53 3.5.3 Translation of full-length positive sense synthetic transcripts.. 59 Discussion 6 2 4.1 Differences in C N V RNA translational efficiencies between rabbit reticulocyte lysates and wheat germ extracts 64 4.1.1 Differential selection of A U G codons between plant and animal ribosomes 64 4.1.2 Endogenous capping activity of wheat germ extracts 67 4.1.3 Differential ability of ribosomes to translate through regions of high secondary structure 68 v 4.2 In vitro translation of other tombusviruses 68 4.3 The putative 92 kDa readthrough protein 69 4.4 Translation of genomic-length synthetic C N V R N A 71 4.4.1 Translation conditions 72 4.4.1.1 Fidelity of translation in rabbit reticulocyte lysates... 72 4.4.1.2 Translation conditions in wheat germ extracts 73 4.4.2 Alternative translation mechanisms 74 4.5 The 0.9 kb subgenomic R N A is a bifunctional mRNA 75 Bibliography 77 v i List of Tables I. C N V clones, subclones and synthetic transcripts 19 II. Sequences surrounding putative A U G codons 65 v i i List of Figures 3.1 Comparison of C N V RNA in vitro translation products in rabbit reticulocyte lysates and wheat germ extracts 27 3.2 Sizes of C N V in vitro translation products 29 3.3 In vitro translation products resulting from the translation of a dilution series of C N V R N A 31 3.4 Time course of in vitro translation of C N V R N A 32 3.5.1 Effect of potassium on in vitro translation products of C N V R N A 35 3.5.2 Effect of magnesium on in vitro translation products of C N V R N A 36 3.6 H A R T studies using a negative sense synthetic transcript corresponding to the entire C N V genome 38 3.7 H A R T studies using increasing concentrations of C N V T x 18/4701(-) 40 3.8 Genomic locations of C N V positive and negative sense synthetic transcripts ... 42 3.9 Genomic location of the 34,600 M r in vitro translation product 44 3.10 Genomic location of the 41,600 M r in vitro translation product 46 3.11 Immunoprecipitation of C N V in vitro translation products 48 3.12 Genomic location of the 24,000 and 21,000 Mr in vitro translation products... 50 3.13 Agarose gel electrophoresis of sucrose gradient fractionated C N V virion RNA. 54 3.14.1 Agarose gel electrophoresis of selected sucrose gradient fractionated R N A samples 56 3.14.2 Northern blot analysis of selected sucrose gradient fractionated R N A samples. 57 3.15 In vitro translation products of selected sucrose gradient fractionated C N V virion R N A samples 59 3.16 In vitro translation of synthetic genomic-length C N V R N A 61 v i i i 4.1 Genomic locations and templates for synthesis of C N V in vitro translation products 63 4.2 Alignment of the nucleotide sequences preceding the putative initiation codons for C N V , TBSV-cherry and CyRSV proteins 70 i x List of Abbreviations A26O - absorbance at 260 nm ATP (dATP) - adenosine triphosphate (deoxy adenosine triphosphate) BE - borate/EDTA B M V - brome mosaic virus B R L - Bethesda Research Laboratories B S A - bovine serum albumin B Y D V - barley yellow dwarf virus C - C e l s i u s ca. - approximately CarMV - carnation mottle virus cDNA - complementary D N A C i (M-Ci) - curie (microcurie) CIP - calf-intestinal phosphatase cm (mm) - centimeter (millimeter) C N V - cucumber necrosis virus CsCl - cesium chloride CTP (dCTP) - cytidine triphosphate (deoxy cytidine triphosphate) CyRSV - cymbidium ringspot virus D N A - deoxyribonucleic acid ds - double stranded DTT - dithiothreitol dTTP - deoxy thymidine triphosphate EDTA - ethylenediaminetetraacetic acid x EtBr - ethidium bromide g (mg, |lg) - gram (milligram, microgram) GTP (dGTP) - guanosine triphosphate (deoxy guanosine triphosphate) H A R T - hybrid-arrested translation hr - hour IgG - immunoglobulin kb - kilobase kDa - kilodalton L B - Luria-Bertani 1 (ml, |il) - litre (millilitre, microlitre) m (cm, mm, nm) - metre (centimetre, millimetre, nanometre) M (mM) - molar (millimolar) MeHgOH - methyl mercuric hydroxide MgCl2 - magnesium chloride min - minute M r - molecular weight mRNA - messenger R N A N. clevelandii - Nicotiana clevelandii NaCl - sodium chloride NaH2P04 - sodium phosphate NaOAc - sodium acetate NaOH - sodium hydroxide N E N - New England Nuclear NET - NaCl/EDTA/Tris-HCl NP40 - Nonidet P40 x i ORF - open reading frame PBS - phosphate buffered saline PEG - polyethylene glycol PVP - polyvinylpyrrolidone R C N M V - red clover necrotic mosaic virus RNA - ribonucleic acid rpm - revolutions per minute rRNA - ribosomal R N A SDS - sodium dodecyl sulfate SDS-PAGE - SDS-polyacrylamide gel electrophoresis ss - single stranded SSC - sodium chloride/trisodium acetate TBS V - tomato bushy stunt virus TES - Tris-HCl/EDTA/SDS T M V - tobacco mosaic virus Tris-HCl - tris(hydroxymethyl)aminomethane-hydrochloric acid tRNA - transfer R N A UTP - uridine triphosphate U V - ultraviolet x i i Acknowledgement Firstly, I would like to sincerely thank my advisor Dr. D'Ann Rochon for her guidance, encouragement and support. Thanks are also extended to Dr. Joan McPherson for acting as my departmental advisor and to Dr. Carl Douglas and Dr. Victor Runeckles for serving on my committee. Finally, I would like to express my appreciation to all those at the Vancouver Research Station of Agriculture Canada for the use of the facilities as well as many helpful discussions. This work was supported by an N S E R C postgraduate scholarship and N S E R C grant number OGP0043840. x i i i Chapter 1 Introduction Knowledge of the organization and expression of plant virus genomes is essential for an understanding of plant virus evolution and biology. The compact size, limited coding capacity, as well as the ease with which some plant viruses can be manipulated provides a unique opportunity to study basic biological problems. Study of the interactions between the information stored on the compact viral genome and the complex eukaryotic host cell may provide insight into basic host cell functions and how these may be altered by the viral pathogen. An understanding of the interactions between host and virus may lead to the development of effective strategies of virus control. As a fundamental step towards this goal, determination of the organization, regulation and expression of the genes encoded in the plant virus genome is of basic importance. 1.1 Plant viruses 1.1.1 General characteristics Plant viruses are diverse in both their particle structure and genome type. Virus particles may be rod-shaped, icosahedral, bacilliform or, in addition, have an outer membrane envelope. Encapsidated within the particle is a genome consisting of one or more molecules of nucleic acid, either R N A or D N A . The nucleic acid may be single-stranded (ss) or double-stranded (ds), circular or linear, and may be of positive or negative polarity. By far, the majority of plant viruses contain positive sense ssRNA genomes (Zaitlin & Hull , 1987); these can be recognized as messenger RNAs (mRNAs) and translated directly by the host cell. 1 Chapter 1. Introduction 2 1.1.2 Genome structure and organization The information required to produce a complete infection by positive sense ssRNA viruses may be contained on a single R N A component (monopartite) or divided among two or more R N A components (multipartite). The individual R N A components of multipartite viruses may be encapsidated together in the same virus particle or separately into different particles. Positive sense ssRNA viruses contain various structures at the 5' and 3' termini of their RNAs. The 5' terminal structures include a m^G^'pppS1 c a p ? a di_ o r triphosphate, or a small covalently linked protein (VpG). The 3' terminal structures include a long polyadenylate sequence, a hydroxyl group, or a tRNA-like structure which may be specifically aminoacylated. Positive sense ssRNA viruses encode both structural and nonstructural proteins. The structural proteins form the virus particle. The nonstructural proteins are involved in various functions such as virus replication, transport and post-translational processing. The organization of the total genome is reflected in the strategy by which the genes are expressed. 1.1.3 Translation strategies Plant viruses have evolved to use the molecular machinery of the eukaryotic cell which they infect. Like eukaryotic cellular mRNAs, plant virus RNAs are recognized by eukaryotic ribosomes and translated. Unlike eukaryotic mRNAs which are generally monocistronic (Shih & Kaesberg, 1973), viral R N A molecules may be multicistronic, ie. contain more than one coding region. According to the scanning model for eukaryotic initiation of translation, however, usually only the 5' proximal coding region of a mRNA is expressed (Kozak, 1978; 1981). The scanning model states that the 40S ribosomal subunit binds to the exposed 5' terminus of the mRNA and migrates downstream to the first A U G codon. If the sequences surrounding the first A U G triplet are Chapter 1. Introduction 3 optimal, the 40S ribosomal subunit is joined by the 60S ribosomal subunit and translation is initiated. Translation is terminated when the 80S ribosome encounters a termination codon afterwhich one or both of the ribosomal subunits dissociate from the transcript. This means of selecting 5' proximal A U G codons for translation initiation is in contrast to that observed in prokaryotic cells. In prokaryotic mRNAs, which often contain more than one cistron, any A U G codon following an exposed ribosome binding site can be selected to initiate translation provided it is in an adequate sequence context (Kozak, 1983). To overcome the restriction of eukaryotic ribosomes in efficiently translating only 5' proximal cistrons, plant virus RNAs have evolved a number of strategies to express the information present in downstream cistrons. At least four different strategies enabling the expression of downstream coding regions have been demonstrated in plant R N A viruses and are briefly described below. Detailed information on the genome expression of plant positive sense ssRNA viruses can be found in review articles by Davies & Hull (1982) and Dougherty & Hiebert (1985). Segmentation of the virus genome The genomes of multipartite viruses are divided among two or more R N A components. Segmentation of the genome places the coding regions for the viral proteins in an optimal 5' location for expression by eukaryotic ribosomes. R N A components which contain only one cistron serve as messengers for a single protein. Multicistronic R N A components must utilize an additional translation strategy for the expression of downstream coding regions (see below). Chapter 1. Introduction Generation of subgenomic mRNAs 4 During infection, viral RNAs may generate one or more subgenomic molecules. The subgenomic RNAs are 3' co-terminal and colinear with genomic R N A and thus contain coding information that is redundant with that present on genomic RNA. Each subgenomic messenger expresses only the gene which is situated at the 5' end of the R N A and is therefore, like genomic RNA, functionally monocistronic. This strategy allows the genome of a multicistronic virus to be expressed through a nested set of 3' co-terminal subgenomic molecules, each set having the coding region for a different viral protein located at its 5' terminus. The formation of subgenomic RNAs may also serve to regulate the timing and level of gene expression. Several models have been proposed to explain how subgenomic mRNAs are synthesized during viral R N A replication. These models include: (1) internal initiation of transcription on negative sense genomic-length molecules; (2) premature termination of transcription during negative strand R N A synthesis and (3) processing by specific nuclease cleavage of genomic-length R N A (Miller et al., 1985). Experimental evidence has revealed that, at least in the case of brome mosaic virus (BMV), subgenomic RNAs are synthesized via internal initiation by the B M V RNA-dependent R N A polymerase (replicase) on the negative strand of genomic R N A (Miller et al., 1985). Readthrough translation Many viral RNAs contain 'leaky' termination codons which, when readthrough by eukaryotic ribosomes, enable translation of downstream portions of the viral genome. Suppression of a termination codon occurs, for example, in tobacco mosaic virus (Pelham, 1978), carnation mottle virus (Harbison et al., 1985) and tomato bushy stunt virus (Quintero et al, 1988) translation. Like Chapter 1. Introduction 5 the generation of subgenomic RNAs, readthrough translation may have the additional role of regulating the timing and level of viral gene expression. Proteolytic processing The entire genome of an R N A virus, or a long portion of it, may be translated as a monocistronic messenger and expressed as a single long polyprotein. The polyprotein is then processed into two or more functional gene products by viral or host encoded proteases or by autocatalytic cleavage. Proteolytic processing occurs in the nepo-, como-, poty- and sobemovirus groups of plant viruses. Ribosomal frameshifting Animal viruses such as retroviruses (Jacks & Varmus, 1985; Jacks et al., 1988) and coronaviruses (Brierley et ah, 1987) utilize ribosomal frameshifting to express overlapping cistrons that are in different translational reading frames. Frameshifting subsequent to translation of a portion of an upstream cistron allows the ribosomes access to a second downstream cistron and results in the production of a fusion protein in addition to the protein which is encoded exclusively by the first cistron. By comparison with corona- and retrovirus RNAs, several plant virus RNAs, including barley yellow dwarf virus (BYDV) genomic R N A (Miller et al, 1988) and red clover necrotic mosaic virus (RCNMV) RNA-1 (Xiong & Lommel, 1989) have also been suggested to utilize translational frameshifting to express a portion of their genome. Internal initiation Direct translation of downstream cistrons by the binding of ribosomes to internal A U G codons has been speculated to occur in plant viruses (Dougherty & Hiebert, 1985) but has never been Chapter 1. Introduction 6 definitively demonstrated. Conclusive evidence for internal initiation has been reported only for poliovirus R N A in which ribosomes have been shown to bind to an internal sequence within the 5' noncoding region of the viral genome (Pelletier & Sonenberg, 1988). Most plant R N A viruses utilize two or more of these translation strategies (excluding internal initiation) to express their genomes. 1.2 Cucumber necrosis virus 1.2.1 General characteristics Cucumber necrosis virus is an isometric plant virus with a 30 nm particle consisting of 180 copies of a single capsid protein of M r ca. 41,000 (Tremaine, 1972). The C N V genome is monopartite and consists of single stranded, positive sense R N A of ca. 4.7 kilobases (kb) (Tremaine, 1972; Rochon & Tremaine, 1989). Systemic infection by C N V is limited to cucumber in nature (McKeen, 1959) and natural spread of the virus is facilitated by zoospores of the fungus, Olpidium radicale (Dias, 1970). The relationship between C N V and other soil-borne isometric plant viruses has been demonstrated through comparison of the double-stranded R N A (dsRNA) intermediates generated upon infection and by nucleic acid hybridization analyses. Results of these studies reveal that C N V is a member of the tombusvirus group (Rochon & Tremaine, 1988). 