AcMNPV PKIP is required for hyperexpression of very late genes and involved in the hyperphosphorylation of the viral basic protein P6.9.

A previous study showed that a mutation in Autographa californica multiple nucleopolyhedrovirus pkip (ac24) led to severe defects in progeny budded virion production and very late gene transcription at non-permissive temperature. To dissect the underlying mechanism, our early study showed that PKIP is associated with nucleocapsid of budded virion and involved in nucleocapsid assembly. However, how pkip affects very late gene transcription has not been determined. In the present study, double-stranded RNA was used to silence pkip expression during virus infection, resulting in the significant reduction of occlusion body production and polyhedrin expression. To find out whether PKIP regulates polyhedrin expression by affecting the transcription of other viral genes for very late gene expression, a comparative transcriptome analysis of viral genes was performed by RNA sequencing and the result showed that silencing pkip specifically down-regulated transcription of very late genes, while the transcription patterns of the viral genes associated with very late gene transcription were not affected. Since PKIP was reported to interact with and stimulate the activity of virus-encoded protein kinase PK1 and PK1 was involved in the hyperphosphorylation of viral basic protein P6.9, which was required for the maximal hyperexpression of very late genes, we sought to determine the association between PKIP and P6.9. Further experiments showed that PKIP interacted with P6.9 during virus infection, and the deletion of pkip resulted in decreased hyperphosphorylation of P6.9. Taken together, our results indicated that PKIP is involved in hyperphosphorylation of P6.9, which in return maybe required for hyperexpression of very late genes.

AcMNPV PKIP is associated with nucleocapsid of budded virions and involved in nucleocapsid assembly.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf24 (pkip) is a unique Alphabaculovirus gene. A previous study showed that a temperature-sensitive mutant of AcMNPV with a mutation in pkip displayed severe defects in progeny budded virion (BV) production and very late gene transcription, however, the underlying mechanism has not been determined. To investigate the function of pkip in the baculovirus replication cycle, we constructed a pkip-knockout AcMNPV bacmid in this study. Our results showed that deletion of pkip led to significant reduction of BV production, while the synthesis of viral DNA and the transcription of early and late genes were not affected. Further examination by transmission electron microscopy analysis showed that deletion of pkip resulted in the formation of massive electron-lucent tubular structures in the nucleus of the infected cells, along with some normal electron-dense nucleocapsids. The pkip-encoded protein PKIP could be detected at late phase during infection and was distributed in both the cytoplasm and nuclei of viruses-infected cells, with a ring pattern near the inner nuclear membrane and punctate distribution in the virogenic stroma area. Biochemical fractionation of virions into nucleocapsid and envelop components showed that PKIP was associated with the nucleocapsid fraction of BV. Taken together, our results indicated that PKIP is associated with nucleocapsids of BV and involved in nucleocapsid assembly, which contributes to the optimal production of BV.

The Autographa californica multiple nucleopolyhedrovirus ac110 gene encodes a new per os infectivity factor.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac110 gene is especially conserved in lepidopteran-specific baculoviruses and is uncharacterized. To investigate the role of ac110 in the baculovirus life cycle, an ac110-knockout (vAc110KO) and a repair (vAc110:HA) viruses were constructed in this study. Budded virion production and occlusion body formation were unaffected in vAc110KO-transfected or infected Sf9 cells. Intrahemocoelic injection of budded virions of vAc110KO killed Trichoplusia ni larvae as efficiently as the repair or the wild-type viruses. However, per os inoculation of occlusion bodies of vAc110KO failed to establish infection in T. ni larvae, while the repair virus was as efficient as the wild-type virus. Treatment with calcofluor white, a reagent that damages the peritrophic membrane, did not rescue the oral infectivity of vAc110KO. These results suggested that Ac110 is a new per os infectivity factor that may play a role after occlusion-derived virions pass through the peritrophic membrane during oral infection.