Characterization of a unique gene of opMNPV and analysis of regulatory motifs involved in basal activity and ie2 trans-activation

Baculovirus early genes are transcribed by host cell transcription factors and serve to regulate the infectious cycle via a cascade effect, and may also play a significant role in viral host range. Opep-2 is one of two unique early genes in the IE1 gene region of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). Opep-2 is transcribed from 1-36 hours post infection as three transcripts, with a single consensus baculovirus early gene initiation motif and three termination sites, corresponding to transcripts of 1.1, 0.98 and 0.88 kb. Opep-2 is regulated by a promoter region that has several recognizable motifs, including a pair of previously unidentified 13 base direct repeats that serve as regulatory motifs, as well as GATA and CACGTG motifs. Transient expression assays in two insect cell lines - Ld652Y and Sf9 - have indicated different minimal promoter requirements for activation by host cell nuclear factors and for trans-activation by the viral transcription factor IE2. This is the first study to investigate the sequence requirements for IE2 trans-activation. No specific sequences are absolutely required for trans-activation by either cell line. Deletion viruses lacking the opep-2 ORF have indicated that opep-2 is not required for infection in tissue culture, and its absence has no effect on virus production or infectivity in either tissue culture or in insects susceptible to the wild type virus. The deletion of opep-2 from the genome has resulted in a change in plaque morphology, and transmission electron microscopy studies have indicated that cells infected with the deletion virus do not lyse as quickly as WT infected cells.Land and Food Systems, Faculty ofGraduat

Characterization of a Unique OpMNPV-Specific Early Gene Not Required for Viral Infection in Tissue Culture

Abstractopep-2is anOrgyia pseudotsugatamulticapsid nucleopolyhedrovirus (OpMNPV) early gene in theie1–ie2gene region for which there is no homolog in either the archetype virus,Autographa californicaMNPV, orBombyx moriNPV.opep-2is transcribed immediately upon infection as three mRNAs which initiate from a early gene motif (TATA-N27-CAGT). The expression of multiple transcripts at very early times postinfection has only been previously described for the baculovirus early geneie1,which produces spliced mRNAs. However, distinct fromie1,the multiple mRNAs ofopep-2are due to multiple termination sites and not splicing. Western blot analysis of steady-state levels of OPEP-2 showed that in OpMNPV-infected Ld652Y cells maximum levels are obtained at 8–12 hr postinfection (p.i.) prior to DNA replication. By 48 hr p.i. OPEP-2 is shut off and is undetectable. To aid in elucidating the function of this OpMNPV-specific gene anopep-2deletion mutant was generated and was compared to wild-type virus to determine if its absence affects viral growth in Ld652Y tissue culture cells