1.2.2 Genome organization The complete nucleotide sequence of the C N V genome has been determined and from this the genome organization deduced (Rochon & Tremaine, 1989). This represented the first complete Chapter 1. Introduction 7 nucleotide sequence of a tombusvirus. The proposed organization is in agreement with that recently suggested for cymbidium ringspot virus (CyRSV), a tombusvirus (Russo et al, 1988; Grieco et al., 1989), and also tomato bushy stunt virus - cherry (TBSV-cherry), a close relative of the type member of the tombusvirus group (Morris & Carrington, 1988; Hillman et al., 1989). The genome of C N V contains five long open reading frames (ORFs) with the capacity to encode proteins of 33,92,41,21 and 20 kilodaltons (kDa). The 92 kDa protein, i f produced, would arise from readthrough of the 33 kDa amber termination codon. The 92 kDa protein is implicated as the viral replicase on the basis of amino acid sequence similarity with the putative replicases of other R N A viruses. The 41 kDa ORF, located immediately downstream of the 92 kDa ORF, is suggested to encode the viral coat protein; its amino acid sequence closely resembles that of other tombusviruses (Riviere & Rochon, 1989). An unusual feature of C N V , and of other members of the tombusvirus group, is the internal, rather than 3' terminal, location of the coat protein coding region (Hillman et al., 1989). The 3' terminus of the C N V genome, instead of containing the coat protein cistron, contains an ORF for a 20 kDa protein which is nested within an ORF for a 21 kDa protein. Neither the function of these two proteins, nor if they are both expressed is known. 1.2.3 Translation strategy In analogy to other tombusviruses such as TBSV (Henriquez et al., 1978; Henriques & Morris, 1979; Morris, 1983; Hayes etal, 1984; Hayes etal, 1988) and CyRSV (Galletelli & Hull, 1985; Burgyan et al., 1986) the translation strategy of C N V might involve the formation of subgenomic R N A species which are 3* co-terminal with genomic RNA. Chapter 1. Introduction 1.3 Thesis objectives 8 This thesis work was undertaken to provide information concerning the proteins produced by C N V in vivo through analyses of the viral proteins synthesized in vitro. Specifically, the thesis objectives are: 1. To determine the size and number of C N V in vitro translation products. 2. To determine the genomic locations of C N V in vitro translation products. 3. To determine the translation strategy by which C N V proteins are produced. Chapter 2 Materials and Methods 2.1 Virus propagation and isolation 2.1.1 Virus propagation C N V was originally obtained from C. D. McKeen at the Agriculture Canada Research Station in Harrow, Ontario and provided for use in the present study by J. H . Tremaine at the Agriculture Canada Vancouver Reasearch Station. For the following work, C N V was propagated in Nicotiana clevelandii which is a systemic host for the virus (Dias & McKeen, 1972). The initial inoculum was previously frozen CNV-infected N. clevelandii leaf tissue which was ground in autoclaved 10 m M sodium phosphate buffer (pH 7.2) using a sterile mortar and pestle. The slurry was rub-inoculated with autoclaved sponge pads onto the leaves of ca. two week old N. clevelandii plants which had been dusted with fine carborundum (Alundum; Norton) to enhance virus infection. Necrotic lesions were observed on the leaves ca. 4 days following inoculation. Leaves exhibiting symptoms were harvested ca. 2 weeks post-inoculation, ground in buffer as described above, and used as fresh inoculum for additional N. clevelandii plants. CNV-infected N. clevelandii leaves exhibiting necrotic lesions, but which were not yet totally necrotic, were again harvested ca. 2 weeks post-inoculation and used for virus purification (see below). 9 Chapter 2. Materials and Methods 10 2.1.2 Virus purification C N V was purified from CNV-infected N. clevelandii leaf tissue by a modification of the pH 5.0 method of Tremaine et al. (1983). This method involves clarification of plant tissue homogenate at pH 5.0 (at which most plant proteins precipitate but the virus remains in solution), polyethylene glycol (PEG) precipitation and differential centrifugation. To purify virus from 200 g of tissue, 300 ml (ie. 1.5 volumes) of 0.1 M sodium acetate (NaOAc) buffer, pH 5.0, containing 5 m M |3-mercaptoethanol was added to the tissue and these were homogenized in a Waring blender. The homogenate was filtered through cheesecloth and the filtrate centrifuged at 10,000 rpm for 15 min at 4 °C in a Sorvall GSA rotor. The pellet was discarded and the supernatant was stirred with 8% PEG for 2 hr at 4 °C. The mixture was then centrifuged as above and the supernatant discarded. The pellet was allowed to resuspend overnight at 4 °C in one-tenth volume 0.1 M NaOAc buffer, pH 5.0. The sample was diluted to a final 25 ml volume with 0.1 M NaOAc buffer, pH 5.0, divided between two Beckman Quick-Seal tubes (16 x 76 mm) and centrifuged at 40,000 rpm in a 70 T i rotor for 2 hr at 4 °C. The supernatant was discarded and the pellets saved at 4 °C for either antisera production or for R N A extraction (see below). Virus purification for antisera production If the virus was to be subsequently used for antisera production, the pellet was resuspended in 3 ml phosphate buffered saline (PBS; 0.15 M NaCl, 10 m M NaH2P04), pH 7.2, and further purified by isopycnic centrifugation using cesium chloride (CsCl) gradients. To the virus suspension was added 11.35 g CsCl (0.45 g CsCl/ml gradient) and the final volume brought to 25 ml with 10 m M PBS, pH 7.2. The CsCl was completely dissolved and the solution was divided between two Beckman Quick-Seal tubes (16 x 76 mm) and centrifuged at 50,000 rpm for 16 hr at Chapter 2. Materials and Methods 11 15 °C in a 70 T i rotor. The virus band, which appeared near the center of each gradient as opalescent material, was withdrawn using a needle and syringe and dialyzed against several changes of PBS over a 4 to 6 hr period and then overnight at 4 °C. The virus preparation was observed under the electron microscope using uranyl acetate as a negative stain and tested for virus purity by Ouchterlony double diffusion (Ouchterlony, 1964) against both tomato bushy stunt virus (TBSV) antisera and C N V antisera provided by J.H. Tremaine. The virus concentration was determined spectrophotometrically using the absorbance at 260 nm determined for T B S V (A26O of a 1 mg/ml solution of TBSV is 4.5; Martelli et al, 1971). The yield of C N V from 200 g of tissue was estimated to be 130 mg. The samples were stored at 4 °C until further use. Virus purification for RNA extraction If the virus was to be subsequently used for R N A extraction, CsCl purification was not used. The virus pellet was resuspended overnight in 3 ml 0.01 M NaOAc buffer, pH 5.0, containing 0.1 M sodium chloride (NaCl) at 4 °C. The sample was then removed of insoluble material by centrifugation in a microfuge at maximum speed for 5 min at 4 °C. The pellet was discarded and the concentration of virus in the supernatant was determined spectrophotometrically as described above. Virus samples were stored at 4 °C until used for R N A extraction. 2.2 Antisera production and antibody purification 2.2.1 Production of polyclonal antisera Antisera to intact virus particles was prepared using 1 ml of purified virus (1 mg/ml) emulsified in 1 ml Freund's incomplete adjuvant (Gibco). A rabbit was immunized by intramuscular injection at Chapter 2. Materials and Methods 12 weekly intervals for 5 weeks and test bleeds were taken after the third week. The rabbit was given a sixth injection 10 days after the fifth one and was sacrificed 1 week later. The blood was allowed to clot for 2 hr at 4 °C after which the serum was decanted and then centrifuged at 4500 rpm in a Sorvall SS34 rotor for 10 min at 4 °C. The final titre of the antisera produced was determined by Ouchterlony double diffusion (Ouchterlony, 1964) and was found to be 1:1024. Aliquots of antisera were stored either with or without the addition of 1% sodium azide at 4 °C or -20 °C or in a 1:1 dilution of glycerol at -20 °C. Prior to immunization, 25 ml pre-immune serum was obtained from the rabbit and stored without preservative at -20 °C or in a 1:1 dilution of glycerol at -20 °C. 2.2.2 Purification of immunoglobulin (IgG) Immunoglobulin (IgG) was purified from C N V antisera by affinity chromatography using Protein A-Sepharose CL-4B (Pharmacia). Protein A-Sepharose CL-4B consists of protein A, which binds specifically to IgG-type antibodies, covalently coupled to a cross-linked matrix, Sepharose CL-4B. For a 2 ml column, ca. 0.5 g freeze-dried Protein A-Sepharose CL-4B powder was swelled and washed in three changes of 0.1 M PBS, pH 7.3 according to manufacturer's instructions. C N V antisera (3 ml) was passed over the column. The column was then washed with ca. 10 volumes of 0.1 M PBS, pH 7.3 to remove unbound material. The IgG was eluted with 8 ml 1.0 M acetic acid and the solution subsequently neutralized with 1.0 M Tris-HCl pH 8.8. To the resulting solution was added 1 volume of 100% ( w/y) ammonium sulfate and the IgG was allowed to precipitate at 4 °C overnight and then pelleted by centrifugation at 10,000 rpm in a Sorvall SS34 rotor for 20 min at 4 °C. The pellet was resuspended in 3 ml PBS buffer and 200 | i l aliquots were stored at -20 °C. Chapter 2. Materials and Methods 13 Determination of IgG protein concentration The concentration of purified IgG was determined using the Coomassie Blue Binding technique of Sedmak & Grossberg (1977) and Spector (1978) as described by Scopes (1982). This technique involves the use of a reagent containing Coomassie blue dye which, when bound to protein, develops a blue color which can be measured spectrophotometrically. The reagent used consisted of 100 mg Coomassie Brilliant Blue G-250 (BioRad) dissolved in 50 ml 95% ethanol and then mixed with 100 ml 85% phosphoric acid. The mixture was diluted to 1 litre with deionized H2O and filtered through Whatman No. 1 filter paper to remove undissolved dye. A 1.5 ml sample containing up to 50 jig IgG protein was added to 1.5 ml of the reagent and mixed well. After 2 min the absorbance at 595 nm was read on a Spectronic 20 (Bausch and Lomb) spectrophotometer. A standard curve was constructed with bovine serum albumin (BSA) to which the samples of unknown concentration were compared. 2.3 Virion RNA extraction 2.3.1 RNA extraction Virion R N A was isolated from purified virus by swelling the virus particles using ethylenediaminetetracetic acid (EDTA) followed by extraction with phenol/chloroform in the presence of sodium dodecyl sulfate (SDS). Purified virus (400 | i l ; 20 mg/ml) was adjusted to 10 m M E D T A and incubated on ice for 5 min. The E D T A chelates calcium ions which are required for protein subunit-subunit interaction in TBSV (Harrison, 1983) and therefore results in swollen (destabilized) virus particles. Virion R N A was extracted from the destabilized virus particles by phenol/chloroform/SDS extraction as follows. To the suspension of swollen virus particles was Chapter 2. Materials and Methods 14 added 200 ^1 redistilled phenol, 200 | i l chloroform/octanol (24:1), 100 0.5 M Tris-HCl pH 8.9, and 25 | i l 20% SDS. The mixture was vortexed for ca. 1 min and then the aqueous and organic phases were separated by centrifugation for 2 min in a microfuge (14,000 x g) at 4 °C. The aqueous layer was removed and set aside on ice while the organic phase was reextracted with 200 | i l of autoclaved cold deionized H2O. The resulting aqueous layer was pooled with that from the previous extraction and to the combined aqueous phases was added 300 (il phenol and 300 | i l chloroform/octanol. The mixture was vortexed and centrifuged as above and the aqueous phase collected. The aqueous phase was further extracted with 600 | i l chloroform/octanol. To the final aqueous phase was added 0.1 volume of 2 M NaOAc, pH 5.8, and 2.5 volumes of absolute ethanol. The solution was mixed well and placed at -70 °C for 30 min or in liquid nitrogen for 5 min to precipitate the RNA. The samples were then centrifuged in a microfuge for 15 min at 4 °C and the supernatant was discarded. The R N A pellet was washed 2 times with 1 ml cold 70% ethanol, dried and resuspended in autoclaved deionized H2O. The R N A quality was determined by electrophoresis through denaturing agarose gels and was quantified with a spectrophotometer (a 1 mg/ml solution of R N A has an A26O of 25). RNA samples were stored at -70 °C. 2.3.2 Denaturing agarose gel electrophoresis of RNA Agarose gel electrophoresis under denaturing conditions using methylmercuric hydroxide (MeHgOH) as the denaturing agent was first described by Bailey & Davidson (1976). Since MeHgOH is toxic and volatile, all manipulations were carried out using gloves and in a chemical fume hood. A l l materials coming in contact with MeMgOH were rinsed or soaked in a 10 m M solution of pVmercaptoethanol to allow for safe disposal. One percent agarose gels were prepared by melting 0.2 g agarose in 18 ml deionized H2O and cooling to ca. 65 °C before the addition of 2 ml 10X B E buffer (10X B E buffer is 400 m M Boric acid/10 m M E D T A brought to pH 7.38 with Chapter 2. Materials and Methods 15 6N NaOH) and 100 | i l 1.0 M MeHgOH (Alfa). The mixture was poured into a BioRad minigel tray and allowed to solidify at room temperature. RNA samples containing between 10 and 100 ng of R N A per band were brought to a volume of 8.5 ul with autoclaved deionized H2O. The RNA was denatured with 4.0 p.1 sample mix (50 | i l autoclaved deionized H2O, 50 | i l 10X B E buffer, 50 ul glycerol, 2.5 | i l 1.0 M MeHgOH and 7.5 | i l saturated bromophenol blue) and allowed to stand at room temperature for 10 min prior to loading onto the gel. Electrophoresis was carried out at 10 volts/cm for 45 to 60 min in I X B E running buffer using a BioRad minigel apparatus. After electrophoresis, the gel was stained in 100 ml of deionized H2O containing 0.5 (ig/ml ethidium bromide (EtBr) and 10 m M (3-mercaptoethanol for 10 min at room temperature. The gel was destained in deionized H2O for 10 min at room temperature and then photographed under ultraviolet light using a Polaroid MP-3 Land camera and Kodak Royal Pan film. 2.4 Analysis of in vitro translation products 2.4.1 In vitro translation In vitro translation in the presense of [35s]-methionine (New England Nuclear; specific activity ca. 1100 Ci/mmole) was carried out using both rabbit reticulocyte lysate cell-free translation systems (Bethesda Research Laboratories and Promega) and wheat germ extract translation systems (Promega and NEN) according to manufacturer's instructions. Translation conditions in wheat germ extracts were optimized with respect to R N A concentration, incubation time, and potassium and magnesium ion concentration (see section 3.2). Translation reactions were incubated at 22 to 25 °C for 2 hr unless otherwise indicated. For translation in the N E N wheat germ system, one-half (2.5 ul) of the recommended (5 u,l) quantity of [35s]-methionine was used. Where Chapter 2. Materials and Methods 16 discussed, wheat germ extract or rabbit reticulocyte lysate translation reactions were supplemented with 800 (ig/ml wheat germ tRNA or calf liver tRNA, respectively. 