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Objective: ORF134 of Bombyx mori nucleopolyhedrovirus (BmNPV) is a homolog of Autographa californica multiple NPV ORF5, but its function is unknown so far. The aim of this study is to characterize BmNPV ORF134 (Bm134). Methods: Transcription, protein expression and subcellular localization of Bm134 in BmN cells and silkworm midguts were analyzed using real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence, respectively. Results: Both the transcription and protein expression of Bm134 were not detected until 24 h post-infection (p.i.) by RT-PCR and Western blot, indicating that Bm134 is a late gene. Western blot revealed that Bm134 encodes an expected 12.4-kDa structural protein that is associated with occlusion-derived virus (ODV), not with budded virus. Immunofluorescence analysis showed that the Bm134 was first detected in the cytoplasm 24 h p.i. and then transported to the nucleus during later infection. Transcripts of Bm134 and the corresponding protein were only detected 48-72 h p.i. in BmNPV-infected larvae of 306, a highly BmNPV-susceptible silkworm strain, but not in the NB strain that is resistant to BmNPV infection. Conclusion: Taken together, our data suggest that Bm134 is a late gene of BmNPV and may function as an ODV structural protein. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

It is unclear how, or to what extent, baculovirus DNA that has been damaged by ultraviolet (UV) light is repaired during infection and replication. In our previous study, expression of Bombyx mori nucleopolyhedrovirus (BmNPV) ORF Bm65, a homolog of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac79, correlated with decreased inactivation of virus by UV irradiation. In the current study, we accumulated more evidence pointing to a role for Bm65 in repair of UV-induced DNA damage. The localization of Bm65 was studied using enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bm65 expression plasmid. The results indicate that Bm65-EGFP accumulates in the nucleus. A host cell reactivation assay showed that Bm65 significantly increased the expression of UV-damaged mCherry reporter gene. An assay measuring cyclobutane pyrimidine dimers (CPDs) in UV-irradiated BmN cells found that CPD quantity was decreased in cells transfected with a Bm65 expression plasmid. We also showed that after UVC treatment, the viability of Bm65-transfected cells was higher than that of egfp-transfected cells. These results suggest that Bm65 may be involved in the repair of baculovirus DNA that has been damaged by UV light. [ABSTRACT FROM AUTHOR]

Silkworms are usually susceptible to the infection of Bombyx mori (B. mori) nucleopolyhedrovirus (BmNPV), which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells) of BmNPV-resistant strains (NB and BC8), the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi), the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses. [ABSTRACT FROM AUTHOR]