Requirement for GP64 to Drive Efficient Budding ofAutographa californicaMulticapsid Nucleopolyhedrovirus

Abstract

Budded virions (BV) ofAutographa californicamulticapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein (GP64) that is present on the plasma membrane of infected cells. GP64 is acquired by virions during budding through the plasma membrane, the final step in assembly of the budded virion at the cell surface. Previous studies (S. A. Monsma, A. G. P. Oomens, and G. W. Blissard (1996).J. Virol.70, 4607–4616) showed that insertional inactivation of the AcMNPVgp64gene resulted in a virus unable to move from cell to cell and nonlethal to orally infectedTrichoplusia nilarvae. To determine whether GP64 is involved in virion budding, we measured BV production from Sf9 cells infected with a gp64null virus. Sf9 cells infected with gp64null virus vAc64−were pulse labeled, and progeny BV were isolated on equilibrium sucrose gradients and quantified. BV production from vAc64−was reduced to ∼2% of that from wild-type AcMNPV. Thus the GP64 protein is important for efficient virion budding. To determine whether the highly charged 7-amino acid cytoplasmic tail domain (CTD) of GP64 was required for virion production, we generated a series of GP64 constructs containing C-terminal truncations or substitutions. Modified forms of GP64 were analyzed in transfected cells and in recombinant viruses in which the wild-typegp64gene was replaced with a modifiedgp64.Deletion of 1–7 amino acids from the CTD did not affect GP64 trimerization, protein transport to the cell surface, or membrane fusion activity. However, deletions of 11 or 14 amino acids, which removed the CTD and portions of the predicted transmembrane (TM) domain, were trimerized but were present at lower levels on the cell surface due to shedding of these truncated proteins. Comparisons of growth curves and quantitative measurements of labeled progeny BV production from recombinant viruses expressing either wild-type or mutant GP64 proteins showed that deletion of the 7-residue CTD only moderately reduced the production of infectious virions (∼50%). However, deletions of the C terminal 11 or 14 amino acids had more substantial effects. Removal of the C terminal 11 amino acids reduced titers of infectious virus by 78–96% and labeled progeny virions were reduced by 91–92%. Removal of 14 amino acids from the C terminus resulted in an ∼98% reduction in progeny BV and a virus that was apparently incapable of efficient propagation in cell culture. Thus the GP64 CTD is not essential for production of infectious BV, but removal of the CTD results in a measurable reduction in budding efficiency. Deletion of the CTD plus small portions of the transmembrane domain resulted in shedding of GP64, reduced surface levels, and a dramatic reduction in the production of BV. Together, these data indicate that GP64 is an important and limiting factor in BV production.