2.4.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Translation products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) through 15% separating gels (0.75 mm) using the discontinuous Laemmli buffer system (Laemmli, 1970) as described by BioRad. Following electrophoresis, gels were gently agitated in 3 changes of fixative (30% methanol/10% acetic acid) for at least 30 min each and fluorographed with Entensify (NEN) following manufacturer's instructions. Gels were dried for 1.5 hr on Whatman 3 M M chromatography paper using a BioRad drier and then exposed to X-ray film at -70 °C. 2.4.3 Protein molecular weight estimates The sizes of C N V in vitro translation products were determined by comparison with [ ^ C ] -methylated proteins (Amersham) and the published sizes of brome mosaic virus (BMV) translation products (Ahlquist et al., 1981). The estimates were made from proteins resolved on 15% SDS-polyacrylamide gels. [1 4C]-methylated proteins of ca. 14,300, 30,000, 46,000 and 69,000 M r and B M V proteins of 20,400 and 32,500 M r were used in the estimates. A graph of the log molecular weight versus electrophoretic mobility was constructed using Cricket Graph, version 1.2 (Cricket software). Regression analysis on the resulting plot, also using Cricket Graph, enabled the sizes of unknown proteins to be estimated. Chapter 2. Materials and Methods 17 2.4.4 Immunoprecipitation Immunoprecipitation of the C N V coat protein was carried out according to a modification of the procedure of Olliver & Boyd (1984). The use of protein A-bearing staphylococci for immunoprecipitation of antigens from cells was first described by Kessler (1981). The following procedure utilized Sepharose-bound protein A attached to C N V antibody to test for the presence of coat protein antigen in in vitro translation reactions. A 10% ( w / v ) solution of Protein A-Sepharose CL-4B (Pharmacia) was swollen, washed three times in NET buffer (150 m M NaCl, 5 m M E D T A , 50 m M Tris-HCl pH 7.4) containing 0.05% Nonidet P40 (NP40, BDH) and resuspended in NET buffer. This Protein A-Sepharose suspension (50 u,l) was pre-incubated with 1.5 | i l normal (pre-immune) serum for 1.5 hr at room temperature, washed three times and brought to a 50 | i l volume with NET buffer containing NP40. This suspension was used to preclear wheat germ extract translation reactions previously programmed with C N V virion RNA. Twenty-five | i l of the suspension was added to 25 | i l translation reaction, incubated for 30 min and centrifuged in an eppendorf microfuge at maximum speed for 2 min. The supernatant was transferred to another tube and the pellet was discarded. The supernatant was again precleared as described above and the pellet discarded. The translation reaction supernatant was then incubated with 300 | i l IgG (1.5 mg/ml) which had previously been bound to Protein A-Sepharose CL-4B (Pharmacia) in the manner described above. The mixture was incubated overnight at 4 °C with slow shaking, centrifuged in an eppendorf microfuge for 2 min and the pellet washed with four changes of NET/NP40. The pellet was finally resuspended in 25 (0.1 of a mixture containing 12.5 | i l NET/NP40 and 12.5 [il 2X Laemmli sample buffer (see section 2.4.2) and the solution heated to 90 °C for 2 min. The immunoprecipitated products were analyzed by SDS-PAGE and fluorography as described in section 2.4.2. Chapter 2. Materials and Methods 18 2.5 CNV synthetic transcripts 2.5.1 CNV clones Recombinant phagemids consisting of cDNA corresponding to C N V R N A inserted into either of the vectors, Bluescript or Bluescribe (Stratagene), were provided by D . M . Rochon. The C N V c D N A clones were used either in the form supplied or used to construct new subclones (see below). Descriptions of C N V cDNA clones and subclones are contained in Table I. 2.5.2 Construction of subclones Additional subclones were constructed from fragments generated by restriction enzyme digestion of phagemid pCNV220A (see Table I) which contains an insert corresponding to almost the entire C N V genome. Restriction enzyme digested D N A was electrophoresed through 1% agarose gels, stained with EtBr and visualized briefly under U V light (302 nm). The bands corresponding to the desired restriction fragments were excised from the gel using a razor blade and the D N A recovered from the gel pieces using GeneClean (BioCan) according to manufacturer's instructions. Purified restriction fragments were either blunt-end or sticky-end ligated into suitably digested, calf-intestinal phosphatase (CIP) treated Bluescript phagemid vector (Stratagene) using a method similar to that outlined by Maniatis et al., (1982). Ligation reactions were carried out in a 10 jxl volume containing 50 m M Tris-HCl pH 7.5, 10 mM MgCl2, 20 m M DTT, 1 m M ATP, 50 ng/ml BSA, 100 ng digested, CIP-treated vector D N A , a 2-3 molar excess of digested insert D N A and 2 units T4 D N A ligase (BRL). Ligation mixtures were incubated for 4 to 6 hr at room temperature and then used to transform competent Escherichia coli DH5cc cells (BRL) (Maniatis et al., 1982; Morrison, 1979). Transformation mixtures were plated onto Luria-Bertani (LB) media containing Table I. DESCRIPTION OF CNV cDNA CLONES, SUBCLONES AND SYNTHETIC TRANSCRIPTS USED IN IN VITRO TRANSLATION STUDIES c D N A Clone Phagemid Site in Vector Genomic Enzyme used Polymerase0 Transcriptd Vector Coordinates3 to Linearizeb pCNV220A e Bluescribe PstI 18-4701 SphI T7 18/4701 (+) Bluescribe PstI 18 - 4701 BamHI T3 18/470K-) Bluescribe PstI 18 - 4701 EcoRV T7 18/1089(+) 3'Exo903T3e Bluescript EcoRl/Pstl 18-910 BamHI T3 18/910C-) 5'Exo451T7e Bluescript EcoRl/PstI 260 - 2156 EcoRI T7 260/2156(+) pSCHincl.55 f Bluescript Smal 2566-4116 Apal T7 2566/4116(+) Bluescript Smal 2566-4116 BamHI T3 2566/4116(-) pSCHind0.67 f Bluescript Smal/Hindin 2566 - 3234 BamHI T3 2566/3234(-) 50Hpal0103« Bluescribe AccI 3634 - 4639 HinoTII T7 3634/4639(+) Bluescribe AccI 3634 - 4639 BamHI T3 3634/4639(-) P K 2 e Bluescribe EcoRI/Smal 1 - 4701 Smal T7 1/4701 (+) a indicates the C N V genomic coordinates to which the cDNA insert corresponds b indicates the enzyme used to linearize template D N A prior to transcription c indicates the phage D N A dependent RNA polymerase used to produce transcripts from linearized template D N A d (+) indicates transcript is of the same polarity as C N V genomic RNA; (-) indicates transcript is of the opposite polarity note: some of the resulting transcripts have short stretches of vector-derived nucleotides at their 5' and 3' termini e indicates cDNA clones and subclones provided by D. M . Rochon f indicates cDNA subclones constructed from pCNV220A for the present study Chapter 2. Materials and Methods 20 ampicillin and the chromogenic substrate Bluogal (BRL) and incubated overnight at 37 °C. Single colonies were selected, inoculated into 3 ml of liquid L B media containing 50 |ig/ml ampicillin, and incubated overnight at 37 °C with vigorous shaking. The cultures were harvested and used to isolate phagemid D N A by the alkaline lysis procedure outlined by Maniatis et al. (1982). Purified phagemid D N A was digested with suitable restriction enzymes to determine the presence and orientation of the desired insert. Large scale preparations of phagemid D N A were obtained using a modified alkaline lysis procedure described by Maniatis et. al. (1982) except that ribonuclease treatment was omitted. Plasmid preparations were purified from contaminating R N A by 2 M lithium chloride precipitation (Siegel et al., 1976) followed by cesium chloride gradient centrifugation (see below) or using Bio-Gel A-150m agarose beads (BioRad) according to manufacturer's instructions. Plasmid D N A was quantified spectrophotometrically (a 1 mg/ml solution of D N A has an A260 of 20) and stored in I X TE buffer ( IX TE buffer is 10 m M Tris-HC1 pH 7.5, and 1 m M EDTA) at -20 °C. 2.5.3 Isolation of supercoiled plasmid DNA Supercoiled plasmid D N A free of contaminating R N A was obtained using CsCl/EtBr gradients originally described by (Holmes & Quigley, 1981) and modified as suggested by (Summers & Smith, 1987). D N A pellets obtained from large-scale plasmid D N A preparations were resuspended in 3.3 ml I X TE buffer. To the D N A was added 3.366 g CsCl (ie. 1.02 g CsCl/ml D N A solution) and 1.65 mg EtBr (ie. 0.5 mg EtBr/ml D N A solution). The mixture was transferred to a Beckman Quick-Seal ultracentrifuge tube (16 x 76 mm), overlayed with mineral oil and centrifuged for ca. 16 hr in a 70Ti fixed angle rotor. The D N A was visualized by fluorescence under ultraviolet illumination (302 nm) and collected through a syringe attached to a large gauge needle inserted into the tube just below the band. The volume of the D N A solution obtained from Chapter 2. Materials and Methods 21 the gradient (ca. 1 ml) was brought to 6 ml with autoclaved deionized H2O. The EtBr was removed from D N A using isoamyl alcohol or n-butanol extraction until the aqueous phase appeared colorless. The volume of the aqueous solution was brought to 5 ml with autoclaved deionized H2O afterwhich 2 volumes of absolute ethanol was added and the solution mixed thoroughly. The D N A was allowed to precipitate overnight at -20 °C or for 10 min at -70 °C and then pelleted at 2500 x g for 20 min at 4 °C. The pellets were washed with 70% ethanol, resuspended in I X TE and stored at -20 °C. 2.5.4 In vitro transcription Synthetic transcripts corresponding to different regions of the C N V genome and of either positive (+) or negative polarity (-) were produced in vitro using the T7 or T3 promoter and Bluescript or Bluescribe plasmids (Stratagene) containing various C N V cDNA inserts. The plasmid templates used for run-off transcription were linearized by selected restriction enzymes that either cleaved within the insert downstream of the region to be transcribed or downstream of the insert in the multiple cloning site of the vector. In vitro transcription reactions were carried out in a 50 (il volume containing 40 m M Tris-HCl pH 7.8, 10 m M NaCl, 6 m M MgCl2, 2 m M spermidine, 0.5 m M each of ATP, CTP, GTP and UTP, 10 m M DTT, 10 units RNAasin (Promega), 10 to 50 units T7 or T3 polymerase (Stratagene or BRL) , and 2.0 fig linearized D N A . The reaction mixtures were incubated at 37 °C for 45 min after which 0.2 units RNAase-free DNAase I (Stratagene) and an additional 10 units of RNAasin were added. The reactions were then incubated at 37 °C for a further 15 min. An aliquot of each reaction was analyzed by electrophoresis through a non-denaturing 1% agarose gel and the remainder extracted with phenol/chloroform and then ethanol precipitated. The amount of R N A transcript produced was determined either spectrophotometrically or by comparison with C N V virion RNA after agarose gel electrophoresis Chapter 2. Materials and Methods 22 and EtBr staining. The latter method for determining transcript concentration was preferred since free ribonucleotides which sometimes co-precipitated with the transcript overestimated concentrations based only on spectrophotometric readings. 2.5.5 Hybrid-arrested translation The hybrid-arrested translation procedure used was similar to that described by Paterson et al. (1977) except that negative sense RNA rather than complementary D N A was used in hybrid arrest assays. Negative sense R N A was used instead of cDNA because synthetic negative sense transcripts can be prepared easily in large quantity and because R N A / R N A hybrids are more stable than c D N A / R N A hybrids. R N A / R N A hybrids were formed using a three fold molar excess of synthetic negative sense R N A over C N V virion RNA. The hybridization was carried out in a 50 \i\ volume containing from 1 jig to 16 |0.g RNA, 80% deionized formamide (Ultrapure, BRL), 10 m M Tris-HCl pH 7.5, and 400 m M NaCl and incubated for 2 hr at 48 °C. After incubation, the mixture was diluted four fold with autoclaved deionized H2O and the nucleic acids were precipitated with 0.1 volume 2 M NaOAc and 2.5 volumes absolute ethanol for 1 hr at -70 °C. The precipitate was pelleted in a microfuge at maximum speed for 15 min, washed twice with 1 ml 70% ethanol and dried in a SpeedVac concentrator (Savant). The pellet was then resuspended in a small volume of autoclaved deionized H2O. A small aliquot of the suspension was analyzed by nondenaturing gel electrophoresis prior to its use for in vitro translation studies described in section 2.4. Chapter 2. Materials and Methods 23 2.6 Analysis of CNV subgenomic RNAs 2.6.1 Log-linear sucrose density gradients Sucrose gradients were prepared by a modification of the procedure of Brakke & Van Pelt (1970). Sucrose solutions of 325, 270, 210, 160, and 100 mg sucrose/ml autoclaved TES buffer (10 m M Tris-HCl pH 7.6, 0.1 m M E D T A and 0.1% SDS; note the SDS is added after autoclaving) were placed in a 60 °C water bath for 30 min. The solutions were cooled to 4 °C and aliquots were then layered in a Beckman SW 27 polyallomer centrifuge tube in the following order from the bottom of the gradient to the top: 5.5 ml 325 mg/ml solution, 11.8 ml 270 mg/ml solution, 7.8 ml 210 mg/ml solution, 5.0 ml 160 mg/ml solution, 4.5 ml 100 mg/ml solution, and 2.2 ml TES buffer. The gradient was allowed to diffuse for 16 hr at 4 °C. C N V R N A (400 |ig) was layered on the gradient after having removed an equivalent volume from the top of the gradient. The gradient was centrifuged in an SW 27 rotor at 24,000 rpm for 26 hr at 6 °C. The gradient was taken down by a gradient collector (Buchler Auto Densi-Flow UC) and fractions of ca. 500 | i l were collected with a Gilson microfractionator. R N A was precipitated from the sucrose fractions by the addition of 0.1 volumes of 2 M NaOAc pH 5.8 and 2.5 volumes of absolute ethanol at -20 °C overnight. The precipitates were then pelleted in a microfuge for 15 min at 4 °C, washed with 1 ml 70% ethanol and the pellets resuspended in 15 ml autoclaved deionized H2O. 2.6.2 Alkaline northern blot The procedure described below is a modification of the alkaline transfer procedure described by Vrati et al. (1987). Agarose gels containing MeHgOH were electrophoresed as described in section 2.3.2, placed in 10 m M P-mercaptoethanol for 15 min to remove mercury and then rinsed Chapter 2. Materials and Methods 24 in deionized H2O. A transfer apparatus essentially as described by Southern (1975) was set up which consisted of an inverted BioRad gel casting tray upon which two sheets of Whatman 3 M M paper were placed. Whatman 3 M M paper was cut to the same width as the tray but exceeded the length of the tray on either side so that it could act as a wick when the tray was placed in transfer solution. Whatman filter paper was saturated with 10 m M NaOH and any bubbles were removed by rolling a pasteur pipet over the saturated paper. The gel was placed on the Whatman paper and any bubbles between the gel and the paper were also removed. Zeta-Probe (BioRad) nylon membrane which had been presoaked in deionized H2O was then applied to the gel and bubbles were removed in the same manner. Three pieces of Whatmann 3 M M paper cut to approximately the same size as the gel were placed on top of the ZetaProbe membrane and then covered with an 8 to 10 cm stack of paper towels cut to approximately the same dimensions. The dish was filled with 10 m M NaOH until the solution covered the ends of the wicks but did not reach the gel tray. The transfer was allowed to continue for 3 to 16 hr at room temperature during which time the stack of NaOH solution-soaked paper towels was replaced as necessary. After transfer, the membrane was removed from the gel, rinsed briefly (less than 5 min) in 2X SSC, 0.1% SDS and blotted lightly with tissue. (2X SSC is 0.3 M NaCl, 0.03 M trisodium citrate) The membrane was either placed in prehybridization buffer immediately or stored dry between two pieces of filter paper in a plastic bag at -20 °C. Membranes were prehybridized in ca. 20 ml of hybridization buffer [50% deionized formamide, 50 m M Tris-HCl pH 7.5, 1 M NaCl, 0.2% BSA, 0.2% PVP, 0.2% Ficoll (Sigma; M.W.