title: A Conserved Glycine Residue Is Required for Proper Functioning of a Baculovirus VP39 Protein.
abstract: The baculovirus VP39 protein is a major nucleocapsid protein essential for viral propagation. However, the critical domains or residues of the VP39 protein have not yet been identified. Here, we performed mutagenesis experiments with Bombyx mori nucleopolyhedrovirus (BmNPV) using 5-bromo-2'-deoxyuridine and isolated a BmNPV mutant that produced fewer occlusion bodies than the wild-type virus. This mutant also produced fewer infectious budded viruses (BVs) than the wild-type virus in both cultured cells andB. morilarvae. Marker rescue experiments using genomic libraries identified a single nucleotide mutation in thevp39gene. This mutation resulted in an amino acid substitution at glycine 276 (Gly-276) to serine, which was required for all the defective phenotypes observed in the mutant. Sequence comparison revealed that this residue is completely conserved among the VP39 proteins of the sequenced alphabaculoviruses, betabaculoviruses, and gammabaculoviruses. Although early viral gene expression was not significantly affected, the level of expression of a late gene,vcath, was reduced. In addition, two of the very late genes were markedly downregulated in cells infected with this mutant. Western blot and quantitative PCR analyses revealed that the BVs produced from cells infected with this mutant contained smaller amounts of the VP39 protein and viral genomic DNA than those produced from wild-type virus-infected cells. Combined with the results of transmission electron microscopy, VP39 Gly-276 can be concluded to be essential for correct nucleocapsid assembly, viral DNA packaging, and viral gene expression, especially of very late genes.IMPORTANCEThe major nucleocapsid protein genevp39is one of the most well-known baculovirus genes. Although several viral and host proteins that interact with the VP39 protein have been identified, the functionally important domains or residues of this protein remain unknown. The present study revealed that the glycine residue at residue 276, which is completely conserved among sequenced alphabaculoviruses, betabaculoviruses, and gammabaculoviruses, is important for the VP39 function, i.e., structural assembly of nucleocapsids and viral DNA packaging. Moreover, our results provide evidence for the link between nucleocapsid formation and the transcription of viral very late genes.
authors.affiliation: Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan katsuma@ss.ab.a.u-tokyo.ac.jp.
authors.forename: Susumu
authors.initials: S
authors.lastname: Katsuma
authors.affiliation: Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Ryuhei
authors.initials: R
authors.lastname: Kokusho
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000254:growth & development / Q000502:physiology* / Q000648:ultrastructure; D015153:Blotting, Western; D012831:Bombyx; D002460:Cell Line; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D020869:Gene Expression Profiling; D015967:Gene Expression Regulation, Viral; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D020125:Mutation, Missense; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D014771:Virion / Q000648:ultrastructure; D014779:Virus Replication
chemical_list: D019590:Nucleocapsid Proteins; D005998:Glycine
keywords: BmNPV; VP39; baculovirus; few polyhedra; nucleocapsid