Characterization of 3'→5' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus

Mikhailov, Victor S.; Marlyev, K. A.; Ataeva, J. O.; Kullyev, P; Atrazhev, Alexey

doi:10.1093/nar/14.9.3841

Cited by 33 publications

(26 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast, dnapol transcription was depressed at all the time points tested. The baculovirus-specific DNA polymerase, originally identified by Mikhailov et al in BmNPV, has a 3'→5' exonuclease activity and is involved in the synthesis of the DNA leading strand and lagging strand, thus ensuring the fidelity of DNA synthesis (Mikhailov et al, 1986). In 2005, Vanarsdall et al found that the replication of the viral genomic DNA is inhibited by the knockout of dnapol (Vanarsdall et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

The effect of BM67 gene deletion on Bombyx mori nuclear polyhedrosis virus replication

Shi¹,

Zhang²,

Xie³

et al. 2015

View full text Add to dashboard Cite

Summary. -Homologs of Bombyx mori nuclear polyhedrosis virus (BmNPV) Bm67 gene ORF67 have been found in the genome of all lepidopteran nuclear polyhedrosis viruses, but their function is still not very clear. In order to analyze it we employed a bacmid harboring the complete BmNPV genome including the Bm67 gene and expressing infectious virus (wtBacmid) for the construction of its Bm67-deficient variant (Bm67-KO-Bacmid) using the Red recombination system and the Bm67-repaired variant (Bm67-Re-Bacmid) using the Bac-to-Bac system. By transfecting BmN cells with these bacmids we demonstrated that the Bm67-deficient virus did not generate infectious virus, while the repaired virus restored its infectivity, indicating that the Bm67 gene is essential for the formation of infectious budding virus (BV). Electron microscopy of BmN cells transfected with the abovementioned bacmids showed many mature rodshaped virus particles in both wtBacmid-and Bm67-Re-Bacmid-transfected cells but none in Bm67-KO-Bacmid-transfected ones. Moreover, the real-time RT-PCR showed that the deletion of Bm67 from wtBacmid significantly reduced the levels of viral genomic DNA and transcripts of viral early genes dnapol, ie-1 and lef-3 but not those of transcripts of late gene vp39 and very late gene p10. The finding that the Bm67-deficient virus generated reduced levels of infectious virus and transcripts of early dnapol gene but not those of late genes indicates that the Bm67 gene is essential for BmNPV replication.

show abstract

Section: Discussionmentioning

confidence: 99%

The effect of BM67 gene deletion on Bombyx mori nuclear polyhedrosis virus replication

Shi¹,

Zhang²,

Xie³

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Biochemical analyses revealed the presence of primase activity by *LEF-1, providing evidence for its role as a primase in baculovirus replication that had been predicted earlier. The list of virally encoded enzymes involved in baculovirus DNA replication now includes a DNA primase in addition to the DNA polymerase (35,39,50) and DNA helicase (32,36) characterized previously. A set of similar viral enzymes are required for genome replication by the Herpesviridae, another family of large DNA viruses (7).…”

