This project cover various aspects of the replication and pathogenesis of enveloped viruses. Enveloped viruses can have RNA or DNA genomes and infamous members of this large group include viruses like rabies, measles, the AIDS virus and herpes virus, etc. A common feature among these viruses is that upon assembly into virus particles, the packaging of the genomes utilizes cellular membranes which represent the viral envelope. The lipid envelope of the virus, together with the specific viral proteins which are inserted, determines in part the cell tropism of the virus. One of the goals of this project during the last FY was to try to understand whether it is possible to alter the tropism of the virus by inserting novel glycoproteins into its envelope. Such novel glycoproteins could potentially be constructed by gene fusion, and could be expressed and inserted into the viral envelope. Viral surface glycoproteins, in general, provide the virus with the ability to specifically bind to cells and to fuse with cellular membranes, thereby facilitating entry into the cytoplasm. In addition, the cytoplasmic domain of the glycoprotein may interact specifically with the viral core during assembly and thereby concentrate the amount of glycoprotein per virus particle. Our recent studies on this project focus on these three functions. We use vesicular stomatitis virus (VSV), a well-characterized enveloped negative strand RNA virus, as a model. Novel genes encoding chimeric glycoproteins are constructed by precise gene fusion using recombinant DNA techniques including polymerase chain reactions. The genes are expressed in vivo using vaccinia viruses recombinants. Earlier, we were able to insert a foreign glycoprotein, the human HIV receptor CD4 as well as a chimeric CD4/G protein, into the envelope of VSV particles, which consists of the ectodomain of CD4 and the transmembrane and cytoplasmic domains of the VSV glycoprotein G. To understand the mechanism of insertion, we have quantitated and compared the efficiency of CD4 vs CD4/G insertion. Although these two proteins were expressed by different vaccinia virus expression systems, their overall amounts were very similar in VSV-infected cell extracts. In comparison, the amount of the VSV G protein was approximately 5-fold higher than the receptor proteins. Packaging of these glycoproteins into VSV particles showed no significant preference for either CD4 or CD4/G, suggesting that the cytoplasmic tail region did not favor the chimeric CD4/G protein. A ratio of 400 G protein trimers bs. 50 CD4 or CD4/G protein per virus particle was determined. Considering the amounts of G protein as compared to CD4/G which were available in cell extracts, these data suggest that the insertion seems to favor the native G protein by about 24 fold. The number of CD4 molecules per virus particle are consistent with earlier immunoprecipitation studies which suggested that every virus particle contained the receptor. The data also explains why it was not possible to inactivate such a large number of G proteins and detect an altered tropism of the virus for env expressing cells. For future studies, a recombinant VSV particle is needed which would eliminate the G protein altogether.
