vlf-1 deletion brought AcMNPV to defect in nucleocapsid formation.
ABSTRACT Recent studies have provided direct evidence that the baculovirus very late factor 1 (VLF-I) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was essential for BV production. To elucidate how vlf-1 deletion blocks BV production we generated a vlf-1 knockout bacmid by ET-recombination technology on AcMNPV bacmid propagated in Escherichia coli. Bacmid DNA transfection and supernatant passage assay revealed that the vlf-1 knockout bacmid was unable to replicate in cell culture, while vlf-1 repair bacmid, which was generated by transposition of the vlf-1 ORF under control of its native promoter into polyhedrin gene locus of vlf-1 knockout bacmid, resumed viral replication ability at wildtype levels. Results of these assays proved the correct construction of the vlf-1 knockout bacmid. Subsequent electron microscopy revealed that the vlf-1 knockout bacmid failed to form nueleocapsid in the nuclei of the transfected cells. Instead, intensely electron-dense virogenic stroma characteristic of viral DNA synthesis were observed. Thus, it is demonstrated for the first time that vlf-1 knockout blocked nucleocapsid formation and the defective nucleocapsid formation resulted in the abolishment of BV and ODV production. Possible roles of vlf-1 in genome processing are suggested and discussed.
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ABSTRACT: Comparative analysis of the complete genome sequences of 13 baculoviruses revealed a core set of 30 genes, 20 of which have known functions. Phylogenetic analyses of these 30 genes yielded a tree with 4 major groups: the genus Granulovirus (GVs), the group I and II lepidopteran nucleopolyhedroviruses (NPVs), and the dipteran NPV, CuniNPV. These major divisions within the family Baculoviridae were also supported by phylogenies based on gene content and gene order. Gene content mapping has revealed the patterns of gene acquisitions and losses that have taken place during baculovirus evolution, and it has highlighted the fluid nature of baculovirus genomes. The identification of shared protein phylogenetic profiles provided evidence for two putative DNA repair systems and for viral proteins specific for infection of lymantrid hosts. Examination of gene order conservation revealed a core gene cluster of four genes, helicase, lef-5, ac96, and 38K(ac98), whose relative positions are conserved in all baculovirus genomes.Annual Review of Entomology 02/2003; 48:211-34. · 13.59 Impact Factor
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ABSTRACT: vlf-1 is a baculovirus gene that regulates very late gene expression (J. R. McLachlin and L. K. Miller, J. Virol., 68, 7746-7756, 1994) and also plays a crucial role in the replication of the budded form of Autographa californica nuclear polyhedrosis virus (AcMNPV) (S. Yang and L. K. Miller, "Expression and mutational analysis of the baculovirus very late factor 1 (vlf-1) gene." Virology, 245, 99-109, 1998). To examine the influence of vlf-1 expression on baculovirus infection, we constructed recombinant viruses that expressed only low levels of VLF-1 and recombinants with vlf-1 under the control of different promoters. Viruses with mutant alleles of vlf-1 that produced low levels of VLF-1 replicated the budded form of the virus normally but produced no occlusion bodies. Thus, a higher concentration of VLF-1 was needed to activate very late gene expression than was needed to support budded virus production. By altering the level and/or timing of vlf-1 expression, the timing of polyhedrin gene (polh) expression, which normally occurs very late in infection, could be advanced or delayed. Early overexpression of vlf-1 increased the level of expression from the polh promoter but caused premature cellular disintegration. The data indicate that VLF-1 is the limiting factor in very late gene expression and that the level of VLF-1 controls the onset of occlusion.Virology 09/1998; 248(1):131-8. · 3.37 Impact Factor
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ABSTRACT: Alkaline nuclease (AN) of the Autographa californica multiple-capsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133) was expressed in recombinant baculovirus as a His(6)-tagged fusion and purified by sequential chromatography on Ni-NTA-agarose, DEAE-Toyopearl, and heparin-Sepharose. At all stages of purification, AcMNPV AN was found to copurify with a 44-kDa polypeptide which was identified as the baculovirus single-stranded DNA (ssDNA)-binding (SSB) protein, LEF-3. Sedimentation analysis in glycerol gradients of highly purified samples suggested that AN and LEF-3 are associated in a complex (designated *AN/L3), predominantly as heterodimers, although oligomeric forms containing both proteins were evident. In reactions with single- or double-stranded 62-mer oligonucleotides that were labeled with (32)P at the 5' or 3' ends, *AN/L3 carried out exonucleolytic hydrolysis of both substrates exclusively in a 5'-->3' direction. Saturation of ssDNA with an excess of LEF-3 prior to the addition of *AN/L3 resulted in a marked decrease in the rate of ssDNA hydrolysis. This suggests that excess LEF-3 may protect ssDNA from digestion by a AN-LEF-3 complex, thus regulating its activity in infected cells. The association of baculovirus AN with the viral SSB LEF-3 and the 5'-->3' exonuclease activity of this complex suggests that AN and LEF-3 may participate in homologous recombination of the baculovirus genome in a manner similar to that of exonuclease (Redalpha) and DNA-binding protein (Redbeta) of the Red-mediated homologous recombination system of bacteriophage lambda.Journal of Virology 02/2003; 77(4):2436-44. · 5.08 Impact Factor