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. 1999 Mar;73(3):2469-80.
doi: 10.1128/JVI.73.3.2469-2480.1999.

A conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for Env-mediated fusion and virus infectivity

K Salzwedel  1 , J T West, E Hunter
Affiliations

A conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for Env-mediated fusion and virus infectivity

K Salzwedel et al. J Virol. 1999 Mar.

Abstract

Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an alpha-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.

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Figures

FIG. 1
FIG. 1
Sequence conservation and predicted structure of the tryptophan-rich region. (A) Schematic representation of gp41, showing the location and sequence variability of the tryptophan-rich region and the overlapping sequences of the 2F5 epitope and DP-178 peptide. Shaded regions indicate the N-terminal fusion peptide and the membrane-spanning domain. The amino acids in the HIV-1 sequence database which occur at each position are denoted underneath the respective amino acid in the HXB2 strain. (B) Alignment of the tryptophan-rich sequence of HIV-1 HXB2 gp41 with homologous regions of HIV-2, SIV, and visna virus glycoproteins. Conserved tryptophan residues are boxed. (C) Helical net projection of the tryptophan-rich region. The outlined region indicates the cluster of conserved hydrophobic residues shown in black.
FIG. 2
FIG. 2
Diagram of mutations and their corresponding fusogenicities. The Env mutations within the tryptophan-rich region are diagrammed in the left panel, juxtaposed to the corresponding name of the mutant and the cell-cell fusion data from a representative experiment. Amino acid changes are underlined, and deletions are denoted by periods. COS-1 cells expressing glycoprotein were mixed 1:10 with HeLa-CD4/LTR-β-gal cells and replated. The cells were stained 24 h later with X-Gal, and blue syncytia were quantitated microscopically. The average number of syncytia per low-power field was determined by counting six nonoverlapping fields per well for each of two wells and averaging the total. The average number of nuclei per syncytium was determined by quantitating 15 syncytia in each of two wells and averaging the total. Wild-Type (A) and Wild-Type (B) indicate results from two separately prepared plasmid clones.
FIG. 3
FIG. 3
Expression and maturation of envelope glycoprotein mutants. SV40-based env expression plasmids were transfected into COS-1 cells. Cells were labeled with [35S]methionine-cysteine, and HIV-1 glycoproteins were immunoprecipitated as described in Materials and Methods from the cell lysate (C) or culture medium (M) by using AIDS patient serum for analysis by SDS-PAGE (8% polyacrylamide). Mock, transfected with wild-type env plasmid lacking a eukaryotic transcriptional promoter.
FIG. 4
FIG. 4
CD4-binding capability of envelope glycoprotein mutants. Radiolabelled HIV-1 glycoproteins were immunoprecipitated from one-half of the cell lysate with CD4-IgG (bottom panel) and from the other half of the lysate with patient serum (top panel). The immunoprecipitates were analyzed by SDS-PAGE (8% polyacrylamide).
FIG. 5
FIG. 5
Biotinylation of envelope glycoprotein mutants expressed on the cell surface. Proteins on the surface of metabolically radiolabeled glycoprotein-expressing cells were biotinylated with NHS-SS-biotin. Viral proteins were isolated by immunoprecipitation with patient serum, and a portion of the immunoprecipitate was analyzed by SDS-PAGE (top panel). The remaining immunoprecipitate was boiled to denature antibodies and incubated with streptavidin-agarose. Following washing, biotinylated proteins were released by reducing agent and analyzed by SDS-PAGE (8% polyacrylamide) (bottom panel). ERRS is a modified HIV-1 glycoprotein containing an endoplasmic reticulum retrieval signal attached to the C terminus of its cytoplasmic tail.
FIG. 6
FIG. 6
Expression of envelope glycoprotein mutants in the context of virus. Radiolabeled viral proteins were immunoprecipitated from COS-1 cells expressing pNL4-3 proviral constructs by using patient serum and analyzed by SDS-PAGE (9% polyacrylamide). The positions of the Pr55gag and p24gag products are indicated to the left.
FIG. 7
FIG. 7
Virus entry and cell-cell fusion in the context of virus. (A) Culture medium from cells expressing virus was filtered and normalized for reverse transcriptase activity. The normalized medium was used to infect duplicate wells (12-well plates) of HeLa-CD4/LTR-β-gal cells. Cells were stained with X-Gal in situ, and the numbers of blue foci (syncytia or single cells) were quantitated microscopically. Virus entry was quantitated as the average number of blue foci per milliliter by first counting the number of blue foci per 16-mm2 field (low power) for six nonoverlapping fields in each of two wells. The average number of blue foci per field was then multiplied by the total number of fields per well, and the result was corrected for the volume of reverse transcriptase-normalized virus supernatant used. (B) Cell-cell fusion was determined as in the legend to Fig. 2, except that proviral constructs were expressed in 293T cells and mixed with HeLa-CD4/LTR-β-gal cells. Wild-Type (XUN) [A] and Wild-Type (XUN) [B] indicate results from two separately prepared pNL4-3/XUN plasmid clones. pNL4-3Δenv, pNL4-3 containing a frameshift mutation in gp120.
FIG. 8
FIG. 8
Incorporation of envelope glycoprotein mutants into virions. Supernatants were collected from metabolically radiolabeled 293T cells expressing pNL4-3 proviral constructs and filtered to remove cellular debris. Virus was pelleted from the filtered supernatants through a sucrose cushion and solubilized in lysis buffer. Viral proteins were immunoprecipitated and analyzed by SDS-PAGE (9% polyacrylamide).
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