Novel HIV-1 therapeutic agents based on virion-targeted viral proteins
The development of Acquired Immune Deficiency Syndrome (AIDS) results from infection with the human immunodeficiency virus (HIV). HIV-1 is a retrovirus, belonging to the lentivirus family, and is characterized by its sexual or blood route of transmission and long clinical latency. The genetic structure of HIV-1 has been well characterized, and is composed of the gag, pol, and env genes common to all retroviruses, two essential regulatory genes, tat and rev, and several auxiliary genes, vpr, vpu, nef, and vif. The Vpr, Vpu and met, non-structural proteins have been found to be associated with the viral particle to a varying extent. The pol gene encodes the reverse transcriptase (RT), protease (PR), and integrase (IN) enzymes, all of which play major roles in the life cycle of the virus. The protease enzyme is an essential component in the process of virus maturation and infectivity. Protease mediates the assembly of the viral core components, which are initially produced as polyprotein precursors, by catalyzing specific cleavages releasing the individual proteins.
Current treatment for infected individuals involves the administration of several drugs, all of which either target the viral reverse transcriptase or protease activities of the virus. Targeting these proteins will inhibit or halt the replication of the virus, the contributing factor to the disease process. The prolonged treatment of virus-infected individuals with these drugs has led to the selection of viruses which exhibit partial to full resistance to treatment due to specific changes in the target enzymes resulting from the low fidelity of the reverse transcription process. In the absence of a successful vaccine to prevent HIV-1 infection, various alternative approaches for inactivating the virus at different stages of the life cycle have been proposed and are being actively investigated. Here we propose antiviral agents “from within” the viral particle, targeted to interfere with the function of the virally encoded protease, preventing the production of infectious virus. We manipulated the virion associated protein Vpr to serve as a pseudosubstrate for protease processing, with hopes that natural substrates will be left unprocessed and virus will remain non-infectious. Likewise, we targeted the dimerization process of protease with other chimeric Vpr proteins, so as to prevent the production of an enzymatically active molecule. To provide proof of the principle, we have shown that viruses generated within the context of chimeric Vpr molecules showed varying levels of inhibition with a chimeric Vpr based on the capsid/p2 cleavage site, 24/2, being 100% effective in preventing the production of infectious virus in single and multiple round replication assays. The chimeric Vpr molecules containing cleavage sites were efficiently processed by protease, and along with the chimeric Vpr proteins generated to interfere with protease dimerization, still allowed for processing of the Gag polyproteins to occur. Furthermore, chimeric Vpr molecules were efficiently incorporated into wild-type virus in the presence of wild-type Vpr when expressed in stably expressing cell-lines. Here we demonstrate that in addition to conventional therapies used to combat HIV-1 in infected individuals, the modifiedVpr protein may serve as an alternative therapeutic molecule in the treatment of HIV-1.