Basic
Science Information

Clinical Information

Cancer
Information

Algarsamy Srinivasan, Ph.D.

Professor, Microbiology & Immunology

Reverse transcriptase (RT) and protease inhibitors are currently being used to treat HIV-1 infections. Termed highly active antiretroviral therapy (HAART), this treatment protocol has made a profound impact on the management of HIV infection as demonstrated by the decline in deaths in individuals with fewer than 100 CD+ cells/ul. However, side effects are associated with these compounds. A major issue with the use of these inhibitors is adherence to the strict timing of administration as the rapid development of resistant isolates has been observed in non-compliant individuals. The emergence of drug resistant viruses, however, will remain a problem with the continued use of antiviral agents that target viral enzymes. Hence, alternative strategies to contain HIV-1 replication are warranted. Towards this goal, an approach to generate a novel anti HIV-1 agent from within the virus has been considered.

It has been shown that HIV-1 incorporates three nonstructural proteins designated Vif, Vpr and Nef into virus particles. Though there is data available regarding the number of molecules of Vif and Nef in the virus particles, this information for Vpr is not clear. We have utilized an epitope-tagging approach to determine the amount of the Vpr in relation to Gag with the rationale that the presence of the same epitope on Gag and Vpr may accurately reflect the ratio of these proteins n the virus particles. For this purpose, chimeric Gag and Vpr expression plasmids were constructed with the Flag epitope (DYKDDDDK). The proteins directed by Gag-FL and Vpr-FL plasmids showed reactivities to the respective antibodies as well as to Flag antibodies. The sequences corresponding to the chimeric protein were then introduced into HIV-1 proviral DNA (NL4-3). In addition, NL4-3 DNA was modified to disrupt Vpr synthesis to determine the extent of incorporation of Vpr-FL expressed in trans. The analysis of virus particles generated by transfection of proviral DNA into RD cells indicated the following: i) Gag-FL protein exhibits sufficient sensitivity as it is amenable for quantitation by p24 antigen assay and immunoblot analysis, ii) The ratio of Gag to Vpr in virus particles, when Vpr-FL is expressed in cis is in the range of 150-200:1, and iii) The expression of Vpr-FL in trans showed efficient incorporation with a Gag to Vpr ratio of 5-7:1. This has prompted us to consider strategies using virion associated protein Vpr for the development of antiviral agents. Chimeric Vpr, being a non-structural viral protein with an added domain, has the potential to interfere with the assembly, maturation, and infectivity of the virus, depending on its fusion partner. To test this, we constructed a chimeric protein in which the coding sequences of the Vpr are linked to the C-terminus of HIV-1 protease required for dimer formation, (Vpr-PC1). We hypothesize that as HIV-1 protease is an obligatory homodimer, the chimeric protein is likely to interfere with the generation of active protease in the virus particle by influencing heterodimer formation. The virus derived from the HIV-1 proviral DNA containing chimeric Vpr registered a reduced level of replication in CEM and CEM X 174 cells in comparison to the wild type Vpr containing viruses. These results suggest that the intravirion display of peptides targeting virally coded enzymes is a powerful approach for developing antiviral agents and for dissecting the dynamic interactions between structural proteins during virus assembly and disassembly.