UBC Theses and Dissertations
Analysis of the orgyia pseudotsugata multiple nucleopolyhedrovirus IE1 acidic activation domain role in viral DNA replication Pathakamuri, Joseph Aja
Baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) transient replication assays have previously identified six genes as essential iel, lef-1, lef-2, lef-3, DNA pol, helicase; three genes as stimulatory ie2, p34, iap-1; and two types of replication origins hrs and non-hrs. One of these essential proteins IE1, has an acidic activation domain (AAD) at the N-terminus that is required for transcriptional transactivation but its role in viral DNA replication was unknown. In this study we have determined that the AAD is also essential for DNA replication. Unlike transcriptional activation, the AAD cannot be replaced with the AcMNPV IE1 and herpesvirus VP 16 AADs for replication. Deletion analysis of the OpMNPV IE 1-AAD revealed the presence of separate sub-domains for replication and transcriptional activation. By substituting OpMNPV lef-1, -2, -3 and helicase proteins (part of the putative replisome) with corresponding AcMNPV factors, the inactive OpMNPV IE1 chimeric protein IE 1-Ac AD, which contains the AcMNPV IE1 AAD, was functional for replication activation. This suggests that AADs interact with viral replication factors in order to maintain viral specificity in replication. The IE 1-Ac AD specificity for the AcMNPV replisome was found to be similar to the native AcMNPV IE1, thus demonstrating that by changing the AAD the specificity of the protein changes. Further studies showed that the role of stimulatory factor OpMNPV IE2 in replication was to maintain the specificity between AAD and the replisome. Absence of IE2 allowed replication from non-specific AAD and replisome but presence of IE2 active replication was observed only between specific AAD and replisome. AcMNPV non-hr origin analysis indicated that it was unable to allow for replication with OpMNPV IE1 and OpMNPV replication proteins; however substituting with AcMNPV LEF-3, POL and HELICASE, resulted in replication. This result reveals the presence of specific interactions between origins and replication factors. Our studies on OpMNPV IEO, the only spliced gene of IE1, showed that IEO is functionally active for replication and can replace IE1. We also performed initial functional analysis of OpMNPV EXONO. To date there is no information on OpMNPV EXONO function other than the fact that it contributes 23aa to the N-terminus of IEO. Our study shows EXONO has a conserved novel ring finger and is expressed as a late gene. However, our preliminary research could not identify any function attributable to EXONO.
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