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.

Binding of Human Immunodeficiency Virus Type 1 Gag to Membrane: Role of the Matrix Amino Terminus

“…Mutations which block or impair HIV-1 Gag myristylation have been reported to disrupt virus assembly and reduce the efficiency of Gag processing (4,16,19,27,34). We previously demonstrated that the 6VR mutation did not impair Gag myristylation (33).…”

Evaluation of Novel Human Immunodeficiency Virus Type 1 Gag DNA Vaccines for Protein Expression in Mammalian Cells and Induction of Immune Responses

“…Immunoblotting and p24 assay. Three days after transfection, cell lysates were prepared as previously described (14). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by using standard methods.…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by using standard methods. Proteins were transferred to nitrocellulose (Schleicher & Schuell) by washing the gel and nitrocellulose in transfer buffer (50 mM NaCl, 1.8 mM EDTA, 10 mM Tris, pH 7.0) and making a sandwich of nitrocellulose sheets with the gel in the middle, wrapping with plastic wrap, and incubating overnight between two glass plates with a 2-kg weight on top, at 4°C (2a, [14][15][16]35). The blots were stained with HIV-1-positive human serum in a PBS solution with 3% nonfat dried milk.…”

Correlation between the immune response elicited in rabbits byenv-recombinant avipox vaccines and the inhibition of HIV-1-specific functions

“…The presence of antibodies against several linear sequences showed the immunogenicity of both CP and FP recombinant vectors. The presence of antibodies against the p55 gag precursor may be ascribed to the use of HIV‐infected cells for challenge: while the main core antigen in cell‐free virus is p24, in infected cells the main core antigen is p55 [27]. Recognition by rabbit CTLs of xenogeneic human E‐line cells would cause their lysis and exposure of the intracellular p55 gag precursor, which would in turn be recognized and presented by antigen‐presenting cells in association with MHC class II molecules.…”