This project was initiated to study several aspects necessary for the design of viral expression vectors which can be targeted to specific cells for gene delivery. Understanding the mechanisms of viral assembly are crucial toward this goal. It is also essential to understand how the envelope of the virus, Whether it is a retrovirus or another envelope virus, could be altered so it could show a specific cell tropism, while maintaining membrane fusion activity. In viral assembly, the viral matrix protein M plays a central role. It brings the genome in contact with the plasma membrane at the site of virus budding. During the past fiscal year, we focused on the role of the matrix protein M of vesicular stomatitis virus (VSV), which is essential for viral assembly and, in part, for viral pathogenesis. Coexpression of the matrix protein with HIV~1 led to inhibition of HIV~1 replication, as indicated by a severe decrease in the level of p24 released and in the number of syncitia. We and others had earlier reported that the M protein can indiscriminately inhibit the expression of genes driven by several different RNA polymerase II promoters. If the cytopathic effect caused by M protein could be made inducible upon infection, for example, by the HIV~1 Tat protein, protection of the cell population may result. The infected cell itself may also be killed, thereby simultaneously clearing the virus from the cell population. To test for this possibility, a temperature~sensitive M protein was cloned under control of the HIV~1 LTR ,and individual cell clones harboring the construct were selected and challenged by transfection with HIV~1 DNA. HIV~1 replication, as evidenced by p24 antigen release and syncytia formation, was severely decreased at the permissive temperature of M (32oC ) as compared to the parental cell line which continued to support HIV~1 replication. In contrast, at the nonpermissive temperature for the M protein (40 0C), the cell clones efficiently supported HIV~1 replication, like the parental cells. The fact, that the cell clones were viable at these temperatures demonstrated that basal levels of the temperature ~ sensitive M expression from the HIV~1 LTR were not toxic to the cell. The expression of M protein at permissive temperature protected the cell population from virus spread. For the M protein to be potentially useful in a gene therapeutic approach, a lack of toxicity at the level of the uninduced wild type M protein expression needs to be demonstrated.
