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Journal of Virology, March 2001, p. 2544-2556, Vol. 75, No. 6
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.6.2544-2556.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

A GP64-Null Baculovirus Pseudotyped with Vesicular Stomatitis Virus G Protein

J. T. Mangor,1 S. A. Monsma,2 M. C. Johnson,3 and G. W. Blissard1,*

Boyce Thompson Institute at Cornell University1 and Department of Molecular Biology and Genetics, Cornell University,3 Ithaca, New York 14853, and Novagen Inc., Madison, Wisconsin 537112

Received 11 September 2000/Accepted 11 December 2000

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc64-, could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9VSV-G) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9VSV-G and Sf9 cells were infected with vAc64-, and cells were monitored for infection and for movement of infection from cell to cell. vAc64- formed plaques on Sf9VSV-G cells but not on Sf9 cells, and plaques formed on Sf9VSV-G cells were observed only after prolonged intervals. Passage and amplification of vAc64- on Sf9VSV-G cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc64- in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc64- was generated at titers of approximately 106 to 107 infectious units (IU)/ml, compared with titers of approximately 108 IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc64- virions in Sf9VSV-G cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc64--derived virus that had acquired the G gene through recombination with Sf9VSV-G cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.

* Corresponding author. Mailing address: Boyce Thompson Institute at Cornell University, Tower Rd., Ithaca, NY 14853. Phone and fax: (607) 254-1366. E-mail: gwb1{at}cornell.edu.

Journal of Virology, March 2001, p. 2544-2556, Vol. 75, No. 6
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.6.2544-2556.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.