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, vAc(64-), 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 (Sf9(VSV-G)) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9(VSV-G) and Sf9 cells were infected with vAc(64-), and cells were monitored for infection and for movement of infection from cell to cell. vAc(64-) formed plaques on Sf9(VSV-G) cells but not on Sf9 cells, and plaques formed on Sf9(VSV-G) cells were observed only after prolonged intervals. Passage and amplification of vAc(64-) on Sf9(VSV-G) cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc(64-) in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc(64-) was generated at titers of approximately 10(6) to 10(7) infectious units (IU)/ml, compared with titers of approximately 10(8) IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc(64-) virions in Sf9(VSV-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 vAc(64-)-derived virus that had acquired the G gene through recombination with Sf9(VSV-G) cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.