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 protease is absolutely required in order to generate infectious particles. This prompted us to consider novel strategies that target viral maturation. Towards this end, we have engineered an HIV-1 virion associated protein, Vpr, to contain protease cleavage signal sequences from Gag and Gag-pol precursor proteins. We previously reported that virus particles derived from HIV-1 proviral DNA, encoding chimeric Vpr, showed a lack of infectivity, depending on the fusion partner. As an extension of that work, the potential of chimeric Vpr as a substrate for HIV-1 protease was tested utilizing an epitope-based 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 resultant products were analyzed by radioimmunoprecipitation using antibodies directed against the Flag epitope. Densitometric analysis of the autoradiograms showed processing to be both rapid and specific. Further, the analysis of virus 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 may provide another avenue for developing antiviral agents.
Serio
D
D
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Singh
S P
SP
Cartas
M A
MA
Weber
I T
IT
Harrison
R W
RW
Louis
J M
JM
Srinivasan
A
A
D000611:Aminobiphenyl Compounds / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology*; D003720:Densitometry; D015195:Drug Design; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D015683:Gene Products, gag / Q000235:genetics; D016336:Gene Products, vpr / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D016333:HIV Protease / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015151:Immunoblotting; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D015531:Radioimmunoprecipitation Assay; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014779:Virus Replication / Q000187:drug effects*; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus; D054325:vpr Gene Products, Human Immunodeficiency Virus
D000611:Aminobiphenyl Compounds; D000998:Antiviral Agents; D000939:Epitopes; D015683:Gene Products, gag; D016336:Gene Products, vpr; D009842:Oligopeptides; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C412836:Ro 26-2812; D054301:gag Gene Products, Human Immunodeficiency Virus; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D054302:pol Gene Products, Human Immunodeficiency Virus; C091950:pr160 gag-pol precursor protein, Human immunodeficiency virus 1; D054325:vpr Gene Products, Human Immunodeficiency Virus; C085706:FLAG peptide; D016333:HIV Protease