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Journal of Virology, February 2006, p. 1724-1733, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1724-1733.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of the Role of Very Late Expression Factor 1 in Baculovirus Capsid Structure and DNA Processing

Adam L. Vanarsdall, Kazuhiro Okano, and George F. Rohrmann*

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804

Received 26 September 2005/ Accepted 15 November 2005

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.

* Corresponding author. Mailing address: Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804. Phone: (541) 737-1793. Fax: (541) 737-0496. E-mail: rohrmanng{at}orst.edu.

Journal of Virology, February 2006, p. 1724-1733, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1724-1733.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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