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. 2008 Jun;82(11):5460-71.
doi: 10.1128/JVI.01049-07. Epub 2008 Mar 19.

An alteration of human immunodeficiency virus gp41 leads to reduced CCR5 dependence and CD4 independence

Brian M Taylor  1 , J Scott Foulke, Robin Flinko, Alonso Heredia, Anthony DeVico, Marvin Reitz
Affiliations

An alteration of human immunodeficiency virus gp41 leads to reduced CCR5 dependence and CD4 independence

Brian M Taylor et al. J Virol. 2008 Jun.

Abstract

Human immunodeficiency virus (HIV) type 1 infection requires functional interactions of the viral surface (gp120) glycoprotein with cell surface CD4 and a chemokine coreceptor (usually CCR5 or CXCR4) and of the viral transmembrane (gp41) glycoprotein with the target cell membrane. Extensive genetic variability, generally in gp120 and the gp41 ectodomain, can result in altered coreceptor use, fusion kinetics, and neutralization sensitivity. Here we describe an R5 HIV variant that, in contrast to its parental virus, infects T-cell lines expressing low levels of cell surface CCR5. This correlated with an ability to infect cells in the absence of CD4, increased sensitivity to a neutralizing antibody recognizing the coreceptor binding site of gp120, and increased resistance to the fusion inhibitor T-20. Surprisingly, these properties were determined by alterations in gp41, including the cytoplasmic tail, a region not previously shown to influence coreceptor use. These data indicate that HIV infection of cells with limiting levels of cell surface CCR5 can be facilitated by gp41 sequences that are not exposed on the envelope ectodomain yet induce allosteric changes in gp120 that facilitate exposure of the CCR5 binding site.

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Figures

FIG. 1.
FIG. 1.
HXBaLm1.2 infects T-cell lines. CEM-SS cells (A) or H9 cells (B) were infected with the indicated viruses or not infected (control). H9 cells (C) or Sup-T1 cells (D) were infected with the indicated viruses without added chemokines or in the presence of CCR5 ligands (MIP-1Ξ² or RANTES) or the CXCR4 ligand SDF. p24 antigen in supernatants was measured by ELISA on the indicated days after infection. Results are from representative experiments. Error bars show standard deviations determined on replicate wells.
FIG. 2.
FIG. 2.
HXBaLm1.2 gp41 DNA sequence. Shown is a comparison of the gp41-coding regions of HXBaLm1.2 and its T-cell-line-competent derived clone HXBaLm6133 with those of the parental virus and wild-type HIV-1(Ba-L). Functional domains are outlined with boxes. FD, fusion domain. Arrows denote predicted sites of palmitoylation. Dashes indicate amino acid identity; letters indicate differences. The numbers above the alignment are the amino acid residue numbers based on HIV-1(Ba-L) gp160.
FIG. 3.
FIG. 3.
T-cell line competence is determined by gp41. The top panel shows a linear representation of env genes of HXBaLm1.2 and derived viruses. Shaded boxes denote HXB2 sequence; white boxes denote Ba-L sequence. gp120 and gp41 are not to scale. FD, fusion domain. The bottom panel shows the ability of these viruses to replicate on the indicated cell lines. (A) Surface expression levels of CCR5 and CD4 on the indicated T-cell lines were determined by flow cytometry using Quantibrite PE microbeads as described in Materials and Methods. (B) Relative levels of infection were judged by medium p24 at 6 days after infection. Infections were performed in triplicate. βˆ’, p24 not detectable; +, 0. 050 to 0.500 ng/ml p24; ++, 0.501 to 0.750 ng/ml p24; +++, β‰₯0.750 ng/ml p24.
FIG. 4.
FIG. 4.
HXBaLm1.2 infects cells expressing low levels of CCR5. (A) Surface expression of CD4 and CCR5 on U87.DOX.CCR5 cells was measured as a function of doxycycline (Dox) concentration by flow cytometry using Quantibrite PE microbeads as described in Materials and Methods. Also shown are the mean channel fluorescence and percent positive cells for CCR5. (B) Infection of U87.DOX.CCR5 cells was measured as a function of cell surface CCR5 expression. Medium p24 antigen was measured at day 3 after infection. Results are from a representative experiment. Standard deviations were determined from triplicate wells. (C) Infection of JC cells expressing different CCR5 levels was measured by p24 at 7 and 10 days after infection. Results are from a representative experiment. Standard deviations were determined from triplicate wells.
FIG. 5.
FIG. 5.
HXBaLm1.2 virions do not incorporate excess gp120. Virions were purified by sucrose cushion and Optiprep (6 to 18% iodixanol) velocity gradient. Protein profiles were analyzed by immunoblotting with anti-gp120 and anti-p24 antibodies. PM1, uninfected negative control; rgp120, purified recombinant gp120 protein. Bands were quantified by densitometric analysis. Shown is the ratio of gp120 to p24 expression for the indicated viruses.
FIG. 6.
FIG. 6.
HXBaLm1.2 is resistant to a fusion inhibitor but not entry inhibitors. PM1 cells were infected by HXBaL or HXBaLm1.2 and infection measured 3 days later by medium p24 levels as described in Materials and Methods. Cells were treated with the indicated concentrations of TAK-779 (A), βˆ’2 RANTES (B), or T-20 (C) as described in Materials and Methods. Results are from representative experiments. Error bars show standard deviations determined from triplicate wells.
FIG. 7.
FIG. 7.
Infection of T-cell lines is CCR5 dependent. T-cell lines were infected with HXB2 or HXBaLm6133 as described in Materials and Methods in the absence or presence of 500 pM or 500 nM TAK-779. Six days after infection, virus production was determined by ELISA of medium p24. (A) SupT1 cells were infected with BaLm6133; (B) SupT1 cells were infected with HXB2; (C) H9 cells were infected with BaLm6133; (D) H9 cells were infected with HXB2. All samples were run in triplicate; error bars show standard deviations.
FIG. 8.
FIG. 8.
HXBaLm1.2 infects cells not expressing CD4. Cells were infected with HXBaL, HXBaLm1.2, HXBaLm6133, or BaL10001 [derived from HIV(BaL) by passage on CD4βˆ’ cells cocultured with progressively decreasing levels of CD4+ cells as described in Materials and Methods]. Infection was monitored by medium p24 at the indicated times after infection. Target cells were BC7/CCR5 CD4-negative cells (A) or Cf2th/synCCR5 CD4-negative cells (B). Results are from representative experiments. Error bars show standard deviations determined from triplicate wells.
FIG. 9.
FIG. 9.
HXBaLm1.2 is more sensitive to sCD4 and the neutralizing MAb 17b. U373/CD4/MAGI cells expressing CCR5 were infected with HXBaL or HXBaLm6133. Virus was treated with sCD4 (A), 17B (B), 2G12 (C), or control IgG (D) as described in Materials and Methods. Ξ²-gal activity was measured at 4 days postinfection. Results are normalized for values from control samples with no antibodies or sCD4, and IC50 were determined. Results are from representative experiments. Error bars show standard deviations determined from triplicate wells. The table shows the calculated IC50, with the 95% confidence intervals for CD4, 17B, and 2G12 indicated in parentheses.
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