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. 2003 Jan;77(1):560-70.
doi: 10.1128/jvi.77.1.560-570.2003.

Concordant modulation of neutralization resistance and high infectivity of the primary human immunodeficiency virus type 1 MN strain and definition of a potential gp41 binding site in gp120

Maria Leavitt  1 , Eun Ju Park, Igor A Sidorov, Dimiter S Dimitrov, Gerald V Quinnan Jr
Affiliations

Concordant modulation of neutralization resistance and high infectivity of the primary human immunodeficiency virus type 1 MN strain and definition of a potential gp41 binding site in gp120

Maria Leavitt et al. J Virol. 2003 Jan.

Abstract

Efforts to develop a vaccine against human immunodeficiency virus type 1 (HIV-1) are complicated by resistance of virus to neutralization. The neutralization resistance phenotype of HIV-1 has been linked to high infectivity. We studied the mechanisms determining this phenotype using clones of the T-cell-line-adapted (TCLA) MN strain (MN-TCLA) and the neutralization-resistant, primary MN strain (MN-P). Mutations in the amino- and carboxy-terminal halves of gp120 and the carboxy terminus of gp41 contributed to the neutralization resistance, high-infectivity phenotype but depended upon sequences in the leucine zipper (LZ) domain of gp41. Among 23 clones constructed to map the contributing mutations, there was a very strong correlation between infectivity and neutralization resistance (R(2) = 0.81; P < 0.0001). Mutations that distinguished the gp120s of MN-P and MN-TCLA clones were clustered in or near the CD4 and coreceptor binding sites and in regions distant from those binding sites. To test the hypothesis that some of these distant mutations may interact with gp41, we determined which of them contributed to high infectivity and whether those mutations modulated gp120-gp41 association in the context of MN-P LZ sequences. In one clone, six mutations in the amino terminus of gp120, at least four of which clustered closely on the inner domain, modulated infectivity. This clone had a gp120-gp41 association phenotype like MN-P: in comparison to MN-TCLA, spontaneous dissociation was low, and dissociation induced by soluble CD4 binding was high. These results identify a region of the gp120 inner domain that may be a binding site for gp41. Our studies clarify mechanisms of primary virus neutralization resistance.

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Figures

FIG. 1.
FIG. 1.
Alignment of amino acid sequences of envelopes of neutralization-sensitive (MN-TCLA), highly neutralization-resistant (MN-P and MN-P14), and partially neutralization-resistant (MN-E6) clones of the HIV-1 MN strain. The bars above the amino acid sequences indicate the approximate locations of regions of the glycoproteins, as follows: C1, C2, C3, and C4, constant regions of gp120; V1/V2, V3, V4, and V5, variable regions of gp120; gp41 regions, fusion peptide (FP), LZ, AH (membrane proximal), TM, and CT. Positions of amino acids are numbered as in MN-P, and amino acids differing from consensus are boxed and shaded. Residues numbers appear in the boxes at locations corresponding to restriction enzyme cleavage sites illustrated in Fig. 2.
FIG. 2.
FIG. 2.
The gp41 LZ interacts functionally with multiple regions of gp120 and gp41 to determine the neutralization resistance, high-infectivity phenotype. (A) Restriction endonuclease cleavage map of the MN-TCLA clone indicating nucleotide positions of cleavage sites for specific enzymes. Below the map are indications of the approximate locations of regions of the glycoproteins, as follows: C1, C2, C3, and C4, constant regions of gp120; V1/V2, V3, V4, and V5, variable regions of gp120; gp41 regions, fusion peptide (FP), LZ, AH (membrane proximal), TM, and CT. (B) Schematic description of a series of chimeric genes (Chim) constructed by exchanging segments of the MN-TCLA and MN-P clones, as described in Materials and Methods. (C) Relative infectivity and neutralization (Neut.) titers obtained for the clones. Relative infectivity was determined as XM/XMN-TCLA (calculated using Excel), where X is the virus dilution that yields a given activity, XM is the value obtained for the particular mutant studied, and XMN-TCLA is the value obtained for MN-TCLA. Best-fit lines were determined by regression analysis of the log-transformed luciferase activity determinations (light units) as a function of inoculum dilutions. y values used for comparisons of MN-TCLA and other clones were selected so as to intersect approximately linear segments of the curves being compared. Neutralization phenotypes of chimeras were determined by using HIV HNS2. Titers are the reciprocal serum dilutions resulting in ≥90% inhibition of infectivity. Each chimeric clone was tested three to five times in comparison to MN-P and MN-TCLA, and geometric mean results are presented.
FIG. 3.
FIG. 3.
Correlation between neutralization resistance and infectivity of viruses pseudotyped with mutant and chimeric MN strain envelope clones. Data shown in Fig. 1 were analyzed in Excel. The null hypothesis of no correlation was rejected with a P of <0.0001.
FIG. 4.
FIG. 4.
Localization of MN-P/MN-TCLA mutations in the atomic structure of HIV-1 gp120 core complexed with CD4 and a neutralizing antibody, 17b. The PDF file is from the Protein Data Bank, 1GC1 (21) and drawn with PCMolecule2 (version 2.0.0; Molecular Ventures, Inc.). The front (A) and back (B) sides of the molecular complex are shown. The gp120 is shown in blue, CD4 is shown in green, and the 17b antibody is shown in yellow (light chain) and in pale blue (heavy chain). The amino acid sequence of gp120 was extracted from the PDF file and aligned with the sequence of MN-TCLA by Clustal W (version 1.7; National Institutes of Health). Mutation sites are colored red and marked as MN-P amino acid/MN-TCLA amino acid followed by the position number of each mutation in the MN-P sequence. Locations of V1/V2 and V3 loops are also indicated.
FIG. 5.
FIG. 5.
Effects of inner-domain and selected outer-domain mutations on infectivity. The restriction enzyme cleavage map of MN-TCLA, shown at top, is described in the legend to Fig. 2. Schematic diagrams of chimeric env genes constructed using fragments of the MN-TCLA and MN-P genes are shown on the left. Stars above the diagrams of chimeras (Chim) indicate the approximate locations of mutations introduced in vitro (see text for description). Relative infectivities of viruses pseudotyped with envelopes encoded by the chimeric genes are shown to the right.
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