Characterization of the role of very late expression factor 1 in baculovirus capsid structure and DNA processing.
Very late expression factor 1 (VLF-1) of Autographa californica multiple nucleopolyhedrovirus is a putative tyrosine recombinase and is required for both very late gene expression and budded virus production. In this report, we show that a vlf-1 knockout bacmid was able to synthesize viral DNA at levels similar to that detected for a gp64 knockout bacmid that served as a noninfectious control virus. Additionally, analysis of replicated bacmid DNA by field-inversion gel electrophoresis indicated that VLF-1 is not required for synthesizing high-molecular-weight intermediates that could be resolved into unit-length genomes when cut at a unique restriction site. However, immunoelectron microscopic analysis revealed that in cells transfected with a vlf-1 knockout bacmid, aberrant tubular structures containing the capsid protein vp39 were observed, suggesting that this virus construct was defective in producing mature capsids. In contrast, rescuing the vlf-1 knockout bacmid construct with a copy of VLF-1 that carries a mutation of a highly conserved tyrosine (Y355F) was sufficient to restore the production of nucleocapsids with a normal appearance, but not infectious virus production. Furthermore, the results of a DNase I protection assay indicated that the DNA packaging efficiency of the VLF-1(Y355F) virus construct was similar to that of the gp64 knockout control. Finally, a recombinant virus containing a functional hemagglutinin epitope-tagged version of VLF-1 was constructed to investigate the association of VLF-1 with the nucleocapsid. Analysis by immunoelectron microscopy of Sf-9 cells infected with this virus showed that VLF-1 localized to an end region of the nucleocapsid. Collectively, these results indicate that VLF-1 is required for normal capsid assembly and serves an essential function during the final stages of the DNA packaging process.
Vanarsdall
Adam L
AL
Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804, USA.
Okano
Kazuhiro
K
Rohrmann
George F
GF
D019943:Amino Acid Substitution; D002213:Capsid / Q000737:chemistry / Q000648:ultrastructure*; D036022:Capsid Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D042003:DNA Packaging; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D017353:Gene Deletion; D016253:Microscopy, Immunoelectron; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017924:Nucleopolyhedroviruses / Q000502:physiology* / Q000648:ultrastructure; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*; D019065:Virus Assembly
C090371:Ac-vlf-1 protein, Autographa californica nucleopolyhedrovirus; D036022:Capsid Proteins; D004279:DNA, Viral; D014157:Transcription Factors; D014764:Viral Proteins