Assembly of membrane glycoproteins studied by phenotypic mixing between mutants of vesicular stomatitis virus and retroviruses

Abstract

Temperature sensitive ( ts) mutations of vesicular stomatitis virus (VSV), Indiana serotype, which belong to complementation group V ( tsV) have been shown to affect the viral envelope glycoprotein, or G protein. When ts V mutants are grown in cells producing avian leukosis viruses, the titers of infectious VSV obtained at the nonpermissive temperature are 10 4-fold higher than in control cells. Cells releasing murine leukemia viruses or avian reticuloendotheliosis virus rescue VSV ts V mutants much less efficiently. The rescued virions have the properties of envelope pseudotypes in that their host range is restricted to that of the helper retrovirus, they are neutralized by anti-retrovirus antibodies but not anti-VSV antibodies, and they are not thermolabile. Sensitive serological techniques, including the use of complement-mediated virolysis, immunoprecipitation, and monoclonal antibody reacting with G protein, show that VSV pseudotypes produced at the nonpermissive temperature have no detectable G protein, whereas VSV particles released from retrovirus infected cells at the permissive temperature have mosaic envelopes bearing both VSV G protein and retrovirus glycoprotein. In mixed infections of Rous sarcoma virus (RSV) and VSV ts V mutants, pseudotype particles with RSV genomes and VSV envelope antigens are produced only at the permissive temperature. In contrast, substantial yields of RSV(VSV) pseudotypes but no VSV(RSV) pesudotypes are obtained at the nonpermissive temperature with VSV carrying mutations in complementation group III, which affect M protein. Thermolabile VSV tsV mutants form RSV(VSV) pseudotypes which also are thermolabile. The kinetics of heat inactivation of G protein function in tsV mutants is the same in VSV particles with unmixed envelopes and with mosaic envelopes. From these studies of phenotypic mixing we draw the following conclusions: (i) The synthesis of functional M protein but not G protein is essential for the maturation of VSV virions. (ii) VSV M protein is not required for the assembly of G protein into retrovirus virions. (iii) The thermolabile nature of tsV VSV mutants is an intrinsic property of the G protein, independent of the type of virion into which it is incorporated and of other viral glycoproteins which may be assembled into the envelope of the same virion.