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 wildtype virus in both cultured cells and B. mori larvae. Marker rescue experiments using genomic libraries identified a single nucleotide mutation in the vp39 gene. 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 gene vp39 is one of the most wellknown 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.KEYWORDS baculovirus, nucleocapsid, BmNPV, VP39, few polyhedra T he Baculoviridae are a large family of viruses that infect insects, particularly those of the order Lepidoptera. Baculoviruses have a large circular, supercoiled, and doublestranded DNA genome packaged into rod-shaped virions (1, 2). They are divided into four genera, i.e., Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus (3). On the basis of molecular phylogenetic studies using genome sequences, alphabaculoviruses can be further subdivided into group I and II nucleopolyhedroviruses (NPVs) (4). NPVs produce two types of virions during their infection cycle.

Baculovirus AC102 Is a Nucleocapsid Protein That Is Crucial for Nuclear Actin Polymerization and Nucleocapsid Morphogenesis

“…Our observations indicate that, in cells infected with the AC102-K66A mutant, expression of the very late protein polyhedrin is also delayed and reduced, and fewer infected cells contain polyhedra compared to WOBpos-infected cells. This phenotype is similar to that recently observed for a virus carrying a point mutation in VP39 (VP39-G276S), which exhibits reduced expression of polyhedrin and fewer polyhedra (37). It was suggested that lower polyhedrin expression in the VP39-G276S mutant might result from the sequestration of viral DNA in aberrant tubular nucleocapsid-like structures, reducing transcription of viral genes, and a similar phenomenon may account for the phenotype of the AC102-K66A mutant.…”

Baculovirus AC102 is a nucleocapsid protein that is crucial for nuclear actin polymerization and nucleocapsid morphogenesis

“…Our observations indicate that, in cells infected with the AC102-K66A mutant, expression of the very late protein polyhedrin is also delayed and reduced, and fewer infected cells contain polyhedra as compared with WOBpos-infected cells. This phenotype is similar to that recently observed for a virus carrying a point mutation in VP39 (VP39-G276S), which exhibits reduced expression of polyhedrin and fewer polyhedra (36). It was suggested that lower polyhedrin expression in the VP39-G276S mutant might result from the sequestration of viral DNA in aberrant tubular nucleocapsid-like structures, and a similar phenomenon may account for the phenotype of the AC102-K66A mutant.…”

Viewing SARS-CoV-2 Nucleocapsid Protein in Terms of Molecular Flexibility

“…In LQ-R, leucine and glutamine account for 14.9% and 12.8%, respectively. In another nucleocapsid protein VP39 of baculovirus, it is known that amino acid substitution at glycine 276 affects nucleocapsid assembly and that this residue is completely conserved among various baculovirus families [ 11 ], suggesting that certain glycine residues are essential for proper function of the nucleocapsid protein. Even though N CoV2 contains 43 glycines in total, which account for 10.3% of 419 residues, there have not been systematic studies on the role of glycine and thus the possible roles of glycine in the N CoV2 function remain unclear.…”