Inhibition of human immunodeficiency virus type 1 virion entry by dominant-negative Hck

Abstract

To study the role of Src family tyrosine kinases in infection with human immunodeficiency virus type 1 (HIV-1), we constructed an Hck mutant, HckN, that hinders signaling from wild-type Hck. HIV-1 produced in HckN-expressing cells was significantly less infectious to HeLa-CD4-LTR-beta-gal (MAGI) cells than HIV-1 produced in mock-transfected cells. The inhibitory effect of HckN was compensated for by the expression of vesicular stomatitis virus G protein. Finally, we found that the HIV-1 produced in the HckN-expressing cells entered into the cells less efficiently than did the control HIV-1. These results suggest that the Src family tyrosine kinases regulate entry of HIV-1 into target cells.

FIG. 1

Inhibition of HIV-1 infectivity by dominant-negative Hck. (A) Structures of the wild-type Hck and of the mutant protein used in this study. (B) HIV-1 proviral DNA, pNL-432, and expression plasmids of HckN, HckN-W93F, HckN-R151S, and CrkII were transfected into 293T cells. Virus stocks harvested at 36 h posttransfection were used to infect MAGI cells. Forty-eight hours later, infected cells were identified by staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside X-Gal). Each bar represents the average of two determinations. (C) 293T cells used to produce virus stock analyzed by immunoblotting with monoclonal antibody against HckN or CrkII.

FIG. 2

Inhibition of the infectivity Nef-deficient HIV-1 by HckN. (A) Proviral HIV-1 DNAs of the wild type (WT; pNL-432) and of the Nef mutant (ΔNef; pNL-432-Xh) transfected with or without the HckN expression vector into 293T cells. We used the virus stocks to infect MAGI cells. (B) Virions collected from the virus stock by ultracentrifugation on a 20% sucrose cushion, separated on an SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with anti-Nef monoclonal antibody.

FIG. 3

Restoration of infectivity by VSV pseudotyping. (A) Proviral DNAs of the wild type (WT) and of the Nef mutant (ΔNef) transfected into 293T cells with vector alone (closed bars), Nef expression vector (open bars), or VSV-G expression vector (shaded bars). Infectivity of the virion was examined as described in the legend to Fig. 1. Each bar represents the average of two determinations. (B) 293T cells used for virus production lysed in lysis buffer, separated on an SDS-polyacrylamide gel, and transferred to a PVDF membrane. VSV-G was detected by anti-G protein monoclonal antibody, followed by an enhanced chemiluminescence detection system (ECL).

FIG. 4

Inhibition of HIV-1 entry by HckN. 293T cells were transfected with wild-type pNL-432 alone (WT), pNL-432 plus pCAGGS-HckN (WT+HckN), pNL-432-Xh (ΔNef), or pNL-432 without the env region [Env(−)]. The viruses produced were used for infection of M8166 (top) and CEMx174 (bottom) cells. The quantities of viruses that entered into the cells were measured by an anti-p24^gag enzyme-linked immunosorbent assay.