= 400,000), 0.1% sodium pyrophosphate, 200 to 500 |ig/ml sheared denatured salmon sperm D N A and 10% sodium dextran sulfate] for 30 min to 2 hr at 42 °C. [ 3 2 P]-labelled denatured nick translated probe (1 to 3 ng probe/ml hybridization solution; see below) was then added to the hybridization buffer and membranes were incubated for 18 to 22 hr at 42 °C. Following incubation, membranes were rinsed in 2X SSC and then washed at room temperature for 15 min in 2X SSC, 0.1% SDS and then at 68 °C for 15 min in 0.2X SSC, 0.1% SDS. The Chapter 2. Materials and Methods 25 membranes were then rinsed in 0.2X SSC, blotted briefly, wrapped in plastic wrap and autoradiographed with the aid of two Lightning Plus intensifying screens (Dupont). 2.6.3 Nick translated probes Nick translated probes were prepared according to a modification of the procedure of Rigby et al. (1977). A 50 ^il reaction contained 50 m M Tris-HCl pH 7.5, 10 m M MgCl2,1 m M DTT, 50 Hg/ml nuclease free B S A (BRL), 0.6 m M each of dCTP, dGTP, and dTTP, 30 to 50 jxCi a [ 3 2 P ] -dATP (ICN, ca. 3000 Ci/mmole), 250 pg DNase I (Sigma), and 5 to 15 units of E. coli D N A Polymerase I (Pharmacia). The reaction was incubated at room temperature for 20 to 30 min and then was stopped by the addition of 1 (il 20% SDS. To remove unincorporated nucleotides, the probe was passed over a miniature Sephadex G-50 (Sigma) column (Maniatis et al., 1982). The column eluant containing the probe was collected and D N A denatured by the addition of an equal volume of a 0.2 M NaOH followed by boiling for 5 min. The probe was quick cooled on ice and added directly to the bag containing the hybridization solution and the prehybridized blot. 2.6.4 RNA size estimates The sizes of R N A species were determined by comparison with R N A markers constructed from cDNA clones by restriction enzyme digestion and in vitro transcription and also by comparison with the known size of C N V genomic R N A (Rochon & Tremaine, 1989). R N A markers of 4.7, 2.5, 1.9, 0.9 and 0.7 kb were electrophoresed through a nondenaturing agarose gel which was then stained with EtBr. Cricket graph version 1.2 (Cricket software) was used as described in section 2.4.3 for protein size estimations to determine the sizes of unknown RNAs. Chapter 3 Results 3.1 Characteristics of CNV in vitro translation products The nucleotide sequence of C N V predicts the synthesis of five large proteins with sizes of ca. 33, 92,41, 21 and 20 kilodaltons (kDa) (Rochon & Tremaine, 1989). As part of an overall laboratory aim to determine which, if any, of the proteins are produced by C N V in vivo, the in vitro translation products directed by C N V virion RNA were analyzed. 3.1.1 Comparison of CNV translation products in rabbit reticulocyte lysates and wheat germ extracts Since both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems are commercially available and routinely used for analyzing in vitro translation products of viral RNAs, the translation products directed by C N V virion RNA in the two systems were compared. C N V virion R N A was translated in either rabbit reticulocyte lysates (Promega) or wheat germ extracts (Promega) in the presence of [35s]-methionine and the protein products were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent fluorography (Fig. 3.1). In rabbit reticulocyte lysates, one major protein was produced (lane 3). In wheat germ extracts, four major proteins were produced (lane 4), one of which comigrated with the single protein product directed by C N V R N A in rabbit reticulocyte lysates. In rabbit reticulocyte lysates, a prominent endogenous protein was detected (lane 2) whereas in wheat germ extracts endogenous proteins were detected but only at a low level (lane 5). The difference in the number of translation products produced in the two systems may reflect a differential selection of A U G initiation codons 26 Chapter 3. Results 27 1 2 3 4 5 6 Fig. 3.1 Comparison of C N V RNA in vitro translation products in rabbit reticulocyte lysates (Promega), lanes 1-3, and wheat germ extracts (Promega), lanes 4 - 6. Products were analyzed by SDS-P A G E (12% gel) and autoradiography. Lane 1, B M V R N A (20 ug/ml); lane 2, no added RNA; lane 3, C N V R N A (160 ug/ml); lane 4, C N V R N A (160 ug/ml); lane 5, no added RNA; lane 6, B M V RNA (20 ug/ml). Chapter 3. Results 28 by the ribosomes of plants and animals (Kozak, 1986b; Lutke et al., 1987) (see section 4.1). The wheat germ extract system was chosen as the system for most of the remaining studies since it seemed reasonable that a plant derived cell-free translation system would more accurately predict C N V in vivo translation products than would an animal derived cell-free translation system. There are also a number of advantages in using the wheat germ extract system over the rabbit reticulocyte lysate system. Wheat germ extracts contain very low levels of endogenous mRNA and therefore, unlike rabbit reticulocyte lysates, do not require nuclease treatment to digest endogenous mRNA (NEN). Wheat germ extracts also contain lower endogenous amino acid levels which might allow for more efficient incorporation of labelled amino acids into translation products than would be possible in the reticulocyte system (NEN). In addition, it has been suggested that the fidelity of translation in wheat germ extracts is greater than in rabbit reticulocyte lysates (Kozak & Shatkin, 1977; Kozak, 1986c) 3.1.2 Size estimation of C N V in vitro translation products [l^C]-labelled protein molecular weight markers (Amersham) and brome mosaic virus (BMV) in vitro translation products (Ahlquist et al., 1981) were used to estimate the sizes of proteins synthesized by C N V virion R N A in wheat germ extracts (Fig. 3.2). To aid in these estimates, regression analyses were performed (plots not shown). It was determined from these analyses that in both rabbit reticulocyte lysates and wheat germ extracts, C N V R N A directed the synthesis of a ca. 34,600 M r protein while, in wheat germ extracts, three additional proteins of ca. 41,600, 24,000 and 20,000 M r were produced. (CNV R N A also directed the synthesis of a few very low molecular weight polypeptides which were not further considered in the present study.) The number and sizes of the proteins produced in wheat germ extracts are similar to those predicted from nucleotide sequence data (Rochon & Tremaine, 1989) with the exception that a 92,000 M r in Chapter 3. Results 29 Fig. 3.2 Sizes of C N V in vitro translation products. Wheat germ extracts (NEN) were programmed with 20 ug/ml B M V R N A (lane 1); no added R N A (lane 2); 160 ug/ml C N V R N A (lane 3). Lane 4 contains [ 1 4 C]-methylated protein markers (Amersham) with M r values (x 103) indicated on the gel. The [14C]-methylated markers between 14.3 x 10 3 and 69.5 x 10 3 M r together with the 20.4 x 10 3 and 32.5 x 10 3 M r B M V proteins were used to estimate the sizes of the C N V in vitro translation products (shown on the gel in M r x 10 3). Regression analyses were performed using Cricket Graph Version 1.2 (Cricket software) (see section 2.4.3 for details). SDS-PAGE was in 15% acrylamide. Chapter 3. Results vitro translation product was not detected. 30 3.2 Optimal in vitro conditions for translation of CNV RNA 3.2.1 Concentration of exogenous RNA To determine the optimal concentration of C N V virion RNA for translation in wheat germ extracts (Promega), in vitro translation reactions were programmed with final C N V R N A concentrations which ranged between 2 and 240 ug/ml. As shown in Fig. 3.3, when C N V R N A is at a final concentration of 180 ug/ml (lane 7), the maximal level of all C N V protein products is observed. It is noted that a high molecular weight protein is present (lanes 6 to 8) which is not detected in those samples programmed with lesser amounts of C N V RNA. By comparison with [^C]-methylated molecular weight markers, the protein is estimated to be between ca. 69,000 and 92,500 Mr. The size of this protein suggests that it may represent the C N V 92 kDa readthrough product predicted from the nucleotide sequence. C N V RNA concentrations of between 120 and 180 ug/ml (ie. 6 to 9 ug C N V R N A in a 50 ul reaction volume) were used for the following translation experiments. 3.2.2 Time course of protein synthesis A time course of the synthesis of C N V proteins in wheat germ extracts (NEN) was carried out to determine the optimal incubation time for translation as well as to investigate the possibility that any translation products might arise from proteolysis and/or R N A degradation. C N V virion R N A was translated for 0, 5, 10, 20, 40, 60, 80 and 120 min, the reaction terminated, and then the products analyzed by SDS-PAGE. Fig. 3.4 shows that the first proteins to be detected in the 5 min incubation time were of ca. 31,000 and 24,000 M r (lane 2). A protein of ca. 34,600 Mr was detected after a 10 min incubation period (lane 3) and proteins of ca. 41,600 and 24,000 M r were Chapter 3. Results 31 Fig. 3.3 In vitro translation products resulting from the translation of a dilution series of C N V RNA. Wheat germ extracts (Promega) were programmed with C N V RNA concentrations of 2 ug/ml (lane 3); 20 ug/ml (lane 4); 60 ug/ml (lane 5); 120 ug/ml (lane 6); 180 ug/ml (lane 7); and 240 ug/ml (lane 8). Lanes 1 and 2 show the in vitro translation products of B M V R N A and no added R N A , respectively. The products were analyzed by SDS-PAGE (15% gel). The approximate sizes (x 10 3 M r ) of the major C N V in vitro translation products are indicated on the right of the gel. Chapter 3. Results 32 Fig. 3.4 Time course of in vitro translation of C N V RNA. Wheat germ extracts (NEN) were programmed with 160 ug/ml C N V RNA. Translation was allowed to proceed for increasing periods of time and then terminated by adding 2X sample buffer and heating the reaction to 90 °C for 2 min. The products were analyzed by SDS-P A G E (15% gel). Lane 1,0 min; lane 2, 5 min; lane 3, 10 min; lane 4, 20 min; lane 5, 40 min; lane 6, 60 min; lane 7, 80 min; lane 8, 120 min. The approximate sizes (x 10 3 M r ) of the major C N V in vitro translation products are indicated on the right of the gel. Chapter 3. Results 33 present after a 20 min incubation period (lane 4). Whereas synthesis of the ca. 31,000 M r protein remained relatively constant following the time of its appearance, synthesis of the ca. 41,600, 34,600, 24,000 and 20,000 M r proteins increased following their time of appearance. After a 60 minute reaction time, translation appeared to reach a level at which no further increase in protein synthesis occurred (lane 6). In addition, the pattern of protein synthesis remained stable over a 2 hr reaction period indicating that proteolysis is probably not responsible for the proteins produced. Translation reactions were allowed to proceed for a period of 2 hr as recommended by the manufacturer. 3.2.3 RNA extraction from wheat germ extracts To determine whether degradation of C N V virion R N A might be responsible for some of the in vitro translation products, C N V R N A was extracted from wheat germ extracts at various time intervals over the course of translation. An aliquot of each sample was denatured with methyl mercuric hydroxide (MeHgOH) and electrophoresed through a nondenaturing agarose gel which was stained with ethidium bromide (EtBr). An aliquot of each sample was also electrophoresed through a denaturing agarose gel which was transferred to ZetaProbe (BioRad) and hybridized with a full-length C N V cDNA clone nick-translated in the presence of [32p]. From these results (not shown), it appeared that C N V R N A remained mostly intact at least through an 80 minute translation period. 3.2.4 Effect of magnesium and potassium ions Optimization of the potassium and magnesium ion concentrations is required for efficient in vitro translation of messenger R N A . The magnesium and potassium ion concentrations in Promega wheat germ extracts (1 m M and 85 m M , respectively) are optimized for efficient translation of Chapter 3. Results 34 B M V RNA. These conditions were used in most of the studies described below since the Promega system was most often used. The N E N wheat germ system also recommends magnesium and potassium ion concentrations for translation of B M V R N A . Optimal ion concentrations for efficient translation of C N V R N A in the N E N system were determined experimentally. The magnesium and potassium ions concentrations recommended for translation of B M V RNA in N E N wheat germ extracts are 2.4 m M and 120 m M , respectively. Fig. 3.5.1 shows the effect of varying the final concentration of potassium ions from 50 m M to 175 m M on the in vitro translation products of C N V R N A under conditions in which the concentration of magnesium ions was held constant at 2.4 mM. Fig. 3.5.2 shows the effect of varying the final concentration of magnesium ions from 1.0 m M to 4.0 m M while the potassium ion concentration remained constant at 120 m M . The final potassium and magnesium ion concentrations selected for translation of C N V R N A in wheat germ extracts (NEN) were 83 m M and 2.2 m M , respectively, because individually, under these conditions in vitro translation of the four major C N V protein products was most efficient. 