Antiviral agent based on the non-structural protein targeting the maturation process of HIV-1: expression and susceptibility of chimeric Vpr as a substrate for cleavage by HIV-1 protease

The processing of precursor proteins (Gag and Gag-pol) by the viral proteas e is absolutely required in order to generate infectious particles. This pr ompted us to consider novel strategies that target viral maturation. Toward s this end, we have engineered an HIV-1 virion associated protein, Vpr, to contain protease cleavage signal sequences from Gag and Gag-pol precursor p roteins. We previously reported that virus particles derived from HIV-1 pro viral DNA, encoding chimeric Vpr, showed a lack of infectivity, depending o n the fusion partner. As an extension of that work, the potential of chimer ic Vpr as a substrate for HIV-1 protease was tested utilizing an epitope-ba sed assay. Chimeric Vpr molecules were modified such that the Flag epitope is removed following cleavage, thus allowing us to determine the efficiency of protease cleavage. Following incubation with the protease, the resultan t products were analyzed by radioimmunoprecipitation using antibodies direc ted against the Flag epitope, Densitometric analysis of the autoradiograms showed processing to be both rapid and specific. Further, the analysis of v irus particles containing chimeric Vpr by immunoblot showed reactivities to antibodies against the Flag epitope similar to the data observed in vitro. These results suggest that the pseudosubstrate approach mag provide anothe r avenue for developing antiviral agents.