Colocalization of Baculovirus IE-1 and Two DNA-Binding Proteins, DBP and LEF-3, to Viral Replication Factories

Abstract

We have recently identified a DNA-binding protein (DBP) from the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) which can destabilize double-stranded DNA (V. S. Mikhailov, A. L. Mikhailova, M. Iwanaga, S. Gomi, and S. Maeda, J. Virol. 72:3107–3116, 1998). DBP was found to be an early gene product that was not present in budded or occlusion-derived virions. In order to characterize the localization of DBP during viral replication, BmNPV-infected BmN cells were examined by immunostaining and confocal microscopy with DBP antibodies. DBP first appeared as diffuse nuclear staining at 4 to 6 h postinfection (p.i.) and then localized to several specific foci within the nucleus at 6 to 8 h p.i. After the onset of viral DNA replication at around 8 h p.i., these foci began to enlarge and eventually occupied more than half of the nucleus by 14 h p.i. After the termination of viral DNA replication at about 20 h p.i., the DBP-stained regions appeared to break down into approximately 100 small foci within the nucleus. At 8 h p.i., the distribution of DBP as well as that of IE-1 or LEF-3 (two proteins involved in baculovirus DNA replication) overlapped well with that of DNA replication sites labeled with bromodeoxyuridine incorporation. Double-staining experiments with IE-1 and DBP or IE-1 and LEF-3 further confirmed that, between 8 and 14 h p.i., the distribution of IE-1 and LEF-3 overlapped with that of DBP. However, IE-1 localized to the specific foci prior to DBP or LEF-3 at 4 h p.i. In the presence of aphidicolin, an inhibitor of DNA synthesis, immature foci containing IE-1, LEF-3, and DBP were observed by 8 h p.i. However, the subsequent enlargement of these foci was completely suppressed, suggesting that the enlargement depended upon viral DNA replication. At 4 h p.i., the number of IE-1 foci correlated with the multiplicity of infection (MOI) between 0.4 and 10. At higher MOIs (e.g., 50), the number of foci plateaued at around 15. These results suggested that there are about 15 preexisting sites per nucleus which are associated with the initiation of viral DNA replication and assembly of viral DNA replication factories.

Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

“…Similar to BmN cells infected with BmNPV (Okano et al, 1999), DBP appeared to be about twice as abundant as LEF-3 in Sf9 cells infected with AcMNPV. The Sf9 cells at 20 hpi contained approximately 3 × 10 7 molecules of DBP and 1.5 × 10 7 molecules of LEF-3.…”

“…Firstly, DBP appeared to be less abundant in Sf9 cells infected with AcMNPV than in BmN cells infected with BmNPV. A quantitative Western blot analysis performed as previously described (Mikhailov et al, 1998) showed that an average Sf9 cell infected with AcMNPV contains approximately 3 × 10 7 molecules of DBP at 20 hpi, significantly lower than the amount of DBP (7 × 10 7 molecules) in the BmN cell infected with BmNPV at 14 hpi (Okano et al, 1999). Secondly, the treatment with high salt released only a minor fraction of DBP from the nuclei.…”

“…It was not found among the structural proteins from budded or occlusion-derived virions (Okano et al, 1999). In nuclei, both LEF-3 and DBP colocalize with IE-1 in viral replication foci (Okano et al, 1999;Mainz et al, 2002;Nagamine et al, 2006), that progressively enlarge during the course of infection eventually occupying more than half of the nucleus (Okano et al, 1999). The resulting electron dense structure is called the virogenic stroma.…”

“…Baculoviruses are infectious for invertebrates and produce two types of virions, budded viruses (BV) that spread the infection within insects and are also infectious for cultured insect cells and occlusion-derived virions (ODV) that mediate transmission between insects. Replication of baculovirus genomes is initiated in a limited number of nuclear foci in close proximity to nuclear domain 10 (ND10) or promyelocytic leukemia protein (PML) domains (Okano et al, 1999;Mainz et al, 2002). Although a rolling circle model was proposed for baculovirus replication (Leisy and Rohrmann, 1993;Oppenheimer and Volkman, 1997), the actual mechanism of replication remains unclear.…”

Detection and Analysis of Early Genes of White Spot Syndrome Virus in Penaeid Shrimp

“…Till date no effective protective method has been developed against WSSV infection, because WSSV are easily spread in the environment of shrimp farms (soil, water, and other materials used for the farm maintenance) [11]. Therefore early detection of WSSV is very useful to shrimp industries.…”