3.3 Hybrid-arrested translation 3.3.1 Use of synthetic negative sense R N A to identify C N V specific in vitro translation products Hybrid-arrested translation (HART) using D N A complementary to mRNA has been shown to be useful for identifying the genomic origin of mRNA translation products (Paterson et al, 1977). A modified hybrid-arrested translation procedure utilizing synthetic R N A complementary to CNV. virion R N A (negative sense RNA) was used to determine the genomic origin of C N V in vitro translation products. Negative sense R N A was chosen instead of D N A for the following reasons. Paterson et al. (1977) used denatured double stranded D N A in the hybridization reaction which Chapter 3. Results 35 Fig. 3.5.1 Effect of potassium on in vitro translation products of C N V R N A in wheat germ extracts (NEN). Potassium acetate concentrations of 175 m M (lane 3), 150 m M (lane 4), 125 m M (lane 5), 100 m M (lane 6), 75 m M (lane 7), and 50 m M (lane 8) were used for the translation of 160 ug/ml C N V R N A . The potassium ion concentration recommended for B M V (120 mM) was used for the translation of B M V R N A and endogenous R N A (lanes 1 and 2, respectively). The magnesium ion concentration was held constant at 2.4 mM. Products were analyzed by SDS-PAGE (15% gel). The numbers to the right of the gel refer to the sizes (x 10 3 M r ) of the major C N V in vitro translation products. Chapter 3. Results 36 Fig. 3.5.2 Effect of magnesium on in vitro translation products of C N V R N A in wheat germ extracts (NEN). Magnesium acetate concentrations of 4 m M (lane 3), 3 m M (lane 4), 2.4 m M (lane 5), 2 m M (lane 6), and 1 m M (lane 7) were used for the translation of 160 ug/ml C N V RNA. The magnesium ion concentration recommended for B M V RNA (2.4 mM) was used for the translation of B M V RNA and endogenous RNA (lanes 1 and 2, respectively). The potassium ion concentration was held constant at 120 m M . Products were analyzed by SDS-PAGE (15% gel). The numbers on the right refer to the sizes (x 10 3 M r ) of the major C N V in vitro translation products. Chapter 3. Results 37 preceded in vitro translation. Hybridization was carried out under conditions which favored mRNA/cDNA hybrids over competing DNA/cDNA hybrids. In the present study, single stranded nucleic acid was chosen to eliminate the possibility of a competitive reaction. R N A was chosen over single stranded D N A because copious amounts of R N A of precise genomic origin can be produced by run-off transcription using cDNA inserted into commercially available transcription vectors [for example, Bluescript (Stratagene)]. To test the possible usefulness of this approach, full-length synthetic negative sense C N V R N A , C N V T x 18/4701 (-) (see Fig. 3.8), was synthesized in vitro and a 3 fold molar excess of the transcript was incubated with C N V virion R N A under hybridization conditions (see section 2.5.5 for details). In control experiments B M V R N A and a 3 fold excess of C N V T x 18/4701 (-) were subjected to hybridization conditions as was C N V virion R N A without the added negative sense RNA. Translation of the resulting mixtures of RNAs demonstrated that full-length negative sense C N V transcripts arrested translation of all proteins normally produced by C N V virion R N A in wheat germ extracts (Fig. 3.6, lane 6). The arrest is specific because the synthesis of proteins by B M V R N A was not altered by C N V negative sense RNA (compare lanes 1 and 5). Also, the arrest is not a result of degradation of C N V virion R N A during incubation because mock hybridized C N V virion R N A produced the same proteins that were produced by untreated C N V virion RNA (compare lanes 2 and 4). Translation of C N V negative sense R N A was shown not to occur (lane 7). These results indicate that negative sense R N A can be used to specifically arrest protein synthesis and verify that the four major proteins synthesized from C N V virion RNA in wheat germ extracts are virus-specific [ie. none of the protein products are a result of translation of encapsidated host R N A as has been reported for another tombusvirus (Hayes et al., 1988)]. Chapter 3. Results 38 1 2 3 4 5 6 7 41.6-34-6-24-20-Fig. 3.6 H A R T studies using a negative sense synthetic transcript corresponding to the entire C N V genome. Wheat germ extracts (Promega) were programmed with 20 ug/ml B M V R N A (lane 1); 120 ug/ml C N V RNA (lane 2); no added RNA (lane 3); 120 ug/ml mock hybridized C N V R N A (lane 4); 20 ug/ml B M V R N A + 60 ug/ml C N V T x 18/4701(-) (lane 5); 120 ug/ul C N V R N A + 360 ug/ml C N V T x 18/4701(-) (lane 6); 360 ug/ml C N V T x 18/4701 (-) (lane 7). Hybridizations were conducted as described in section 2.5.5. In vitro translation products were analyzed by SDS-PAGE (15% gel) and autoradiography. The numbers on the left of the gel refer to the sizes (x 10 3 M r ) of the major C N V in vitro translation products. Chapter 3. Results 3.3.2 Optimal molar amounts of CNV negative sense RNA 39 For further hybrid-arrest experiments, it was considered desirable to determine the optimal amount of synthetic negative sense R N A required to completely arrest the translation of C N V virion RNA. Increasing concentrations of CNVTx 18/4701(-) were incubated with a constant amount of C N V virion R N A under hybridization conditions. The amount of negative sense R N A varied from 0.1 to 3.0 fold of the molar amount of C N V virion RNA. Translation indicated that a 3.0 fold molar excess of synthetic negative sense R N A was sufficient to arrest the synthesis of all CNV-directed proteins (Fig. 3.7, lane 5). Hybridization of C N V virion R N A using 1.0, 0.3 and 0.1 fold molar amounts of synthetic negative sense RNA resulted in incomplete hybrid-arrested translation (lanes 6 through 8). 3.4 Genomic location and identification of CNV in vitro translation products A number of experimental approaches were taken to obtain information concerning the genomic origins of C N V in vitro translation products. These were: (1) hybrid-arrested translation; (2) in vitro translation of synthetic positive sense C N V transcripts; (3) immunoprecipitation of in vitro translation products with C N V polyclonal antisera and (4) in vitro translation of size-fractionated C N V virion R N A . Fig. 3.8 is a diagrammatic representation of the genomic locations of C N V -specific positive and negative sense synthetic transcripts used in the experiments described below. Chapter 3. Results 40 1 2 3 4 5 6 7 8 Fig. 3.7 H A R T studies using increasing concentrations of CNVTx 18/4701(-). Wheat germ extracts (Promega) were programmed with 20 ug/ml B M V R N A (lane 1); 120 ug/ml C N V R N A (lane 2); no added RNA (lane 3); 120 ug/ml mock hybridized C N V R N A (lane 4); 120 ug/ml C N V R N A + 360 ug/ml CNVTx 18/4701(-) (lane 5); 120 ug/ml C N V RNA + 120 ug/ml CNVTx 18/4701(-) (lane 6); 120 ug/ml C N V R N A + 40 ug/ml C N V T x 18/4701(-) (lane 7); 120 ug/ml C N V R N A + 12 ug/ml CNVTx 18/4701 (-) (lane 8). In vitro translation products were analyzed by SDS-PAGE (15% gel) and autoradiography. The numbers on the left of the gel refer to the sizes (x 10 3 M r ) of the major C N V in vitro translation products. Chapter 3. Results 3.4.1 Genomic location of the 34,600 Mr protein coding region 41 Hybrid-arrested translation To determine which proteins arise from the 5' proximal region of the C N V genome, a synthetic negative sense transcript corresponding to this region was used in hybrid-arrested translation studies. C N V virion R N A was incubated with a 3 fold molar excess of C N V T x 18/910(-) (see Fig. 3.8) and the resulting hybrids were translated in wheat germ extracts. Also subjected to hybridization conditions in control experiments were B M V R N A together with CNVTx 18/910(-) as well as C N V virion R N A to which CNVTx 18/910(-) was not added. The proteins produced from in vitro translation of these RNAs are shown in Fig. 3.9. As seen in lane 6, prior incubation of C N V virion R N A with CNVTx 18/910(-) resulted in a dramatic reduction of the ca. 34,600 M r polypeptide but had little or no affect on the other C N V proteins. (Visual comparison of the background protein indicated that less protein was loaded into lane 6 than was loaded into lane 5) The transcript did not affect the translation of B M V RNA (lane 4) giving further support for the specificity of the reaction. In addition, C N V R N A incubated without added negative sense transcript (lane 5) produced the same number and amount of proteins that untreated C N V R N A produced (lane 2) demonstrating that the loss of the 34,600 M r protein was not due to degradation of its template during the hybridization reaction. These results indicate that the ca. 34,600 M r C N V in vitro translation product is derived from the 5' proximal region of C N V RNA. It is noted that translation of the negative sense transcript, C N V T x 18/910(-), gave rise to several low molecular weight polypeptides (lane 8). Analysis of the C N V nucleotide sequence revealed that C N V T x 18/910(-) has the capacity to encode a number of low molecular weight proteins (analysis not shown). Therefore, although hybrid-arrested translation using negative sense synthetic transcripts can be useful for determining the genomic origin of a template, control SYNTHETIC CNV TRANSCRIPTS ^CNVTx 3634/4639 (-CNVTx 3634/4639 fc-) CNVTx 2566/3234 (-) ^CNVTx 18/910 (-) CNVTx 2566/4116 (-) CNVTx 18/1090 (+)^ CNVTx 2566/4116 (+) CNVTx 260/2156 (+) • CNVTx 18/4701 (-) CNVTx 18/4701 (+) CNVTx PK2 (+) • amber codon CNV GENOMIC RNA ORF1 33kDa ORF2 ORF3 ORF4 152 1042 92kDa 152 (putative replicase) 2608 41 kDa 2628 (coat protein) 3770 20kDa PREDICTED PROTEIN PRODUCTS 3832 4353 21 kDa 3 8 0 0 4 3 7 2 o P H o c Fig. 3.8 Genomic locations of CNV positive and negative sense synthetic transcripts. The CNV synthetic transcripts were produced by in vitro transcription of cDNA clones with inserts corresponding to selected regions of the CNV genome (see section 2.5 for details). The sizes and locations of the CNV open reading frames (ORFs) and predicted protein products were determined by Rochon & Tremaine (1989). to Chapter 3. Results 43 experiments should be run to determine the template activity of a negative sense RNA. Translation of positive sense transcripts Translation of a positive sense transcript covering the 5' region of C N V RNA, CNVTx 18/1090(+) (see Fig. 3.8), resulted in the synthesis of one protein which comigrated with the C N V ca. 34,600 M r translation product as well as another protein which had a slighdy faster mobility (Fig. 3.9, lane 7). The synthesis of a ca. 34,600 M r protein from this template together with results of hybrid-arrested translation studies demonstrate that the ca. 34,600 M r protein is derived from the 5' proximal region of the C N V genome. One possible origin of the smaller product was suggested through the translation of another positive sense transcript, C N V T x 260/2156(+) (see Fig. 3.8), whose coding region overlaps that of CNVTx 18/1090(+) but which is truncated at its 5' terminus. Translation of CNVTx 260/2156(+) (Fig. 3.9, lane 9) produced a protein that comigrated with the ca. 31,000 M r protein produced by CNVTx 18/1090(+). As will be discussed, the smaller ca. 31,000 M r protein might arise from aberrant ('leaky') translation (Kozak, 1981) from A U G codons which are downstream from the 33 kDa A U G codon. Together these results demonstrate that the 34,600 M r C N V in vitro translation product originates from the 5' region of the C N V genome. Since analysis of the nucleotide sequence revealed that this region has the capacity to encode proteins no larger than 33 kDa (Rochon & Tremaine, 1989), it is proposed that the 34,600 M r C N V in vitro translation product is the 33 kDa protein predicted from the nucleotide sequence. For clarity, however, this translation product will be referred to as the 34,600 M r protein in the remaining results sections. Fig. 3.9 Genomic location of the 34,600 M r in vitro translation product. In vitro translation was in wheat germ extracts (Promega) programmed with 20 ug/ml B M V RNA (lane 1); 120 ug/ml C N V R N A (lane 2); no added RNA (lane 3); 20 ug/ml B M V R N A + 60 ug/ml CNVTx 18/910 (-) (lane 4); 120 ug/ml mock hybridized C N V R N A (lane 5); 120 ug/ml C N V RNA + 70 ug/ml C N V T x 18/910(-) (lane 6); 240 ug/ml CNVTx 18/1090(+) (lane 7); 240 ug/ml CNVTx 18/91CK-) (lane 8). The products were analyzed by SDS-PAGE in a 15% gel. In a separate experiment, 80 ug/ml CNVTx 260/2156(+) was translated in wheat germ extracts and the products analyzed by SDS-PAGE in a 12% gel (lane 9). The 31 x 10 3 M r band is shown aligned with the 31 x 10 3 M r band in C N V R N A as it was in the original. The sizes (x 10 3 M r ) of the major C N V in vitro translation products are indicated on the left of the gel. Chapter 3. Results 3.4.2 Genomic location of the 41,600 M r protein coding region 45 Hybrid-arrested translation Since it seemed reasonable that the 41,600 M r in vitro translation product might arise from the 41 kDa C N V ORF, a synthetic negative sense transcript, C N V T x 2566/4116(-) (see Fig. 3.8), covering the 41 kDa protein coding region was synthesized and used in hybrid-arrested translation studies with C N V virion RNA. Fig. 3.10 (lane 6) shows that this transcript arrested the translation of the ca. 41,600 M r protein. The transcript also arrested the translation of the ca. 20,000 M r protein. The arrest of the ca. 20,000 M r protein might be due to the overlap of C N V T x 2566/4116(-) with the putative A U G initiation codon (nucleotides 3832-3834) for the predicted C N V 20 kDa ORF. To test this possibility, a shorter transcript, CNVTx 2566/3234(-) (see Fig. 3.8), which does not overlap with this A U G was used in hybrid-arrested translation studies. Fig. 3.10 (lane 9) shows that CNVTx 2566/3234(-) arrested the translation of the ca. 41,600 M r protein but did not affect translation of the ca. 20,000 M r protein. These hybrid-arrested translation studies therefore suggest that the C N V 41,600 M r protein indeed corresponds to the 41 kDa ORF predicted from the nucleotide sequence. It is noted that CNVTx 2566/3234(-) may also partially prevent synthesis of the 34,600 M r protein (compare the relative intensities of the 34,600 M r protein and the 24,000 and 20,000 M r proteins in lane 5 with those in lane 9). If this is so, it is possible that hybridization of this transcript to the 41,600 M r coding region non-specifically interferes slightly with translation of a far upstream cistron (see also section 3.4.3). Chapter 3. Results 46 Fig. 3.10 Genomic location of the 41,600 M r in vitro translation product. In vitro translation was in wheat germ extracts (Promega) programmed with 20 ug/ml B M V R N A (lane 1); 120 ug/ml C N V R N A (lane 2); no added RNA (lane 3); 20 ug/ml B M V R N A + 60 ug/ml CNVTx 2566/4116(-) (lane 4); 120 ug/ml mock hybridized C N V R N A (lane 5); 120 ug/ml C N V R N A + 120 ug/ml C N V T x 2566/4116(-) (lane 6); 240 ug/ml CNVTx 2566/4116(+) (lane 7); 240 ug/ml CNVTx 2566/4116(-) (lane 8). In a separate experiment 120 ug/ml C N V RNA was incubated under hybridization conditions with 120 ug/ml CNVTx 2566/3234(-) and translated in wheat germ extracts (lane 9). The products were analyzed by SDS-PAGE in a 15% gel. The sizes (x 103 M r ) of the major C N V in vitro translation products are indicated on the left of the gel. Chapter 3. Results Translation of positive sense transcripts 47 The in vitro translation products of the positive sense transcript, CNVTx 2566/4116(+) (see Fig. 8), are shown in Fig. 3.10 (lane 7). A protein that comigrated with the C N V ca. 41,600 M r product can be seen, however, the protein is faint indicating that this transcript is not very effective in producing the 41 kDa product. The weak messenger activity of CNVTx 2566/4116(+) is likely due to the fact that the transcript contained 5' proximal A U G codons which were followed shortly by in-frame termination codons (particularly since the short reading frame set by one A U G triplet terminated after the start of the 41 kDa ORF). The insertion of upstream A U G triplets has been demonstrated to significantly reduce or completely eliminate protein synthesis from downstream coding regions (Kozak, 1984c). Immunoprecipitation Tha amino acid sequence of the C N V 41 kDa ORF indicates that it encodes the C N V coat protein (Rochon & Tremaine, 1989). To determine if the 41,600 M r in vitro translation product is the coat protein, C N V in vitro translation products were immunoprecipitated with C N V IgG (which was prepared against intact viral particles) bound to Protein A-Sepharose CL-4B (Pharmacia). Fig. 3.11 shows that the 41,600 Mr protein is precipitated (lane 3), however, bands corresponding to other C N V translation products are also precipitated but not as efficiently as the 41,600 M r protein. In control experiments (not shown), a small but significant amount of non-specific binding of C N V in vitro translation products to both C N V antiserum and Protein A-Sepharose CL-4B (Pharmacia) also occurred. Although not conclusive, the enrichment of the 41,600 M r protein over the other C N V in vitro translation products following immunoprecipitation suggests that the C N V 41,600 Mr protein is the C N V coat protein. Chapter 3. Results 48 Fig. 3.11 Immunoprecipitation of C N V in vitro translation products using antisera to C N V virions. Wheat germ extracts (NEN) were programmed with B M V R N A (lane 1) and 120 ug/ml C N V R N A (lane 2). In lane 3, C N V in vitro translation products were immunoprecipitated with C N V IgG bound to Protein A-Sephadex (Pharmacia) (see section 2.4.4 for details). Products were analyzed by SDS-PAGE in a 15% gel and autoradiography. The size (x 10 3 M r ) of the C N V coat protein is indicated on the right of the gel. Chapter 3. Results 49 The results of hybrid-arrested translation and translation of positive sense transcripts suggest that the 41,600 M r in vitro translation product is derived from the 41 kDa ORF predicted from the C N V nucleotide sequence. In addition, the immunoprecipitation studies provide further support that the 41 kDa ORF is the C N V coat protein coding region. 