AEGiS-11IAC: Regulation of HIV-1 replication by mutants of the IFN inducible protein kinase PKR.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

Regulation of HIV-1 replication by mutants of the IFN inducible protein kinase PKR.

Int Conf AIDS 1996 Jul 7-12; 11:440 (abstract no. Pub.A.1016)
Koromilas AE, Nagai K, Li S, Hiscott J, Sonenberg N, Wainberg M; McGill University, Montreal, Canada. Fax: (514) 340-7576. E-mail: mdak@musica.mcgill.ca.

OBJECTIVE: PKR is an interferon (IFN) inducible serine-threonine kinase which regulates protein synthesis. Activation of PKR by viruses results in phosphorylation of the translation initiation factor eIF-2, a modification that causes inhibition of protein synthesis. HIV-1 infection results in a significant decrease in the amount of cellular PKR and this represents an important mechanism in HIV pathogenesis. The objective of our research is to examine the molecular mechanisms by which PKR controls HIV-1 gene expression and pathogenesis. To this end we have used cell systems permissive to HIV-1 infection and replication, in which PKR activity was impaired by the expression of PKR mutants.

METHODS: Jurkat cells (CD4+ T cells) expressing PKR mutants were established by transfection. HIV-1 replication was determined by measuring the levels of reverse transcriptase activity (RT) and/or p24 HIV protein (ELISA) in culture supernatants. HIV-1 protein expression was determined by immunoblotting with specific monoclonal antibodies to viral antigens. HIV-1 provirus formation and RNA expression was determined by semi-quantitative PCR.

RESULTS: Expression of PKR mutants with a defect either in catalytic or RNA binding activity induced HIV-1 gene expression and replication. The catalytically inactive mutant of PKR facilitated HIV-1 gene expression at the translational level. In contrast, the RNA mutant of PKR had no effect on translation. HIV-1 provirus formation was induced in cells expressing the RNA mutant of PKR only. Virus assembly and release was not affected by either of the mutants.

CONCLUSIONS: PKR activation plays an important role in HIV-1 gene expression and replication. Studies with mutants of PKR revealed that PKR is implicated in different stages of HIV-1 life cycle: HIV-1 translation and HIV-1 provirus formation. Further studies on the molecular mechanisms that PKR regulates HIV-1 expression will be of great importance to devise biological strategies to combat HIV-1 replication and HIV-1 pathogenesis.

Keywords: AEGIS, eIF-2 Kinase, HIV-1, Virus Replication, Eukaryotic Initiation Factor-2, Protein-Serine-Threonine Kinases, HIV Infections, Mutation, Interferons, Antigens, CD4, RNA-Directed DNA Polymerase, Jurkat Cells, Phosphorylation, RNA, Enzyme Activation, T-Lymphocytes, Proviruses, Human, metabolism, virology, genetics, ICA11

960707
PubA1016