Bombyx mori Nucleopolyhedrovirus p26 Is Associated with Viral Late Stage Replication

SIMPLE SUMMARY: p26 is conserved among all completely sequenced Lepidoptera baculoviruses, and some baculoviruses even have two copies of p26 (p26a and p26b), which suggested that p26 may have a basic role in the baculovirus infection cycle. p26 may be transcribed by the host RNA polymerase II in both early and late infection. Here, protein analyses showed that Bombyx mori nucleopolyhedrovirus (BmNPV) p26 levels were very low amounts during the early phases of infection, which then increased and then declined during the late infection phase. Thus, BmNPV p26 might have functions in both the cytoplasm and nucleus. Previous p26 knockout study indicated that p26 may be an auxiliary gene that does not influence key aspects of viral replication or transmission, and RNAi response to p26 may somewhat regulate viral replication. Therefore, in order to maintain low p26 expression and measure BmNPV p26 function, a RNAi-based knockdown method was chosen. The results indicated that high p26 expression during the middle interval is necessary for late-stage viral replication. Since p26 is not essential for baculovirus replication and transmission, it would be interesting to investigate whether p26 is involved in regulating host innate immune response. ABSTRACT: Bombyx mori nucleopolyhedrovirus (BmNPV) p26 is conserved among all Lepidoptera baculoviruses that have been completely sequenced thus far, and some baculoviruses even have two copies of p26, which suggested that p26 may play an important role in the virus infection cycle. This study aimed to characterize BmNPV p26. We found that BmNPV p26 transcripts were detectable as early as 3 h post-infection (hpi), and the transcript levels rapidly increased starting from 12 hpi. Western blot analysis using an anti-p26 polyclonal antibody demonstrated that the corresponding protein was also detectable from 6 hpi in BmNPV-infected cell lysates. Immunofluorescence analysis demonstrated that p26 was mainly dispersed in the infected cell cytoplasm, whereas the over-expressed fusion protein EGFP-p26 also accumulated in the nucleus. These results indicated that p26 is an early BmNPV gene and has functions both in the cytoplasm and the nucleus. RNAi-based knockdown of p26 could produce infectious virus and normal-appearing virions but decreased budded virus (BV) production in BmNPV-infected cells at 72 hpi. Moreover, the results of further quantitative PCR (Q-PCR) analysis indicated that the gp64 and p74 transcripts levels decreased significantly. These results indicated that BmNPV p26 may be associated with BmNPV replication during the late infection stage.