Functional Analysis Of Helicoverpa Armigera Nucleopolyhedrovirus (HearNPV) Orf81

Helicoverpa armigera nucleopolyhedrovirus （HearNPV） is one of the most studiedmodel virus in Group II baculoviruses. HearNPV orf81, ha81, is489nt long and encods aprotein of approximately19.0kDa. It has a typical late gene promoter motif, ATAAG, about129nt upstream from the translation start codon. Analyses with the InterProScan programrevealed that HA81shares31%–70%amino acid identity with its homologs in baculoviruses,and these homologs constitute the DUF682baculovirus protein family, which the functionsare unknown. HzAM1cells were infected with wildtype HearNPV，ha81-specific transcriptwas detected at12hours post infection (hpi) and can be detected until96hpi while the proteinwas detected from12to96hpi by Western blot analysis. These results show that ha81was alate gene. Immunofluorescence analysis showed that the protein mainly localizes in thecytoplasm.To clarify the function of ha81in the HearNPV life cycle, ha81-knockout and repairedbacmids were generated via Red homologous recombination. HzAM1cells were transfectedwith HaBacHZ8-PG, HaBac Δ81-PG, and HaBacRep81-PG bacmid DNA, and then thebudded virus (BV) production of the ha81-null virus was completely blocked. While thevHaBacRep81-PG showed a steady increase in BV production similar to that of thevHaBacHZ8-PG. Electron microscopy showed that the deletion of ha81had no effect onnucleocapsid assembly. Thus, ha81is required for the egress of nucleocapsids from thenucleus to the cytoplasm, which explains why BV production was completely blocked.Quantitative PCR analysis showed that viral DNA replication was initiated at the sametime among the three viruses, from0to12hours post transfection (hpt), and showed similarpatterns of increase by24hpt. However, the viral DNA levels differed greatly between theHaBacΔ81-PG-and HaBacRep81-PG-transfected cells and between the HaBacΔ81-PG-andHaBacHZ8-PG-transfected cells at24–96hpt, which could be attributable to secondaryinfections in the HaBacHZ8-PG-and HaBacRep81-PG-transfected cells. Consequently, qPCRanalysis showed that ha81is not required for viral DNA replication.To understand whether the transcription of other genes was affected by the deletion of ha81, the transcription of several well-characterized viral genes was investigated in theha81-knockout HearNPV mutant. No obvious changes were observed at the transcriptionlevel, except in the odv-e25gene downstream from ha81. Further studies revealed that at leasttwo different transcripts were existed for odv-e25, and one of the transcripts contained ha81was disrupted by the ha81knockout. The transcription of odv-e25in the vHaBacΔ81-PG waspromoted by its independent promoter.In conclusion, ha81is an essential late gene required for BV production, which involvedin the egress of nucleocapsids from the nucleus, but ha81deletion does not affect viral DNAreplication and transcription but blocks BV production.