A bipartite membrane-binding signal in the human immunodeficiency virus type 1 matrix protein is required for the proteolytic processing of Gag precursors in a cell type-dependent manner

Abstract

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4(+) T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4(+) T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.

FIG. 1

Cell type-dependent proteolytic processing of unmyristylated HIV-1 Gag proteins. COS cells (A and B) or HeLa cells (C) were mock transfected (lane 1) or transfected with the myristylation-negative mutant Myr− (lanes 2) or the wild-type Myr+ (lanes 3) proviral plasmid. At 72 h after transfection, cells were lysed in radioimmunoprecipitation assay lysis buffer and separated by SDS–12% PAGE and then transferred simultaneously to two nitrocellulose filters. Viral proteins were visualized by immunoblotting with an HIV-1-positive human serum (A and C) or a polyclonal anti-CAp24 antiserum (B).

FIG. 2

A polybasic domain at the N termini of HIV-1 Gag proteins is required for processing of Gag precursors in a cell type-dependent manner. COS (lanes 1 to 3) or HeLa (lanes 4 to 6) cells were mock transfected (lanes 1 and 4) or transfected with wild-type plasmid HXB2R3 (lanes 2 and 5) or mutant plasmid B5 (lanes 3 and 6). At 72 h posttransfection, cell lysates were separated by SDS-PAGE and viral proteins were visualized by immunoblot analysis with an HIV-1-positive human serum (A) or a polyclonal anti-CAp24 antiserum (B). M, molecular weight standards.

FIG. 3

Defect in proteolytic processing of HIV-1 Gag precursors in Myr− CD4⁺ T cells. Myr+ and Myr− CD4⁺ T-cell lines were generated as described in Materials and Methods. (A and B) Cell lysates from uninfected SupT-1 (lane 1), Myr−/SupT-1 (lane 2), and Myr+/SupT-1 (lane 3) cell lines were prepared by lysing of cells in radioimmunoprecipitation assay (RIPA) lysis buffer. The cell lysates were separated by SDS–12% PAGE and transferred to two nitrocellulose filters and then immunoblotted with an HIV-1-positive human serum (A) or with a monoclonal anti-RTp66/p51 antibody (B). (C) The supernatants from uninfected SupT-1 (lane 1), Myr−/SupT-1 (lane 2), and Myr+/SupT-1 (lane 3) cell lines were harvested after 48 h of incubation with fresh complete medium. The virion-associated proteins from harvested supernatants were concentrated by ultracentrifugation and analyzed by SDS-PAGE and immunoblotting with an HIV-1-positive human serum. (D) Cell lysates from uninfected H9 (lane 1), Myr−/H9 (lane 2), and Myr+/H9 (lane 3) cell lines were prepared by lysing the cells in RIPA lysis buffer. The cell lysates were separate by SDS–12% PAGE and the immunoblotted with an HIV-1-positive human serum.

FIG. 4

Coimmunoprecipitation analysis. (A and B) Cell lysates from uninfected (lanes 1), Myr−/SupT-1 (lanes 2), Myr+/SupT-1 (lanes 3), and Pr160/SupT-1 (lanes 4) cells were analyzed by SDS-PAGE and immunoblotting with an HIV-1-positive human serum (A) or with a monoclonal anti-RTp66/p51 antibody (B). (C, D, and E) Cell lysates from uninfected (lanes 1), Myr−/SupT-1 (lanes 2 and 5), Myr+/SupT-1 (lanes 3 and 6), and Pr160/SupT-1 (lanes 4) cells were prepared by lysing of cells in PBS containing 1% Triton X-100, followed by immunprecipitation with a polyclonal anti-p6 antiserum (lanes 1 to 4) or without antiserum (lanes 5 and 6) overnight at 4°C. The immunoprecipitated materials were separated by SDS–12% PAGE and transferred to two nitrocellulose filters. Viral proteins on the filters were visualized by immunoblotting with an HIV-1-positive human serum (C), and the same filter was then exposed for a longer period of time (D). The other filter was probed with monoclonal anti-RTp66/p51 antibody (E) to visualize the Pr160^Gag-Pol precursors.

FIG. 5

Ex vivo rescue of the processing defect in unmyristylated Gag precursors by cotransfection with myristylated Pr55^Gag proteins. HeLa cells were mock transfected (lanes 1), transfected with the wild-type Myr+ (lanes 2), mutant Myr− (lanes 3), or ΔPol (lanes 4) construct, or cotransfected with the Myr+ and ΔPol (lane 5) or Myr− and ΔPol (lane 6) constructs. At 72 h after transfection cell lysates were analyzed by SDS-PAGE and immunoblotting with an HIV-1-positive human serum (A) or polyclonal anti-CAp24 antiserum (B).

FIG. 6

Proteolytic processing of unmyristylated HIV-1 Gag proteins in COS cells is inhibited by HIV-1 protease inhibitor. COS cells were mock transfected (lanes 1 and 5) or transfected with the myristylation-negative mutant Myr− (lanes 2 and 6), the HXB2Pr-Neo (lanes 3 and 7), or the wild-type Myr+ (lanes 4 and 8) proviral plasmid. Half of the transfected cells were treated with 20 μM saquinavir (lanes 1 to 4). At 72 h after transfection, cells were lysed in radioimmunoprecipitation assay lysis buffer and separated by SDS–12% PAGE and then transferred to nitrocellulose filters. Viral proteins were visualized by immunoblotting with an HIV-1-positive human serum.