Effect of Extension of the Cytoplasmic Domain of Human Immunodeficiency Type 1 Virus Transmembrane Protein gp41 on Virus Replication

Abstract

The biological significance of the presence of a long cytoplasmic domain in the envelope (Env) transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) is still not fully understood. Here we examined the effects of cytoplasmic tail elongation on virus replication and characterized the role of the C-terminal cytoplasmic tail in interactions with the Gag protein. Extensions with six and nine His residues but not with fewer than six His residues were found to severely inhibit virus replication through decreased Env electrophoretic mobility and reduced Env incorporation compared to the wild-type virus. These two mutants also exhibited distinct N glycosylation and reduced cell surface expression. An extension of six other residues had no deleterious effect on infectivity, even though some mutants showed reduced Env incorporation into the virus and/or decreased cell surface expression. We further show that these elongated cytoplasmic tails in a format of the glutathione S-transferase fusion protein still interacted effectively with the Gag protein. In addition, the immediate C terminus of the cytoplasmic tail was not directly involved in interactions with Gag, but the region containing the last 13 to 43 residues from the C terminus was critical for Env-Gag interactions. Taken together, our results demonstrate that HIV-1 Env can tolerate extension at its C terminus to a certain degree without loss of virus infectivity and Env-Gag interactions. However, extended elongation in the cytoplasmic tail may impair virus infectivity, Env cell surface expression, and Env incorporation into the virus.

Wild-Type-Like Viral Replication Potential of Human Immunodeficiency Virus Type 1 Envelope Mutants Lacking Palmitoylation Signals

“…CEM-SS, PM1, and H938 cells were cultured in RPMI 1640 supplemented with 10% FBS. Hybridomas 902, Chessie 8, and 183 (clone H12-5C), which produce mouse monoclonal antibodies (MAbs) reactive with HIV-1 gp120, gp41, and p24, respectively, and sheep anti-gp120 were as previously described (9). The SIM2 hybridoma secreted a MAb that specifically recognizes human CD4.…”

“…Transfected HeLa cells grown in 6-cm petri dishes were metabolically labeled with [ 35 S]methionine overnight, and labeled cells were surface biotinylated with 0.25 mg/ml of membrane-impermeable sulfo-N-hydroxysuccinimide-biotin. The levels of total and cell surface Env expressions were then assessed as previously described (9,25). For palmitoylation studies, a set of transfected dishes was labeled with […”

“…Two days after transfection, cells were divided into two portions. One portion of cells was used to determine total Env expression, and another portion of cells was processed for Env cell surface expression by fluorescence-activated cell sorter (FACS) analysis according to a procedure described previously (9).…”

“…To examine viral protein expression of these three Cys-to-Ser mutants in CD4 ϩ T cells, which are natural target cells for HIV-1 infection, a high-level transient HIV-1 expression system based on pseudotyping with the VSV G protein (4, 31) was employed. This method has been shown to effectively express viral proteins in suspended T cells (9,25,40), which are usually transfected less efficiently than is required for biochemical analyses when other commonly used transfection methods are used. Equal amounts of cell-free VSV G-transcomplemented viruses obtained from each cotransfection and after normalization by RT activity were used to challenge CEM-SS cells.…”

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

“…Mutagenesis analyses suggest that the matrix substitution L49D destabilizes the GP120-GP41 interaction, but this impairment can be rescued by a Y710S substitution at the GP41CT (84). The last 13 to 43 amino acid positions in the GP41CT are critical for the GP41 Env -matrix Gag interaction (73). In addition, GP41CT mutations may confer resistance to HIV protease inhibitors (PIs) (85), a mechanism which is described in detail below.…”