3.4.3 Genomic location of the 20,000 and 24,000 M r protein coding regions Translation of positive sense transcripts Translation of a synthetic positive sense transcript, C N V T x 3634/4639(+) (see Fig. 3.8), corresponding to the 3' terminus of the C N V genome resulted in the synthesis of proteins that comigrated with the ca. 20,000 and 24,000 M r proteins produced by C N V virion R N A in wheat germ extracts (Fig. 3.12, lane 7). This result very strongly suggests that the ca. 24,000 and 20,000 M r proteins are derived from the 3' terminus of the C N V genome and likely correspond to the 21 kDa and 20 kDa proteins predicted from the nucleotide sequence, respectively. For consistency, these in vitro translation products will be referred to as the ca. 24,000 and 20,000 M r proteins in the remaining results sections. Hybrid-arrested translation was also tested as a means for determining which in vitro translation products arise from the 3' terminal region of the C N V genome. A synthetic negative sense transcript, C N V T x 3634/4639(-) (see Fig. 3.8), corresponding to this region was synthesized and used in H A R T experiments as described previously. The results showed that this transcript diminished the translation of all four major C N V proteins (Fig. 3.12, lane 6). The arrest of all C N V in vitro translation products was not entirely non-specific, however, because this negative sense transcript did not reduce translation of B M V R N A (compare lanes 1 and 4). The unusual behaviour of this transcript in reducing all C N V in vitro translation products is not Chapter 3. Results 50 Fig. 3.12 Genomic location of the 24,000 and 21,000 M r in vitro translation products. Wheat germ extracts (Promega) were programmed with 20 ug/ml B M V R N A (lane 1); 120 ug/ml C N V R N A (lane 2); no added R N A (lane 3); 20 ug/ml B M V R N A + 60 ug/ml C N V T x 3634/4639(-) (lane 4); 120 ug/ul mock hybridized C N V R N A (lane 5), 120 ug/ml C N V R N A + 80 ug/ml C N V T x 3634/4639(-) (lane 6), 240 ug/ml CNVTx 3634/4639(+) (lane 7), 240 ug/ml C N V T x 3634/4639(-) (lane 8). The products were analyzed by SDS-PAGE in a 15% gel and autoradiography. The sizes (x 10 3 M r ) of the major C N V in vitro translation products are indicated on the left of the gel. Chapter 3. Results 51 understood at this time. It is possible that the 3' terminal region of the C N V genome has a regulatory role in translation which the antisense R N A used this experiment prevented. A regulatory role for the 3' terminus has been suggested for carnation mottle virus RNA (Saloman et al., 1978). Alternatively, the binding of synthetic transcripts to C N V virion R N A might affect the R N A secondary structure at sites other than those which are hybridized and, in turn, affect the efficiency of translation from these regions. The involvement of secondary structure in the regulation of translation by antisense R N A at sites away from those which are directly affected has been demonstrated in prokaryotic systems (for recent reviews, see Inouye, 1988 and Simons, 1988). This type of translational regulation could represent a problem in the use of hybrid-arrested translation assays with multicistronic viral RNAs. The results of the positive sense translation experiments indicate that the ca. 24,000 and 20,000 Mr C N V in vitro translation products are derived from the 3' terminal coding region(s) of C N V R N A . It is not known if the two proteins are translated from different reading frames as is predicted from the nucleotide sequence or if the ca. 20,000 M r protein is produced from initiation of translation at a downstream A U G codon within the reading frame for the ca. 24,000 Mr protein. Alternatively, the ca. 20,000 M r protein could be a premature termination product or degradation product of the ca. 24,000 M r protein. Results from the time course of protein synthesis (see section 3.2.2), however, did not supply any evidence for the occurrence of proteolytic processing or protein degradation. Analysis of the nucleotide sequence from which C N V T x 3634/4639(+) was transcribed shows that it does not have the capacity to encode a protein the size of the ca. 24,000 M r C N V in vitro translation product (results not shown; see Rochon & Tremaine, 1989). It is therefore proposed that at least one, and possibly both, of the 21 and 20 kDa proteins are translated in vitro from the same R N A derived from the 3' terminus of the C N V genome. Chapter 3. Results 3.5 Natural template(s) for the expression of CNV proteins 52 The translation strategy of C N V is thought to involve the formation of 3' co-terminal subgenomic RNAs with only the 5' proximal gene being expressed from the genomic-length R N A species. Since eukaryotic ribosomes can access only 5* proximal cistrons, the strategy of forming subgenomic RNAs allows translation of otherwise closed cistrons. Analysis of dsRNA species (which may represent the replicative forms of genomic and subgenomic RNAs) from C N V -infected plants revealed the presence of two prominent dsRNAs of ca. 2.1 and 1.0 kb (Rochon & Tremaine, 1988). In addition, northern blots of R N A extracted from CNV-infected leaves which were probed with random-primed cDNA made to C N V R N A also provided evidence for the existence of subgenomic RNAs (Rochon, D. M . , personal communication). In the present study, it was of interest to determine whether C N V subgenomic RNAs are encapsidated and, if so, whether they act as templates for the expression of downstream cistrons in the C N V genome. To answer these questions, C N V R N A purified from virus particles was size-fractionated on a linear-log sucrose gradient and selected fractions were translated in vitro. The results of these experiments are described below. 3.5.1 Sucrose gradient fractionation of C N V virion R N A Agarose gel electrophoresis of fractionated CNV virion RNA Fig. 3.13 shows aliquots of sucrose-gradient fractionated C N V virion R N A which were denatured with methyl mercuric hydroxide (MeHgOH), electrophoresed through a non-denaturing agarose gel and stained with ethidium bromide (EtBr). These results demonstrate that C N V virion RNA preparations contain many discrete-sized, less than full-length R N A species which cannot easily be detected in unfractionated virion R N A (refer to Fig. 3.14). Some of these species might represent Chapter 3. Results 53 degradative products of viral RNA or viral-specific subgenomic RNAs. Alternatively, some these species might represent defective RNAs which are known to be generated during tombusvirus infections (Hillman et al., 1987). Therefore, further experiments were conducted to determine if these less than full-length R N A species could serve as templates for C N V in vitro translation products. Northern blot analysis of fractionated CNV RNA Fig. 3.14.1 shows an EtBr-stained, MeHgOH-containing denaturing agarose gel of selected samples taken from every sixth sucrose fraction beginning with fraction #32 through to fraction #74. A northern blot was prepared using samples from these fractions which were resolved on a MeHgOH-containing denaturing gel and transferred to ZetaProbe (BioRad). To detect the presence of 3' co-terminal subgenomic RNAs, the blot was hybridized with a [32p]-labelled probe made by nick-translation of a c D N A clone corresponding to the 3' terminus of the C N V genome. Fig. 3.14.2 shows that C N V virion R N A preparations contain, in addition to genomic sized R N A , several 3* co-terminal, less than genomic-sized R N A molecules of ca. 2.3 kb, 1.5 kb, 1.0 kb and 0.5 kb. The subgenomic-sized RNAs were further analyzed for messenger R N A activity in wheat germ and rabbit reticulocyte systems. 3.5.2 In vitro translation of sucrose gradient fractionated C N V R N A In vitro translation of fractionated CNV RNA in wheat germ extracts Samples from selected sucrose gradient fractions were translated in wheat germ extracts and the resulting products are shown in Fig. 3.15. Fraction #32, which contained an R N A species of ca. 0.5 kb, did not have messenger activity (lane 4). The 0.5 kb R N A species is very likely a 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Fig. 3.13 Agarose gel electrophoresis of sucrose gradient fractionated C N V virion R N A . Lanes 2 - 39 contain samples of every other fraction from fraction #2 to fraction #39 (numbered from the top to the bottom of the gradient). Samples were denatured with MeHgOH, electrophoresed through a 1% nondenaturing agarose gel and stained with EtBr. The sizes (in kb) of R N A markers in lanes 1 and 40 are indicated on the side of the gel. Chapter 3. Results 55 defective interfering R N A (Rochon, D. M . , unpublished observations) similar to that reported by Hillman et al. (1987) and, if so, would not have coding capacity. Fractions #38 and #44, which contained R N A species of ca. 1.0 kb, produced proteins of ca. 20,000 and 24,000 M r (lanes 5 and 6). Thus, it appears that the ca. 1.0 kb subgenomic RNA(s) serves as the template for the ca. 24,000 and 20,000 M r proteins. Fractions #50 through #74 contained, in addition to ca. 1.0 kb R N A molecules, species of ca. 1.5 and 2.3 kb. These fractions gave rise to proteins of ca. 20,000 and 24,000 M r as well a protein of ca. 41,600 M r (lanes 7 to 11). Thus, either the ca. 1.5 kb species or the ca. 2.3 kb species or both could serve as mRNAs for the 41,600 M r protein. However, since the 1.5 kb RNA species is 3' co-terminal with genomic RNA, it would not contain the entire 41 kDa ORF and so could not produce the 41,600 M r protein. In addition, fraction #56, which contained the ca. 2.3 kb subgenomic in the greatest abundance (see Fig. 3.14.2), produced the ca. 41,600 Mr translation product most efficiently (lane 8). It is, therefore, concluded that the ca. 2.3 kb subgenomic R N A serves as the template for the 41,600 M r in vitro translation product. The only fraction which contained an efficient template for the synthesis of the ca. 34,600 M r product was fraction #74 (lane 11) which contained full-length genomic R N A (4.7 kb). Therefore, it is concluded that genomic R N A serves as the template for the ca. 34,600 M r product. The results of these translation experiments demonstrate the involvement of encapsidated subgenomic R N A species in the synthesis of the ca. 41,600, 24,000 and 20,000 M r C N V in vitro translation products. These results, however, cannot exclude the involvement of genomic-length R N A in the translation of downstream cistrons since a sucrose gradient fraction containing exclusively genomic-length R N A was not obtained. This result will be discussed further below. Chapter 3. Results 56 1 2 3 4 5 6 7 8 9 10 Fig. 3.14.1 Agarose gel electrophoresis of selected sucrose gradient fractionated R N A samples. Equal volumes of samples of sucrose gradient fractions were denatured with MeHgOH, electrophoresed through a 1% agarose gel containing 5 m M MeHgOH, and stained with EtBr. Lane 1 contains RNA markers (sizes in kb shown on the left of the gel). Lanes 2 to 9 contain samples from sucrose fractions #32, #38, #44, #50, #56, #62, #68, and #74, respectively. Lane 10 contains unfractionated C N V virion RNA (500 ng). Chapter 3. Results 57 1 2 3 4 5 6 7 8 9 10 Fig. 3.14.2 Northern blot analysis of selected sucrose gradient fractionated RNA samples. Samples were electrophoresed through a 1% agarose gel containing 5 m M MeHgOH, transferred to ZetaProbe (BioRad) and hybridized to a [32P]-labelled cDNA probe corresponding to the 3' terminus of the C N V genome. Lanes 2 to 9 contain sucrose fractions #32, #38, #44, #50, #56, #62, #68 and #74, respectively. Lane 10 contains unfractionated C N V virion RNA (500 ng). The sizes to the right of the gel refer to the sizes (in kb) of 3' co-terminal C N V RNA species estimated using the R N A markers shown in Fig. 3.14.2. Chapter 3. Results 58 In vitro translation of fractionated CNV RNA in rabbit reticulocyte lysates Translation of samples from the above fractions in rabbit reticulocyte lysates resulted in the production of only the ca. 34,600 M r protein (not shown). The synthesis of a single ca. 34,600 M r protein in rabbit reticulocyte lysates is similar to previous results (see section 3.1.1) and suggests, as before, that the translation initiation signals for several cistrons of this plant virus are not recognized by rabbit reticulocyte ribosomes. 