Section: Discussionmentioning

confidence: 99%

Baculovirus Replication Factor LEF-1 Is a DNA Primase

Mikhailov

Rohrmann

2002

J Virol

View full text Add to dashboard Cite

The baculovirus replication factors LEF-1 and LEF-2 of the Autographa californica multinucleocapsid nucleopolyhedrovirus were overexpressed as fusions containing a hemagglutinin (HA) epitope and a HIS 6 tag using recombinant baculoviruses. LEF-1 was purified to near homogeneity and found to have primase activity in an indirect assay employing Escherichia coli DNA polymerase I (Klenow enzyme) and poly(dT) template. The LEF-1 primase products were also directly characterized by electrophoresis in 20% polyacrylamide-8 M urea gels and agarose gels. Primer synthesis was time dependent, and products of several hundred nucleotides or more were observed from the M13 single-stranded DNA (ssDNA) template. The LEF-1 primase was absolutely dependent on divalent cations (Mg 2؉ ), and optimal activity was supported by 10 mM MgCl 2 . An alkaline pH (8.8 to 9.4) was optimal, whereas monovalent salt (KCl) was inhibitory. Mutation of an invariant aspartic acid in a putative primase domain caused LEF-1 activity to be abolished. Upon ultracentrifugation in glycerol gradients, LEF-1 was found to have a sedimentation coefficient of 3S that is consistent with its being present as a monomer. Elution profiles of LEF-1 and LEF-2 from ssDNA-cellulose and DEAE resin suggested that LEF-2 may bind to both DNA and LEF-1.The Baculoviridae are a large and diverse family of rodshaped, enveloped, occluded viruses that are pathogenic for invertebrates, particularly members of the Insecta. They have been reported from over 600 species, most of which are members of the Lepidoptera, Diptera, and Hymenoptera (34). Two genera of baculoviruses have been characterized, and they include the nucleopolyhedroviruses (NPVs) (47), which have numerous virions within large polyhedron-shaped occlusion bodies, and the granuloviruses (52), which commonly have a single virion within small granular occlusion bodies. Baculovirus genomes consist of double-stranded, circular, supercoiled DNA of 100 to 180 kb, depending on the strain of virus (20). Although evidence suggests that baculovirus genomes may replicate via a rolling-circle intermediate (31,42), the mechanisms of initiation, elongation, processing, and maturation have not been determined.Baculovirus DNA replication has been shown to be associated with discrete replication factories in the nuclei of infected cells (41). In addition, a conserved set of genes that are essential or highly stimulatory for transient DNA replication have been identified for Autographa californica multinucleocapsid NPV (AcMNPV) (26, 33), Orgyia pseudotsugata MNPV (OpMNPV) (reviewed in reference 1), and Lymantria dispar MNPV (44). These include genes encoding a DNA polymerase homolog, a DNA helicase homolog, ie-1, a transactivator of early gene transcription, and late expression factors (LEFs) encoded by lef-1, -2, and -3. The DNA polymerase and DNA helicase homologs were subsequently shown to have activities associated with these enzymes (35,36). In addition, lef-3 encodes a product with the properties of a single-stranded DNA (SSB) bi...

show abstract

“…Pupae of the silkworm B. mori were supplied by Dr P. KuUyev (Institute of Zoology, Ashkhabad, Turkmenistan) shortly after larval-pupal ecdysis. BmNPV was prepared from polyhedra and was injected into the pupae as described previously (Onodera et al, 1965;Mikhailov et al, 1986). Infected and uninfected pupae were incubated at 25 °C and sampled daily for biochemical analysis.…”

Section: Methodsmentioning

confidence: 99%

“…However, little is known about ICSP synthesis in whole insects. We have used silkworm pupae infected with BmNPV as a stock for preparative purification of some ICSPs (Mikhailov et al, 1986;Voronova et al, 1991). Preliminary experiments suggested that early phase pupae can be used successfully for the expression of foreign genes under the control of the promoter of the very late viral genes whose products are abundant late in infection.…”

Section: Introductionmentioning

confidence: 99%

Protein synthesis in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus: resistance to viral infection acquired during pupal period

Mikhailov

Zemskov

Abramova

1992

Journal of General Virology

Self Cite

View full text Add to dashboard Cite

Protein synthesis has been studied in pupae of the silkworm Bombyx mori (Bm) infected with nuclear polyhedrosis virus (BmNPV) at various stages of the pupal period. Nascent proteins were labelled by injection of [3sS]methionine into pupae and then analysed by SDS-PAGE. Temporal regulation of synthesis of infected cell-specific proteins (ICSPs) in pupae was demonstrated by electrophoretic analysis of the proteins labelled at different times post-infection (p.i.). The rate of ICSP synthesis reached a maximum at 4 to 5 days p.i., exceeding the rate of synthesis of cellular proteins in uninfected pupae by about twofold. The viral proteins pl0 and polyhedrin were the most abundant products synthesized late in the infection. Both proteins were found to be associated with the nuclear matrix after fractionation of nuclei from infected pupae. Two virus-induced phosphoproteins, pp35 and ppB, were found to be the major acceptors of labelled phosphate from [~-32p]ATP during in vitro phosphorylation of proteins in pupal homogenates, nuclei and nuclear extracts. These proteins had electrophoretic mobilities comparable to those of structural phosphoproteins of BmNPV virions with Mrs of 35K and 11K to 16K, respectively. The latter polypeptide was identified as the major DNA-binding protein of the virus. The susceptibility of silkworms to BmNPV decreased markedly during the pupal period. Following injection of BmNPV all young pupae acquired polyhedrosis and finally died whereas most of the older pupae did not exhibit disease and completed metamorphosis normally. Moreover, the later in the pupal period the silkworms were infected, the lower the production of polyhedrin in diseased pupae.

show abstract

Characterization of 3'→5' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus

Cited by 33 publications

References 23 publications

The effect of BM67 gene deletion on Bombyx mori nuclear polyhedrosis virus replication

The effect of BM67 gene deletion on Bombyx mori nuclear polyhedrosis virus replication

Baculovirus Replication Factor LEF-1 Is a DNA Primase

Protein synthesis in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus: resistance to viral infection acquired during pupal period

Contact Info

Product

Resources

About