Effect of Point Mutations in the N Terminus of the Lentivirus Lytic Peptide-1 Sequence of Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41 on Env Stability

Lin

Chen

2005

J Virol

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Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4 ؉ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T-and M-tropic mutants also productively replicated in human primary CD4 ؉ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.

Section: Methodsmentioning

confidence: 99%

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

Lin

Chen

2005

J Virol

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“…For the construction of cytoplasmic tail elongation mutants, an NruI site was first introduced in between the C-terminal Leu-856 codon and the stop codon of the env gene of the HXB2RU3 provirus (44) by oligonucleotide-directed, site-specific mutagenesis with PCR overlap extension as previously described (27). Oligonucleotides 5Ј-TTGGAAAGGATTTTGCTATCGCGATAAGATGGGTGGCAA GTGGTC-3Ј (sense) and 5Ј-CACTTGCCACCCATCTTATCGCGATAGCAA AATCCTTTCCAAGCCCTG-3Ј (antisense) (sequences in italic mark the NruI recognition site) were used as the internal paired primers to generate the NruI site.…”

Section: Methodsmentioning

confidence: 99%

“…The three highly conserved amphipathic ␣-helical segments, designated lentiviral lytic peptide 1 (LLP)-1, LLP-2, and LLP-3, located in the C terminus of the cytoplasmic tail are thought to be associated with the inner surfaces of viral and cellular membranes (33,45). We previously showed that the multimerization potential and membrane binding ability of the cytoplasmic tail may play a crucial role in virus replication (3,5,7,28) and that the N-terminal segment of LLP-1 contains a structural determinant critical for modulating Env stability (27).…”

mentioning

confidence: 99%

“…Metabolic labeling with [ 35 S]methionine and chase with excess unlabeled methionine were performed as previously described (27). For cell surface biotinylation, transfected cells were labeled with [ 35 S]methionine for 3 h and then biotinylated with 0.25 mg of membrane impermeable sulfo-N-hydroxysuccinimide-biotin per ml on ice for 30 min as previously described (37).…”

mentioning

confidence: 99%

“…Cell and virus lysates obtained from transfection or VSV G pseudotype infection were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting with murine Mabs 902, Chessie 8, and 183 as previously described (27). Virus production was measured by reverse transcriptase activity as previously described (4).…”

mentioning

confidence: 99%

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

Wang

Lin

et al. 2004

J Virol

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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.

The dominant-negative action of a fusion protein containing the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 in virus replication

Chen

2007

J Biomed Sci

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We previously described a novel mode of downregulation of human immunodeficiency virus type 1 (HIV-1) Gag expression by a cytoplasmic domain fusion protein of the envelope (Env) transmembrane protein, beta-galactosidase (beta-gal)/706-856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli beta-gal. In the present study, we showed that this mediator conferred a dose-dependent dominant interference with virus infectivity. In the context of an HIV-1 provirus, this inhibitor downregulated steady-state Env expression. Paradoxically, Env overexpression suppressed beta-gal/706-856-mediatd Gag downregulation. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag, Env, and beta-gal/706-856 had stable interactions and formed aggregated complexes in perinuclear regions. Moreover, Env overexpression hindered colocalization of Gag with beta-gal/706-856 in the perinuclear region. Further cytoplasmic domain mapping analyses showed a correlation between the ability of cytoplasmic subdomains to downregulate Gag expression and the ability of these subdomains to stably interact with Gag. These studies show that redirection of Gag from its cytoplasmic synthesis site to a perinuclear compartment is a prerequisite for beta-gal/706-856-mediated Gag downregulation. The results also illustrate that the dynamic interplay among Gag, Env, and beta-gal/706-856 can modulate Gag and Env expression, thus controlling HIV-1 infection.