3.5.3 Translation of full-length positive sense synthetic transcripts Translation of sucrose gradient fractionated C N V virion RNA in wheat germ extracts demonstrated that subgenomic R N A molecules directed the synthesis of proteins originating from downstream cistrons in the C N V genome. However, because all of the fractions contained less than full-length R N A species (see Fig. 3.14.2), the possibility that translation of downstream cistrons on genomic as well as on subgenomic R N A molecules might occur could not be excluded. To eliminate the possibility that C N V genomic R N A serves as a template for the ca. 41,600, 24,000 and 20,000 M r proteins, full-length synthetic positive sense transcripts, C N V T x 18/4701(+) and PK2 (see Fig. 3.8), were translated in wheat germ extracts. C N V T x 18/4701 (+) is missing 18 viral R N A nucleotides at the 5' terminus and instead contains ca. 20 additional guanosine residues which were added during the cDNA cloning procedure (see Rochon & Tremaine, 1989). PK2 is an infectious synthetic C N V transcript (Rochon, D. M . , personal communication) and has the same 5' terminus as C N V genomic R N A plus four 5' non-viral nucleotides. Fig. 3.16 (lane 4) shows that PK2 directs the synthesis of a single ca. 34,600 M r protein in wheat germ extracts (Promega). Since PK2 does not contain subgenomic RNAs, it can be deduced that the synthesis of the ca. 41,600, 24,000 and 20,000 M r C N V in vitro translation products is directed by subgenomic RNAs. The Chapter 3. Results 59 1 2 3 4 5 6 7 8 9 10 11 Fig. 3.15 In vitro translation products of selected sucrose gradient fractionated C N V virion RNA samples. Wheat germ extracts (Promega) were programmed with B M V RNA (lane 1); C N V virion RNA (160 ug/ml) (lane 2); no added RNA (lane 3); equal volumes of RNA from sucrose fractions #32, #38, #44, #50, #56, #62, #68, and #74 (lanes 4 to 10, respectively). SDS-PAGE was in 15% acrylamide. The sizes of the major C N V in vitro translation products are indicated on the right of the gel in M r x 103. Chapter 3. Results 60 synthesis of a single 34,600 Mr protein, however, was not consistently observed in all batches of wheat germ tested. Fig. 3.16 (lane 5) shows that C N V T x 18/4701(+) [as well as PK2 (gel not shown)] directed the synthesis of ca. 41,600, 24,000 and 20,000 M r proteins in addition to a ca. 34,600 M r protein in wheat germ extracts (Promega). These results would suggest that C N V genomic R N A can serve as a template for the expression of all the C N V in vitro translation products. It is unlikely that the proteins observed in Fig. 3.16 are all derived from initiation of translation at successive A U G codons present in the 5' proximal coding region of C N V since in vitro translation of a transcript corresponding solely to this region (see Fig. 3.8) did not produce a similar result. The reason for such a discrepancy in these in vitro translation experiments is not known at this time but will be discussed further (see section 4.4). Chapter 3. Results 61 Fig. 3.16 In vitro translation of synthetic genomic-length C N V RNA. Wheat germ extracts (Promega) were programmed with 20 ug/ml B M V RNA (lane 1); 120 ug/ml C N V virion RNA (lane 2); no added RNA (lane 3); 240 ug/ml PK2 (lane 4); 240 ug/ml CNVTx 18/4701 (+) translated in a separate experiment (lane 5). In vitro translation products were analyzed by SDS-PAGE in 15% acrylamide. The sizes on the sides of the gel refer to the sizes (x 103 M r ) of the major C N V in vitro translation products. Chapter 4 Discussion In vitro translation of C N V virion RNA resulted in the synthesis of a single protein of 33 kDa in rabbit reticulocyte lysates whereas four proteins of 41, 33,21, and 20 kDa were produced in wheat germ extracts. Using hybrid-arrested translation and translation of positive sense transcripts in wheat germ extracts, the 33 kDa protein coding region was mapped to the 5' terminus of C N V R N A and the 41, 21, and 20 kDa protein coding regions were mapped to downstream regions in the C N V genome. In wheat germ extracts, the 33 kDa protein was translated from full-length genomic R N A while the 41, 21 and 20 kDa proteins were produced from subgenomic RNAs or possibly also from unusual translation of downstream cistrons on genomic-length R N A (discussed below). Fig. 4.1 summarizes the C N V in vitro translation products, their genomic locations and their templates for synthesis. In rabbit reticulocyte lysates, only the 5' proximal cistron of C N V genomic RNA, but not the subgenomic RNAs, is recognized and translated whereas in wheat germ extracts, both genomic and subgenomic RNAs are recognized and translated efficiently. Several explanations to account for the difference in translational efficiencies of C N V virion RNA in wheat germ extracts and rabbit reticulocyte lysates are presented below. The number, sizes, and genomic locations of C N V in vitro translation products are in agreement with predictions based on the C N V nucleotide sequence data of Rochon & Tremaine (1989) with the exception that synthesis of the 92 kDa readthrough protein was not conclusively demonstrated. The lack of readthrough protein synthesis in vitro is also discussed further. 62 amber codon CNV GENOMIC RNA V///////yy//////////////////A 4.7 kb ORF1 ORF2 CNV SUBGENOMIC RNAs ORF3 ORF5 2.1 kb 0.9 kb ORF4 33kDa CNV PROTEINS 152 1042 92kDa 152 (putative replicase) - i 2608 41 kDa 2628 ( C O a t P r o t e i n ) 3770 20kDa 3832 4353 21 kDa r 3800 4372 n n 2 c o 3 Fig. 4.1 Genomic locations and templates for synthesis of CNV in vitro translation products. The size of CNV genomic R N A was determined by Rochon & Tremaine (1989). The sizes of the CNV subgenomic RNAs shown here are those determined by primer extension analyses (Rochon, personal communication). The sizes and locations of the CNV open reading frames (ORFs) are from Rochon & Tremaine (1989). Chapter 4. Discussion 4.1 Differences in C N V R N A translational efficiencies between rabbit reticulocyte lysates and wheat germ extracts 64 4.1.1 Differential selection of AUG codons between plant and animal ribosomes Analysis of plant and animal mRNA sequences and results of mutagenesis studies conducted both in vivo and in vitro have revealed differences in the consensus sequences surrounding the A U G initiation codons of plant and animals (Kozak, 1984a, 1986b; Liitke, 1987). In animals, the consensus sequence has been identified as C ^ / n C C A U G G . The nucleotide 3 positions upstream of the A U G initiation codon (preferably adenine) is implicated as an important regulator of translational efficiency (Kozak, 1984b). In plants, the consensus sequence for initiation is A A C A A U G G C , however the nucleotide at position -3 (although also preferably adenine) does not appear to modulate the efficiency of initiation (Liitke, 1987). The selection of intitiation codons in plants may be influenced to a greater extent by the nucleotides in position +4 and position +5 relative to the A (position +1) of the A U G triplet, possibly in a manner similar to that demonstrated for the nucleotide in position -3 in animal mRNA (Liitke, 1987). Table II compares the sequences surrounding the putative initiation codons for the C N V 41, 33, 21, and 20 kDa proteins with the consensus sequences established for either plant or animal mRNAs. This comparison reveals that the sequences surrounding the putative A U G codons for the 41 and 20 kDa proteins are in almost optimal context for translation by either plant or animal ribosomes. It is therefore not immediately obvious why the subgenomics for these products are translated more efficiently in wheat germ extracts than in rabbit reticulocyte lysates. The sequence surrounding the putative 33 kDa initiation codon is not in an optimal context for translation by animal ribosomes although the purine 3 nucleotides upstream of the A U G codon is favored over a pyrimidine in this position. In rabbit reticulocyte lysates, however, the 33 kDa Chapter 4. Discussion 65 Table II. S E Q U E N C E S S U R R O U N D I N G P U T A T I V E A U G I N I T I A T I O N CODONS O F C N V IN VITRO T R A N S L A T I O N P R O D U C T S C N V protein Nucleotides matching consensus sequence in animalsa (CACCAUGG) Nucleotides matching consensus sequence in plants0 (AACAAUGGC) 33 kDa * * * C G A C A U G G * C G A C A U G G A 41 kDa * * * * C A C A A U G G C A C A A U G G C 21 kDa * * A U U C A U G G * * A U U C A U G G A 20 kDa * * * * A A C C A U G G * * * * A A C C A U G G A a from Kozak, 1986b b from Lutke, 1987 * indicates identity with the consensus sequence surrounding the A U G initiation codon Chapter 4. Discussion 66 protein is translated much more efficiently from genomic R N A than the 41 or 20 kDa proteins are from subgenomic RNAs. Also, since the presence of mRNA competition has been reported to have no effect in wheat germ extracts but to increase the differences observed in translational efficiencies in reticulocyte lysates (Liitke, 1987), the latter system might be expected to translate the subgenomic RNAs (having near optimal initiation sequences) with greater efficiency than genomic R N A (having a less than optimal initiation sequence). The sequence surrounding the putative initiation codon for the 21 kDa protein is in a poor context for translation by animal ribosomes and thus its synthesis might not be expected in rabbit reticulocyte lysates. The lack of synthesis of the 20 kDa protein in rabbit reticulocyte lysates could then be rationalized if production of the 20 kDa protein were dependent upon translation of the upstream 21 kDa protein coding region (as would be the case i f the 20 kDa protein were produced via leaky translation; see section 4.5). The lack of synthesis of the 41 kDa protein in rabbit reticulocyte lysates, however, cannot be correlated with the sequences surrounding the putative A U G initiation codon for this protein. This could be due to a lack of understanding of the sequences surrounding initiation codons or indicate that sequences further upstream or downstream of the initiation codons might also modulate translational efficiency. Eukaryotic mRNAs have not been demonstrated to contain a conserved ribosome binding site analogous to the Shine-Dalgarno site (Shine & Dalgarno, 1974) in prokaryotic mRNAs. The Shine-Dalgarno site is a polypurine sequence located upstream of the A U G initiation codon in bacterial mRNAs which is complementary to the sequence at the 3' end of 16S ribosomal RNA (rRNA). This site, therefore, enables direct binding of prokaryotic ribosomes to mRNA and places the ribosomes ca. 7-10 nucleotides upstream of the translational start site, precluding any significant ribosomal scanning. In contrast to prokaryotic initiation, the 40S subunit of eukaryotic ribosomes binds at the 5' end of mRNA (facilitated by the presence of a m^G cap) and migrates down the transcript, which might suggest that the presence of an internal ribosome binding site is Chapter 4. Discussion 67 not necessary. Despite the differences between prokaryotic and eukaryotic initiation mechanisms, the 18S rRNA sequence in eukaryotes has been examined for sequences that are complementary to those surrounding the A U G initiation codons of eukaryotic mRNAs. Several models of ribosome binding have been proposed which involve interaction between the 5' untranslated region of eukaryotic mRNAs and purine-rich sequences (Hagenbuchle et al., 1978; Azad & Deacon, 1980) or noncontiguous sequences (Sargan etal., 1982; Kozak, 1986b) in 18S rRNA. Mutagenesis studies on the sequences upstream of the A U G initiation codon have revealed the optimal context for initiation to be ( G C C ) G C C A / G C C A U G G (Kozak, 1987a). Since this sequence is internally repetitious, it was suggested that the 3 nucleotide motif, A / Q C C , might phase scanning by directing the 40S ribosomal subunit to read nucleotide triplets in the same frame as the A U G codon and downstream coding region (Kozak, 1987a). The sequences upstream of the A U G initiation codon might alternatively determine the R N A secondary structure to an extent that might affect the migration of the ribosomal subunit (Kozak, 1986a). Although there is no conclusive evidence for the existence of a eukaryotic ribosome binding site located upstream of the A U G initiation codon, these regions may differentially influence the efficiency of recognition or translation of mRNAs between plant and animal ribosomes. The nucleotides preceding the putative A U G codons of the C N V 41, 21 and 20 kDa proteins are shown in Fig. 4.2 (see section 4.2). 4.1.2 Endogenous capping activity of wheat germ extracts The presence of a low level endogenous capping activity present in wheat germ extracts (Paterson & Rosenberg, 1979) but not in rabbit reticulocyte lysates might also influence the efficiency of translation of C N V R N A in vitro. The presence of a 5' terminal m^G cap is known to greatly increase the efficiency of translation of mRNA although the mechanism by which translation is enhanced is not completely understood (Shatkin, 1976; Kozak, 1983). Many viral RNAs are not capped, however, and it is uncertain how these messengers lacking a cap are translated with such Chapter 4. Discussion 68 efficiency (Kozak, 1983). It is not known whether C N V genomic or subgenomic RNAs are capped, however, the absence of a cap on C N V subgenomic RNAs might explain why these RNAs are not translated in rabbit reticulocyte lysates. In wheat germ extracts, the low level of endogenous capping activity might enhance translation of uncapped C N V subgenomic RNAs. 4.1.3 Differential ability of ribosomes to translate through regions of high secondary structure A high degree of secondary structure in the 5* untranslated region of mRNA has been demonstrated to inhibit initiation of translation by eukaryotic ribosomes (Kozak, 1986a). An experiment to examine the possible effect of C N V secondary structure on translational efficiency was conducted in both wheat germ extracts and rabbit reticulocyte lysates. Because R N A was routinely denatured by heating for 10 min at 67 °C prior to translation in wheat germ extracts but not in rabbit reticulocyte lysates (as recommended by Promega), it was possible that the prior heat treatment could be responsible for the observed differences in translation products between the two systems. However, both for translation of C N V virion R N A and the synthetic transcript, CNVTx 18/4701 (+), heating had no effect on the number or the sizes of translation products in either wheat germ or rabbit reticulocyte lysates (results not shown). Thus, secondary structure of C N V RNA does not appear to play a role in the differential translation by the two translation systems. 4.2 In vitro translation of other tombusviruses In contrast to the results found for CNV, translation of other tombusviruses including cymbidium ringspot virus (CyRSV) and the type strain of tomato bushy stunt (TBSV) resulted in the synthesis of a number of protein products in rabbit reticulocyte lysates. Translation of size-fractionated CyRSV RNA (Burgyan et al, 1986; Russo et al., 1988) in rabbit reticulocyte lysates indicated that Chapter 4. Discussion 69 genomic-length R N A acts as the template for the synthesis of a single protein (40 kDa) while three proteins (43, 34 and 22 kDa) are synthesised from less than genomic-length species. Similarly, translation of sucrose gradient fractionated TBSV virion R N A species (Quintero etal, 1988) or hybrid-selected T B S V R N A species (Hayes et al, 1988) demonstrated that both genomic and subgenomic RNAs may act as templates for the expression of TBSV proteins in rabbit reticulocyte lysates. The startpoints for CyRSV (Grieco et al, 1989a), TBSV-cherry (Hillman et al, 1989) and C N V (Rochon, D. M . , personal communication) subgenomic RNAs have been mapped using primer extension analyses. Fig. 4.2 shows a comparison of the nucleotide sequences upstream of the putatve A U G codons for the 41, 21 and 20 kDa ORFs of C N V to analogous regions in the CyRSV and TBSV-cherry genomes. Although extensive similarity exists between these regions, some dissimilarity is observed, particularly in the region upstream of the 41 kDa ORF in all three viruses. The differences in these upstream regions may account for the differential translation of these RNAs in rabbit reticulocyte lysates and wheat germ extracts. Since 5' noncoding sequences likely play a negative regulatory role in translation (Kozak, 1986d), it is possible that sequences present in C N V R N A , but not in CyRSV or T B S V R N A might inhibit translation in rabbit reticulocyte lysates. 