The Identification of a Small Molecule Compound That Reduces HIV-1 Nef-Mediated Viral Infectivity Enhancement

Abstract

Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of viral production itself. However, the infectivity of the viruses produced in the presence of 2c was significantly lower than that of control viruses. Importantly, an inhibitory effect was observed with Nef+ wild-type viruses, but not with viruses produced in the absence of Nef or in the presence of proline-rich PxxP motif-disrupted Nef, both of which displayed significantly reduced intrinsic infectivity. Meanwhile, the overexpression of the SH3 domain of the tyrosine kinase Hck, which binds to a PxxP motif in Nef, also reduced viral infectivity. Importantly, 2c inhibited Hck SH3-Nef binding, which was more marked when Nef was pre-incubated with 2c prior to its incubation with Hck, indicating that both Hck SH3 and 2c directly bind to Nef and that their binding sites overlap. These results imply that both 2c and the Hck SH3 domain inhibit the interaction of Nef with an unidentified host protein and thereby reduce Nef-mediated infectivity enhancement. The first inhibitory compound 2c is therefore a valuable chemical probe for revealing the underlying molecular mechanism by which Nef enhances the infectivity of HIV-1.

Potential Role of the Formation of Tunneling Nanotubes in HIV-1 Spread in Macrophages

“…The WT virus replicated more efficiently in MDMs than DNef virus (Fig. 1B, 1C) as reported previously (30). However, even when the replication of these two viruses was adjusted to equivalent levels (25 and 100 ng/ml input Gag for WT and DNef viruses, respectively; see Fig.…”

“…rHIV-1 was prepared as described previously (30). HEK293 cells cultured with DMEM-10% FCS were used as viral producer cells.…”

M-CSF Inhibits Anti–HIV-1 Activity of IL-32, but They Enhance M2-like Phenotypes of Macrophages

“…HIV-1 viruses were prepared as described previously (29,30). HEK293 cells (Invitrogen) were maintained in DMEM-10% FCS and used as viral producer cells.…”

“…After 6 h of transfection, the culture medium was replaced with fresh medium, and the cells were cultured for an additional 48 h. Then the supernatants containing the viruses were clarified by centrifugation, analyzed for their p24 HIV-1 Gag protein concentrations by ELISA (RETROtek p24 Ag ELISA kit; ZeptoMetrix, Buffalo, NY), and kept at 270˚C before use. The HIV-1 infection was performed essentially as described previously (29,30). Macrophages cultured on 24-well tissue culture plates were incubated with 250 ml of the supernatants of 293 cells containing HIV-1 viruses (the number of viruses was normalized by adjusting the p24 Gag protein concentrations to 100 ng/ml) for 2 h at 37˚C.…”

“…Then the cells were washed twice with PBS to remove any unbound viruses. Half of the culture medium was replaced with complete medium every 3 d (29). In a selected experiment (see Fig.…”