During the past year this study continued to focus on the role of the viral matrix protein M of vesicular stomatitis virus (VSV) in cytopathogenesis and in viral assembly. We had previously reported that the VSV M protein is responsible for cytopathic effects which involve the disorganization of the cytoskeleton after VSV infections. Other investigators also found that the matrix protein, which remains the only protein of this cytoplasmic virus found in cell nuclei is able to inhibit cellular transcription. We have studied this property of the M protein after isolation of HIV-1 susceptible cell clones which encode either a wild type or a temperature sensitive M protein under the control of the inducible HIV-1 LTR promoter. Cell clones which encode this protein were infected with HIV-1 as well as with HIV-2. Gene expression and replication of both viruses were drastically inhibited by wild type M protein after transactivation with the HIV Tat1 and Tat2 proteins, respectively. Although HIV-1 was able to establish a provirus in these cells at the same efficiency as in cells without M protein, subsequent viral gene expression and spread were drastically inhibited in M expressing cell lines. Since basal levels of wild type VSV M protein were not toxic to the cell, it appeared to function as an inducible antiviral. The mechanism for the inhibition of cellular transcription by the VSV M protein was further studied in the context of interferon inducible genes which are turned off after VSV infections. A novel nuclear DNA binding protein was detected in a gel shift assay after VSV infections. Binding was detected with a specific interferon stimulated response element sequence. In collaboration with Dr. Ozato's group, we found that the induction of this protein binding was dependent on VSV gene expression and replication. With one of our temperature M protein mutants, this binding was temperature sensitive, which indicated that the VSV M protein may play a role in that shift. Supershift experiments did not identify M protein as part of the DNA/protein complex itself. Further experiments are in progress to examine the potential role of M protein in inducing this new DNA binding activity as well as its role in the shut off of cellular transcription. These studies will allow important insights into the mechanism of VSV induced cytopathogenesis as well as into specific mechanisms of gene regulation, possibly involving new negative cellular transcription factors.