4.3 The putative 92 kDa readthrough protein The 92 kDa protein predicted from nucleotide sequence data is implicated as the C N V replicase on the basis of amino acid sequence similarity with other viral replicases (Rochon & Tremaine, 1989). The observed similarity is 3' of the 33 kDa amber termination codon, in particular, in a region surrounding and including a glycine-aspartate-aspartate tripeptide which has been found in the replicases and putative replicases of many plant and animal viruses. These observations suggest that the region downstream of the amber codon is expressed. The most obvious means for the expression of this portion of the genome is via readthrough translation since a subgenomic Chapter 4. Discussion C N V 33 kDa TBSV 33 kDa CyRSV 33 kDa C N V 41 kDa TBSV 41 kDa CyRSV 41 kDa C N V 21/20 kDa TBSV 21/20 kDa CyRSV 22/19 kDa G A U A A A U U G U A A C U U C C A G U A A A C G A - C G A C A U G G A U A A A U U G U A A C U U C C A A C A A A U A A G C G A C A U G G A U A A A U U G U A A C U U C C U G C A A A U A A G C A A - A U G — • G A C C A A G C A A A C A C A A A C A C A - A U G G A C C A A G A A U A C A C A C A C G C A G G A U A G A C A C A U G G A C C A A A U A C A C A - A U G * * * * * * * * * *(21kDa) - K J A A U C U A A C C A A U U C A U G G A A C A A G A C C A G U U C A U G G A A C C U A A C C A U U U C A U G (21kDa)* * * * * * * * * * * * * * * * * * * * * * * * **(20kDa) A U G G A U A C U G A A U A C G A A C A A G U C A A U A A A C C A U G A U G G A U A C U G A A U A C G A A C A A G U C A A U A A A C C A U G  A U G G A C A C U G A A U A C C A A C A A G U U A A U A A A C C A U G Fig. 4.2 Alignment of the nucleotide sequences preceding the putative initiation codons for C N V , TBSV-cherry and CyRSV proteins. The upstream 30-31 nucleotides of the 33 kDa proteins for the 3 viruses are aligned. Nucleotide sequences upstream of the other proteins are aligned from subgenomic startpoint (denoted by an arrow) to putative A U G initiation codon (shown underlined). The sequences upstream of the 20 kDa (or 19 kDa protein in the case of CyRSV) are aligned from the putative A U G codon of the 21 (or 22) kDa protein to the putative A U G codon for the 20 (or 19) kDa protein, inclusively. Since the template for the C N V 21 and 20 kDa proteins were determined in this study to be the same, it is assumed that they are also the same for analogous proteins in TBSV-cherry and CyRSV. The nucleotides which are the same in all 3 sequences (excluding the A U G codon) are starred. The C N V and CyRSV sequences were obtained from Rochon & Tremaine (1989) and Grieco et al. (1989), respectively. The TBSV-cherry sequence was obtained from Hillman et al. (1987; 1989). The startpoints for the C N V , CyRSV and T B S V subgenomic RNAs were determined by Rochon (personal communication), Grieco et al. (1989a) and Hillman et al. (1989). Chapter 4. Discussion 71 corresponding to this region was not found. [In addition, hybrid-arrested translation using an antisense R N A corresponding to nucleotides 663 through 1599 did not affect the synthesis of C N V in vitro translation products (results not shown).] Readthrough of leaky termination codons is utilized by many R N A viruses for translation of downstream cistrons. One well studied case is that of tobacco mosaic virus (TMV) in which the addition of purified yeast amber suppressor tRNA increased readthrough translation from less than 10% to nearly 70% (Pelham, 1978). Similarly, the addition of calf liver tRNA in rabbit reticulocyte lysates resulted in the synthesis of the 90 kDa putative replicase of TBSV (Quintero et al., 1988). Synthesis of the predicted 92 kDa protein of C N V was not observed, even with the addition of either calf liver tRNA in rabbit reticulocyte lysates or of wheat germ tRNA in wheat germ extracts (results not shown). The addition of supraoptimal concentrations of magnesium ions, which resulted in increased readthrough in T M V (Pelham, 1978), also failed to induce readthrough of the predicted C N V 92 kDa protein (see Fig. 5b). The lack of synthesis of the 92 kDa protein might be due to an insufficient supply of tRNA or to a lack of the appropriate suppressor tRNA in the tRNA preparation used. The latter suggestion is likely in the case of wheat germ extracts since tyrosine-specific tRNAs, which from tobacco have been shown to suppress termination at amber codons (Beier et al., 1984a,b), are highly modified in wheat germ and are not able to suppress (Bienz & Kubli , 1981). Alternatively, the R N A sequence surrounding the termination codon (Pelham, 1978) might reduce the efficiency of readthrough of the C N V amber termination codon. 4.4 Translation of genomic-length synthetic CNV RNA Translation of synthetic genomic-length C N V transcripts resulted in the synthesis of 33, 41, 21 and 20 kDa proteins or, alternatively, only a 33 kDa protein (Fig. 3.16). The latter observation is Chapter 4. Discussion 72 one that conforms to the restrictions predicted for eukaryotic ribosomes in only translating 5' cistrons and not translating internal cistrons-directly (Kozak, 1978). The production of four proteins that comigrated with the four major in vitro translation products directed by C N V virion R N A suggests the occurrence of aberrant protein synthesis due either to in vitro translation conditions or to the utilization of an alternative translation strategy (discussed below). 4.4.1 Translation conditions 4.4.1.1 Fidelity of translation in rabbit reticulocyte lysates In vitro translation studies conducted in rabbit reticulocyte lysates have raised some questions regarding the fidelity of initiation of translation in this system (Gaillione et al., 1981; Parker et al., 1986). Kozak (1986c) has suggested that translation in reticulocyte lysates may result in the synthesis of products arising from internal locations due to reticulocyte ribosomes initiating inappropriately at each successive A U G codon. Recent studies have indicated that high fidelity translation in rabbit reticulocytes may, however, be obtained under certain conditions (Dasso & Jackson 1989). These conditions include the use of: (1) potassium ions in the form of potassium chloride rather than potassioum acetate; (2) potassium ion concentrations of ca. 20 m M above the level which gives maximum protein synthesis; (3) capped rather than uncapped mRNAs and (4) low mRNA concentrations. It was suggested that increasing mRNA levels may out-titre the capacity of some endogenous reticulocyte factors that are critical for accurate translation and lead to increasingly leaky scanning (Dasso & Jackson, 1989). The results of translation of C N V virion R N A in rabbit reticulocyte lysates, however, did not indicate a lack of fidelity of translation since only a single translation product was produced by C N V virion R N A in this system. Chapter 4. Discussion 4.4.1.2 Translation conditions in wheat germ extracts 73 In contrast to the situation for rabbit reticulocyte lysates, wheat germ ribosomes have been suggested to initiate only at authentic, 5' proximal A U G codons (Kozak & Shatkin, 1977; Kozak, 1986c). However, wheat germ extracts have also been shown to be sensitive to reaction conditions (Kozak, 1979), and might undergo aberrant translation if translation conditions are not adjusted properly as has been shown for rabbit reticulocytes (Dasso & Jackson, 1989). Although there is no indication of aberrant translation of C N V virion R N A in wheat.germ extracts, the results obtained from translation of full-length positive sense synthetic transcripts in wheat germ extracts (see section 3.5.2) warrants a closer investigation of translation conditions. The translation conditions recommended to achieve high fidelity translation in rabbit reticulocyte lysates, while likely different from those for wheat germ extracts, may have some general application to the present study. For accurate translation in rabbit reticulocyte lysates, potassium acetate and not potassium chloride was recommended as the source of potassium ions (see above). This recommendation may not apply to wheat germ extracts since the use of potassium acetate in all presently available commercial wheat germ extract systems has not appeared to have led to aberrant translation. Also, the endogenous capping activity present in wheat germ extracts might preclude the use of precapped R N A for translation in wheat germ extracts as was recommended for translation in rabbit reticulocyte lysates. However, in the present study, the effect of high mRNA concentrations for translation in wheat germ extracts should be investigated since in rabbit reticulocyte lysates, high levels resulted in increased leaky scanning (Dasso & Jackson, 1989) (discussed below). In addition to the conditions investigated by Dasso & Jackson (1989), the presence or absence of certain translational factors or differences in ribosomal age might possibly contribute to the variation observed in the translation products directed by C N V synthetic positive sense transcripts in different batches of wheat germ extracts. Chapter 4. Discussion 4.4.2 Alternative translation mechanisms 74 Alternative mechanisms that enable ribosomes to reach internally located A U G codons have been proposed which might explain the production of four proteins from translation of synthetic genomic-length C N V transcripts. These include: (1) leaky scanning (Kozak, 1981); (2) termination-reinitation (Kozak, 1984c) and (3) internal initiation (Pelletier & Sonenberg, 1988). The first two of these mechanisms, leaky scanning and termination-reinitation, are essentially modifications of the scanning hypothesis which were proposed largely to accommodate cases of dual initiation in viral mRNAs (Kozak, 1986c). Leaky scanning operates when the 5' proximal A U G codon lies in an unfavorable context for initiation by eukaryotic ribosomes (Kozak, 1981). In such cases, some ribosomes may not initiate at the first A U G codon but scan past it and initiate instead at a second downstream A U G codon. This second A U G codon usually, but not always, lies in a stronger context than the first A U G codon. Termination-reinitiation occurs i f an ORF is preceded by an upstream A U G codon which lies in a favorable context but is followed by an in-frame termination codon (Kozak, 1984c). In these cases, the ribosomes may reinitiate translation at a downstream A U G codon after translating the upstream 'minicistron'. Although the efficiency of reintiation at downstream cistrons is usually low, efficiency may be improved as the intercistronic sequence is lengthened (Kozak, 1987b). Direct binding of ribosomes to internal sequences to enable access to internally located cistrons has also been proposed for viral RNAs. This has been shown to occur in naturally uncapped poliovirus R N A where ribosomes bind to an internal sequence within the 5' noncoding region to initiate translation by a cap-independent mechanism (Pelletier & Sonenberg, 1988). In addition to the above mechanisms, a hybrid mechanism involving both ribosome scanning and direct internal ribosome binding has been proposed. In this hybrid mechanism, 98% of the ribosomes scan from the 5' end of the mRNA to locate A U G codons and the remaining 2% bind to A U G codons directly without previous scanning (Kozak, 1986d). It is interesting that many of the deviations from the generally accepted scanning Chapter 4. Discussion 75 model for initiation of translation are derived from studies of viral R N A . Insufficient data, however, has been collected to speculate as to whether these or any other novel mechanisms operate during the translation of either authentic or synthetic C N V RNA. 4.5 The 0.9 kb subgenomic RNA is a bifunctional mRNA The work described in this thesis demonstrates that C N V virion RNA contains a subgenomic RNA species that can direct the synthesis of two translation products in vitro. Northern blot analysis of sucrose fractionated C N V virion R N A showed that several discrete-sized less than full-length 3' co-terminal virus-specific RNA species are present in virion R N A preparations. Translation of sucrose gradient fractions containing subgenomic RNAs of ca. 1.0 kb demonstrated that proteins of ca. 21 and 20 kDa were synthesised by this template in wheat germ extracts. Similarly, proteins of ca. 21 and 20 kDa were produced in wheat germ extracts from a single synthetic positive sense transcript corresponding to the 3' terminal coding region of the C N V genome. Identification of the precise genomic locations of the subgenomic RNAs on the C N V genome by primer extension analysis indicated that only one subgenomic species in the 1.0 kb size range was present (Rochon, D. M . , personal communication). These results therefore suggest that the 1.0 kb subgenomic mRNA is bifunctional, ie. capable of encoding two distinct proteins from the same mRNA molecule. Further work will be required to demonstrate that the 20 kDa protein is not related to the 21 kDa protein and to determine whether both proteins are produced in vivo. Since these 3' terminal nested ORFs are conserved among all tombusviruses that have been sequenced (Hillman et al, 1989; Grieco et al, 1989a; Rochon & Tremaine, 1989), these coding regions are very likely functional in vivo. A number of other cases in which two proteins are synthesized from extensively overlapping reading frames have been discovered in animal virus RNAs and are described by Chapter 4. Discussion 76 Kozak (1986c). Turnip yellow mosaic virus (Morch et al, 1988; Weiland & Dreher, 1989) and members of the luteovirus group (eg. B Y D V ; Miller etal, 1988; Murphy etal, 1989) have also been suggested to encode two proteins from overlapping or nested open reading frames. The bifunctional mRNAs described by Kozak (1986c) conform best to translation via the leaky scanning mechanism. This mechanism is also the most likely for translation of the 21 and 20 kDa C N V in vitro translation products since the putative A U G codon for the 21 kDa protein is in suboptimal context while that for the 20 kDa protein is in near optimal context for translation by eukaryotic ribosomes (see Table II). If this strategy of expression is utilized, it might serve not only to allow ribosomes access to the second coding region but also to regulate the amount of protein produced. The potential for the existence of bifunctional plant viral RNAs may consitute further evidence for the compact and versatile nature of plant virus genomes. Bibliography [I] Ahlquist, P., Luckow, V. , and Kaesberg, P. (1981). Complete nucleotide sequence of brome mosaic virus R N A 3. Journal of Molecular Biology, 153:23-38. [2] Azad, A . A . and Deacon, N . J. (1980). The 3'-terminal primary structure of five eukaryotic 18S rRNAs determined by the direct chemical method of sequencing. 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