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

Abstract

There has been increasing interest in the identification of novel HIV entry inhibitors. For the discovery of these entry inhibitors, robust surrogate anti-HIV assays are highly desired. The authors report a novel anti-HIV assay system using Moloney murine leukemia viruses (MMLVs) pseudotyped with cytoplasmic tail-truncated HIV envelope protein gp140. These pseudotyped MMLV-HIVgp140 viral particles carry luciferase transcripts; therefore, robust luciferase signal can be detected in cells infected by these pseudotypes. Polycationic agent polybrene and spinoculation markedly enhanced the infection efficiency of these pseudotypes. It was demonstrated that the tropism of these pseudotypes is dependent on the pseudotyped HIV envelope proteins. MMLV viruses pseudotyped with gp140 from an R5 HIV virus specifically infect CCR5-expressing cells, and viruses pseudotyped with gp140 from an X4 HIV virus specifically infect CXCR4-expressing cells. Furthermore, CCR5 antagonists inhibited only MMLV-gp140(R5) infections, and CXCR4 antagonists inhibited only MMLV-gp140(X4) infections. A variety of known HIV entry inhibitors were tested in both R5- and X4-dependent pseudotype antiviral assays, and the IC₅₀ values generated were consistent with published results. The pseudotype antiviral assay was also used in the characterization of hundreds of novel CCR5 antagonists. The IC₅₀ values determined in this assay were compared with those determined in HIV antiviral and cell-cell fusion (CCF) assays, and good correlation was found between pseudotype antiviral assay and HIV antiviral assay (R ² = 0.9) or CCF assay (R ² = 0.8).