Toward the Development of a Virus-Cell-Based Assay for the Discovery of Novel Compounds against Human Immunodeficiency Virus Type 1

Abstract

The emergence of human immunodeficiency virus type 1 (HIV-1) strains resistant to highly active antiretroviral therapy necessitates continued drug discovery for the treatment of HIV-1 infection. Most current drug discovery strategies focus upon a single aspect of HIV-1 replication. A virus-cell-based assay, which can be adapted to high-throughput screening, would allow the screening of multiple targets simultaneously. HIV-1-based vector systems mimic the HIV-1 life cycle without yielding replication-competent virus, making them potentially important tools for the development of safe, wide-ranging, rapid, and cost-effective assays amenable to high-throughput screening. Since replication of vector virus is typically restricted to a single cycle, a crucial question is whether such an assay provides the needed sensitivity to detect potential HIV-1 inhibitors. With a stable, inducible vector virus-producing cell line, the inhibitory effects of four reverse transcriptase inhibitors (zidovudine, stavudine, lamivudine, and didanosine) and one protease inhibitor (indinavir) were assessed. It was found that HIV-1 vector virus titer was inhibited in a single cycle of replication up to 300-fold without affecting cell viability, indicating that the assay provides the necessary sensitivity for identifying antiviral molecules. Thus, it seems likely that HIV-1-derived vector systems can be utilized in a novel fashion to facilitate the development of a safe, efficient method for screening compound libraries for anti-HIV-1 activity.Currently, the principal regimen for the treatment of individuals infected with human immunodeficiency virus type 1 (HIV-1) involves highly active antiretroviral therapy, which typically includes a combination of a protease inhibitor and a nucleoside and/or nonnucleoside reverse transcriptase (RT) inhibitor. HIV-1 protease inhibitors such as indinavir, ritonavir, and saquinavir prevent proteolytic processing of immature viral polyproteins to produce mature infectious virions (37) Nucleoside RT inhibitors such as zidovudine, didanosine, lamivudine, and stavudine lack the 3Ј-OH moiety on the ribose sugar and act as chain terminators when incorporated into the elongating DNA chain by the HIV-1 RT, while the nonnucleoside RT inhibitors such as efavirenz, delavirdine, and nevirapine represent a class of diverse polycyclic compounds that bind to a site near the catalytic domain of RT and interfere with polymerase activity (26).Highly active antiretroviral therapy has greatly decreased morbidity and mortality for millions of HIV-1-infected individuals over the past several years. However, 10 to 50% of pa-

Development of a Moloney Murine Leukemia Virus–Based Pseudotype Anti-HIV Assay Suitable for Accurate and Rapid Evaluation of HIV Entry Inhibitors

“…Therefore, it can be used as a surrogate antiviral assay for routine identification and characterization of novel HIV entry inhibitors. Although the HIV-HIVenv pseudotype single-cycle antiviral assay can be used for the detection of all anti-HIV agents, 17,45 this MMLV-based anti-HIV assay system is specifically designed for HIV entry inhibitors. Therefore, this novel MMLV-HIVenv antiviral assay system can be used for HTS of novel inhibitors that target various viral entry steps.…”

HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors

“…The concept of using HIV-1 reporter viruses to monitor HIV-1 replication was first introduced using a replication competent HIV-1 reporter virus containing the chloramphenicol acetyltransferase gene in place of HIV-1 Nef sequences. Cells are then infected with the recombinant reporter virus and virus replication is quantified by measuring the expression of the virally encoded reporter gene (Adelson et al, 2003;Dey & Berger, 2003). For reporter cell assays, the target cells of interest are engineered to contain a reporter gene, which is activated upon viral infection.…”

High-Throughput Human Immunodeficiency Virus Type 1 (HIV-1) Full Replication Assay That Includes HIV-1 Vif as an Antiviral Target

“…Several different antiviral assay formats that could potentially be used for antiviral high-throughput screening have been proposed in the literature (1,5,6,7,9,20,27,29,31,32,36). Despite this, the execution of an antiviral HTS is not trivial, and few have been successfully achieved on an industrial scale (Ͼ10 6 compounds).…”

“…Both BMS-488043 (3,14,21,38) and PA-457 (11,19), two novel target HIV-1 inhibitors that recently entered clinical development, were originally identified by using antiviral screens. Several different HIV-1 antiviral assay formats have been described that could be adapted for medium-to high-throughput screening (1,5,6,7,9,20,27,29,31,32,36), and many of these assays utilize either a reporter virus or a reporter cell to measure HIV-1 replication. In reporter virus assays, a reporter gene is introduced into the virus genome, usually in place of a viral gene not required for replication in the target cells of interest.…”

“…In reporter virus assays, a reporter gene is introduced into the virus genome, usually in place of a viral gene not required for replication in the target cells of interest. The cells are then infected with the recombinant reporter virus, and virus replication is quantified by measuring the expression of the virusencoded reporter gene (1,5,29,31). In reporter cell assays, the target cells of interest are engineered to contain a reporter gene, which is activated upon viral infection.…”