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Identification of the LWYIK motif located in the human immunodeficiency virus type 1 transmembrane gp41 protein as a distinct determinant for viral infection.

ABSTRACT: The highly conserved LWYIK motif located immediately proximal to the membrane-spanning domain of the gp41 transmembrane protein of human immunodeficiency virus type 1 has been proposed as being important for the surface envelope (Env) glycoprotein's association with lipid rafts and gp41-mediated membrane fusion. Here we employed substitution and deletion mutagenesis to understand the role of this motif in the virus life cycle. None of the mutants examined affected the synthesis, precursor processing, CD4 binding, oligomerization, or cell surface expression of the Env, nor did they alter Env incorporation into the virus. All of the mutants, particularly the DeltaYI, DeltaIK, and DeltaLWYIK mutants, in which the indicated residues were deleted, exhibited greatly reduced one-cycle viral replication and the Env trans-complementation ability. All of these deletion mutant proteins were still localized in the lipid rafts. With the exception of the Trp-to-Ala (WA) mutant, which exhibited reduced viral infectivity albeit with normal membrane fusion, all mutants displayed loss of some or almost all of the membrane fusion ability. Although these deletion mutants partially inhibited in trans wild-type (WT) Env-mediated fusion, they were more effective in dominantly interfering with WT Env-mediated viral entry when coexpressed with the WT Env, implying a role of this motif in postfusion events as well. Both T20 and L43L peptides derived from the two gp41 extracellular C- and N-terminal alpha-helical heptad repeats, respectively, inhibited WT and DeltaLWYIK Env-mediated viral entry with comparable efficacies. Biotin-tagged T20 effectively captured both the fusion-active, prehairpin intermediates of WT and mutant gp41 upon CD4 activation. Env without the deletion of the LWYIK motif still effectively mediated lipid mixing but inhibited content mixing. Our study demonstrates that the immediate membrane-proximal LWYIK motif acts as a unique and distinct determinant located in the gp41 C-terminal ectodomain by promoting enlargement of fusion pores and postfusion activities.

SUBMITTER: Chen SS

PROVIDER: S-EPMC2612407 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Identification of the LWYIK motif located in the human immunodeficiency virus type 1 transmembrane gp41 protein as a distinct determinant for viral infection.

Chen Steve S-L SS Yang Polung P Ke Po-Yuan PY Li Hsiao-Fen HF Chan Woan-Eng WE Chang Ding-Kwo DK Chuang Chin-Kai CK Tsai Yu Y Huang Shu-Chen SC

Journal of virology 20081105 2

The highly conserved LWYIK motif located immediately proximal to the membrane-spanning domain of the gp41 transmembrane protein of human immunodeficiency virus type 1 has been proposed as being important for the surface envelope (Env) glycoprotein's association with lipid rafts and gp41-mediated membrane fusion. Here we employed substitution and deletion mutagenesis to understand the role of this motif in the virus life cycle. None of the mutants examined affected the synthesis, precursor proces ...[more]

PMID: 18987155

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Project description:Human immunodeficiency virus (HIV) is membrane-enveloped, and an initial infection step is joining/fusion of viral and cell membranes. This step is catalyzed by gp41, which is a single-pass integral viral membrane protein. The protein contains an ∼170-residue ectodomain located outside the virus that is important for fusion and includes the fusion peptide (FP), N-helix, loop, C-helix, and viral membrane-proximal external region (MPER). The virion initially has noncovalent complexes between three gp41 ectodomains and three gp120 proteins. A gp120 contains ∼500 residues and functions to identify target T-cells and macrophages via binding to specific protein receptors of the target cell membrane. gp120 moves away from the gp41 ectodomain, and the ectodomain is thought to bind to the target cell membrane and mediate membrane fusion. The secondary and tertiary structures of the ectodomain are different in the initial complex with gp120 and the final state without gp120. There is not yet imaging of gp41 during fusion, so the temporal relationship between the gp41 and membrane structures is not known. This study describes biophysical and functional characterization of large gp41 constructs that include the ectodomain and transmembrane domain (TM). Significant fusion is observed of both neutral and anionic vesicles at neutral pH, which reflects the expected conditions of HIV/cell fusion. Fusion is enhanced by the FP, which in HIV/cell fusion likely contacts the host membrane, and the MPER and TM, which respectively interfacially contact and traverse the HIV membrane. Initial contact with vesicles is made by protein trimers that are in a native oligomeric state that reflects the initial complex with gp120 and also is commonly observed for the ectodomain without gp120. Circular dichroism data support helical structure for the N-helix, C-helix, and MPER and nonhelical structure for the FP and loop. Distributions of monomer, trimer, and hexamer states are observed by size-exclusion chromatography (SEC), with dependences on solubilizing detergent and construct. These SEC and other data are integrated into a refined working model of HIV/cell fusion that includes dissociation of the ectodomain into gp41 monomers followed by folding into hairpins that appose the two membranes, and subsequent fusion catalysis by trimers and hexamers of hairpins. The monomer and oligomer gp41 states may therefore satisfy dual requirements for HIV entry of membrane apposition and fusion.

Project description:The transmembrane subunit (TM) of human immunodeficiency virus type 1 (HIV-1) envelope protein contains four well-conserved sites for the attachment of N-linked carbohydrates. To study the contribution of these N-glycans to the function of TM, we systematically mutated the sites individually and in all combinations and measured the effects of each on viral replication in culture. The mutants were derived from SHIV-KB9, a simian immunodeficiency virus/HIV chimera with an envelope sequence that originated from a primary HIV-1 isolate. The attachment site mutants were generated by replacing the asparagine codon of each N-X-S/T motif with a glutamine codon. The mobilities of the variant transmembrane proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that all four sites are utilized for carbohydrate attachment. Transfection of various cell lines with the resulting panel of mutant viral constructs revealed that the N-glycan attachment sites are largely dispensable for viral replication. Fourteen of the 15 mutants were replication competent, although the kinetics of replication varied depending on the mutant and the cell type. The four single mutants (g1, g2, g3, and g4) and all six double mutants (g12, g13, g14, g23, g24, and g34) replicated in both human and rhesus monkey T-cell lines, as well as in primary rhesus peripheral blood mononuclear cells. Three of the four triple mutants (g124, g134, and g234) replicated in all cell types tested. The triple mutant g123 replicated poorly in immortalized rhesus monkey T cells (221 cells) and did not replicate detectably in CEMx174 cells. However, at 3 weeks posttransfection of 221 cells, a variant of g123 emerged with a new N-glycan attachment site which compensated for the loss of sites 1, 2, and 3 and resulted in replication kinetics similar to those of the parental virus. The quadruple mutant (g1234) did not replicate in any cell line tested, and the g1234 envelope protein was nonfunctional in a quantitative cell-cell fusion assay. The synthesis and processing of the quadruple mutant envelope protein appeared similar in transient assays to those of the parental SHIV-KB9 envelope. Given their high degree of conservation, the four N-linked carbohydrate attachment sites on the external domain of gp41 are surprisingly dispensable for viral replication. The viral variants described in this report should prove useful for investigation of the contribution of carbohydrate moieties on gp41 to recognition by antibodies, shielding from antibody-mediated neutralization, and structure-function relationships.

Dataset Information

Identification of the LWYIK motif located in the human immunodeficiency virus type 1 transmembrane gp41 protein as a distinct determinant for viral infection.

Publications

Identification of the LWYIK motif located in the human immunodeficiency virus type 1 transmembrane gp41 protein as a distinct determinant for viral infection.

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