INHIBITION OF HUMAN AND SIMIAN IMMUNODEFICIENCY VIRUS PROTEASE FUNCTION BY TARGETING VPX-PROTEASE-MUTANT FUSION PROTEIN INTO VIRAL PARTICLES

The human immunodeficiency virus type 1 (HIV-1) Vpr and HIV-2 Vpx prot eins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have be en exploited to incorporate foreign proteins into virions by expressio n as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995), To explore the possibility of utilizin g Vpx and Vpr to target dominant negative mutants of the HIV Pol prote ins into virions, we fused HIV-2 Vpx with an enzymatically defective p rotease (PR) mutant. Using a vector system to facilitate transient coe xpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein w as expressed and packaged efficiently into HIV-2 and simian immunodefi ciency virus virions. Immunoblot analysis of purified virions demonstr ated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-char acterized active-site PR inhibitor. The incomplete processing of Gag a nd Gag/Pol was consistent with a 25-fold reduction in virion infectivi ty, The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions, Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were norm al with respect to the processing of Gag protein and the ability to in fect and replicate in vitro. These results indicate that VpxPR(M) spec ifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign pr oteins into virions via fusion with Vpx can inhibit HN replication. Th e use of accessory proteins as vehicles to deliver deleterious protein s to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antir etroviral strategies, The ability to package PR by expression in tuans , independent of the Gag/Pol precursor, also represents a novel approa ch that may be exploited to study the function of the Pol proteins.