Functional analysis of the P34 regulatory gene of the baculovirus OpMNPV
Creators: name: Huijskens, Ilse
Publisher: name: University of British Columbia
Year: 2010

description: World crops losses attributable to insect pests present a considerable economic problem. Baculoviridae represent a great alternative to pesticides since they are capable of a high speed to kill and do not threaten non-target organisms or the environment. Baculoviruses also exhibit a narrow host range which allows them to infect only a single or a small group of insects. However, the mechanisms behind host range determination by baculoviruses have not been identified to date. Furthermore, no host proteins have been reported to be involved in insect susceptibility, while several viral proteins have been identified to have a function in host range, virulence a and virus replication. This study investigates the interaction between the Orgyia pseudotsugata nucleopolyhedrovirus (OpMNPV) regulatory protein P34 and host proteins and determines the role of host proteins in viral replication. The p34 gene is a homolog of AcMNPV pe38 but is unique as it is the only baculovirus gene that is known to produce two gene products by alternative transcriptional initiation at early and late times post-infection. The early 1.1 kb p34 transcript initiates from an early promoter and is translated as a 34 kDa protein called P34. The late transcipt is a 0.7 kb mRNA expressed from a late promoter that is internal to the p34 ORF and is translated as a 20 kDa N-terminally truncated protein named P34Δ. P34 possesses a number of distinct protein domains including an N - terminal basic region, a RING finger, acidic, glutamine rich and leucine zipper domains. P34A consists of the acidic, glutamine rich and leucine zipper domains. Previous studies classified P34 as a regulatory protein that activates viral transcription and replication; however the role of P34Δ has yet to be determined. This study presents new data about the role and mode of action of P34 and P34Δ during OpMNPV infection. More precisely, we show that P34 can function as an E3 ubiquitin ligase. In addition, P34 forms a homodimer, which is dependent upon all domains, and also interacts with P34Δ to form heterodimers. Confocal microscopy indicated that P34 is distributed in punctate nuclear foci in Ld652Y cells and colocalizes with P34Δ. Studies were also performed to identify the interactions of P34 with cellular factors. A yeast 2-hybrid screen was conducted with P34 (as bait) and a cDNA library of OpMNPV infected Ld652Y cells to identify proteins that interact with the P34 baculovirus regulatory protein. To date this is the first study that identified interactions between cellular proteins and a baculovirus protein and showed that they are required for infection. Two cellular proteins, LdUEV and LdSUGl, which are homologs of proteins known to participate in the ubiquitination pathway, were shown to bind to P34 but not P34Δ. The role of these two cellular proteins in the OpMNPV infection cycle was further analysed using gene silencing (RNAi) studies. This study extends the basic understanding of how baculoviruses interact with the host cell, which is essential for determining the molecular basis for virulence and viral host range.