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Bombyx mori Nucleopolyhedrovirus p26 Is Associated with Viral Late Stage Replication.

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.

SUBMITTER: Ge JQ

PROVIDER: S-EPMC8396461 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A DNA-binding protein (DBP) [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05), indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (p<0.01). The transcriptional level of dbp-ko-Bacmid early gene lef-3, ie-1, dnapol, late gene vp39 and very late gene p10 were statistically significantly lower than dbp-re-Bacmid and wtBacmid (p<0.01). The results presented are based on Western blot analysis, which indicated that the lack of dbp gene would lead to low expressions of lef3, vp39, and p10. In conclusion, dbp was not only essential for early viral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle.

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Bombyx mori Nucleopolyhedrovirus p26 Is Associated with Viral Late Stage Replication.

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