Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 May;74(9):4433-40.
doi: 10.1128/jvi.74.9.4433-4440.2000.

Envelope glycoprotein determinants of increased fusogenicity in a pathogenic simian-human immunodeficiency virus (SHIV-KB9) passaged in vivo

B Etemad-Moghadam  1 , Y Sun, E K Nicholson, M Fernandes, K Liou, R Gomila, J Lee, J Sodroski
Affiliations

Envelope glycoprotein determinants of increased fusogenicity in a pathogenic simian-human immunodeficiency virus (SHIV-KB9) passaged in vivo

B Etemad-Moghadam et al. J Virol. 2000 May.

Abstract

Changes in the envelope glycoprotein ectodomains of a nonpathogenic simian-human immunodeficiency virus (SHIV-89.6) that was serially passaged in vivo have been shown to be responsible for the increased pathogenicity of the resulting virus, SHIV-KB9 (G. B. Karlsson, et al., J. Exp. Med. 188:1159-1171, 1998). The 12 amino acid changes in the envelope glycoprotein ectodomains resulted in increased chemokine receptor-binding and syncytium-forming abilities. Here we identify the envelope glycoprotein determinants of these properties. A single amino acid change in the gp120 third variable (V3) loop was both necessary and sufficient for the observed increase in the binding of the SHIV-KB9 gp120 glycoprotein to the CCR5 chemokine receptor. The increased syncytium-forming ability of SHIV-KB9 involved, in addition to the V3 loop change, changes in the second conserved (C2) region of gp120 (residue 225) and in the gp41 ectodomain (residues 564 and 567). The C2 and gp41 ectodomain changes influenced syncytium formation in a cooperative manner. Changes in the V1/V2 gp120 variable loops exerted a negative effect on syncytium formation and chemokine receptor binding, supporting a previously described role of these changes in immune evasion. The definition of the passage-associated changes that determine the efficiency of chemokine receptor binding and membrane fusogenicity will allow evaluation of the contribution of these properties to in vivo CD4-positive lymphocyte depletion.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Amino acid sequences of the 89.6 and KB9 envelope glycoproteins. The identity and position of the amino acid residues that were altered during animal passage of SHIV-89.6 are indicated. The locations of the amino acid residues within one of the variable (V) or conserved (C) regions of the gp120 envelope glycoprotein are also indicated. Numbering of envelope glycoprotein residues is according to current convention (33).
FIG. 2
FIG. 2
Syncytium-forming ability of envelope glycoprotein recombinants. 293T cells, transiently expressing the 89.6, KB9, or recombinant envelope glycoproteins, were cocultivated with either CEMx174 (A), SupT1 (B), or CD4+-enriched human PBMC (C) for 6 h at 37°C. The number of syncytia was scored and normalized to that observed for the wild-type 89.6 envelope glycoproteins (assigned a value of 1). The latter value corresponds to an average of 150, 200, and 112 syncytia/ml in assays performed with CEMx174, SupT1, and CD4+-enriched human PBMC, respectively. The number of syncytia observed with the negative control (ΔKS) was close to zero when either CEMx174 or SupT1 cell lines were used as targets and was 10-fold lower than that associated with the 89.6 envelope glycoproteins when CD4+-enriched human PBMC were used as target cells. The mean values and standard deviations for at least two independent experiments are shown.
FIG. 3
FIG. 3
Ability of selected amino acid changes to enhance syncytium formation. (A) Selected passage-associated changes were introduced singly, or in combination, into the 89.6 envelope glycoproteins. The syncytium-forming ability of these envelope glycoprotein variants was investigated. (B) The syncytium-forming ability of KB9 recombinant envelope glycoproteins with selected reversions, alone or in combination, was tested. Syncytium formation analysis was performed with CEMx174 cells as targets, and the number of syncytia was normalized to that observed for the 89.6 envelope glycoproteins (assigned a value of 1).
FIG. 4
FIG. 4
Effect of chemokine receptor usage on syncytium formation. The stable Cf2Th-luciferase cell line used in the assay was made by cotransfection of Cf2Th canine thymocytes with the pLTR-Luc plasmid, which expresses luciferase under the control of the HIV-1 long terminal repeat (61) and a plasmid conferring resistance to Neomycin. Stable transfectants were selected with 0.8 mg of Neomycin per ml. The Cf2Th-luciferase cell line was then transiently cotransfected with a pCEP4 plasmid encoding CD4 and a pcDNA3 plasmid encoding either CCR5 or CXCR4 (8). Approximately 36 h after transfection, the cells were used as targets in the syncytium formation assays by cocultivating them with 293T cells expressing the envelope glycoproteins and Tat. Luciferase activity was quantified by using the Luciferase Assay System (Promega). A defective envelope glycoprotein (ΔKS) was used as a negative control to account for any baseline luciferase activity in the Cf2Th-luciferase cells and for potential effects of Tat secreted from the transfected 293T cells. The background luciferase activity observed with ΔKS was 10- and 50-fold lower with the CCR5 and CXCR4-expressing cells, respectively. Luciferase activity in the lysate was calculated and normalized to that observed for the 89.6 envelope glycoproteins.
FIG. 5
FIG. 5
CCR5-binding ability of soluble recombinant envelope glycoproteins. Equivalent amounts of recombinant soluble gp120 envelope glycoproteins labelled with [35S]cysteine and [35S]methionine were preincubated with 0.5 μg of sCD4 per ml for 1 h at room temperature. Cf2Th-CCR5 cells (106 cells/well in six-well plates), which stably express human CCR5 (17), were overlayed with the gp120-sCD4 mixture and were incubated at 37°C for 2 h. The cells were subsequently washed with phosphate-buffered saline plus 2% fetal calf serum and were lysed with NP-40 buffer. The cell lysates were then immunoprecipitated with serum from an HIV-1-infected individual, and the amount of precipitated gp120 was determined by densitometry of sodium dodecyl sulfate-polyacrylamide gels. As a control, the binding assay was also performed with the 89.6 gp120 glycoprotein in the absence of sCD4 (Mock). Two independent clones of each recombinant envelope glycoprotein were used in the analysis, and the experiments were carried out at least twice.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alkhatib G, Combadiere C, Broder C C, Feng Y, Kennedy P E, Murphy P M, Berger E A. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science. 1996;272:1955–1958. - PubMed
    1. Barre-Sinoussi F, Chermann J C, Rey F, Nugeyre M T, Chamaret S, Gruest J, Dauguet C, Axler-Blin C, Vezinet-Brun F, Rouzioux C, Rozenbaum W, Montagnier L. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS) Science. 1983;220:868–871. - PubMed
    1. Brand D, Srinivasan K, Sodroski J. Determinants of human immunodeficiency virus type 1 entry in the CDR2 loop of the CD4 glycoprotein. J Virol. 1995;69:166–171. - PMC - PubMed
    1. Cao J, Park I W, Cooper A, Sodroski J. Molecular determinants of acute single-cell lysis by human immunodeficiency virus type 1. J Virol. 1996;70:1340–1354. - PMC - PubMed
    1. Chan D C, Fass D, Berger J M, Kim P S. Core structure of gp41 from the HIV envelope glycoprotein. Cell. 1997;89:263–273. - PubMed

MeSH terms