The AcMNPV pp31 gene is not essential for productive AcMNPV replication or late gene transcription but appears to increase levels of most viral transcripts.
The pp31 gene of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes a phosphorylated DNA binding protein that associates with virogenic stroma in the nuclei of infected cells. Prior studies of pp31 by transient late expression assays suggested that pp31 may play an important role in transcription of AcMNPV late genes [Todd, J. W., Passarelli, A. L., and Miller, L. K. (1995). Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J. Virol. 69, 968-974] although genetic studies of the closely related BmNPV pp31 gene suggested that pp31 may be dispensable [Gomi, S., Zhou, C. E., Yih, W., Majima, K., and Maeda, S. (1997). Deletion analysis of four of eighteen late gene expression factor gene homologues of the baculovirus, BmNPV. Virology 230 (1), 35-47]. In the current study, we examined the role of the pp31 gene in the context of the AcMNPV genome during infection. We used a BACmid-based system to generate a pp31 knockout in the AcMNPV genome. The pp31 knockout was subsequently rescued by reinserting the pp31 gene into the polyhedrin locus of the same virus genome. We found that pp31 was not essential for viral replication although the absence of pp31 resulted in a lower viral titer. Analysis of viral DNA replication in the absence of pp31 showed that the kinetics of viral DNA replication were unaffected. An AcMNPV oligonucleotide microarray was used to compare gene expression from all AcMNPV genes in the presence or absence of pp31. In the absence of pp31, a modest reduction in transcripts was detected for many viral genes (99 genes) while no substantial increase or decrease was observed for 43 genes. Transcripts from 6 genes (p6.9, ORF 97, ORF 60, ORF 98, ORF 102 and chitinase) were reduced by 66% or more compared to the levels detected from the control virus. Microarray results were further examined by qPCR analysis of selected genes. In combination, these data show that deletion of the pp31 gene was not lethal and did not appear to affect viral DNA replication but resulted in an apparent modest down-regulation of a subset of AcMNPV genes that included both early and late genes.
Yamagishi
Junya
J
Boyce Thompson Institute at Cornell University, Ithaca, NY 14853, USA.
Burnett
Erik D
ED
Harwood
Steven H
SH
Blissard
Gary W
GW
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009036:Moths / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D014764:Viral Proteins; C078031:phosphoprotein pp31, Baculovirus