Cytolysis by CCR5-using human immunodeficiency virus type 1 envelope glycoproteins is dependent on membrane fusion and can be inhibited by high levels of CD4 expression

Abstract

T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.

FIG. 1.

Receptor expression on cell lines used in the study. Cf2Th cell lines and primary human CD4⁺ peripheral blood lymphocytes were stained for expression of the CD4 glycoprotein and/or the CCR5 glycoprotein. Cf2Th-CD4^hi/CCR5 and Cf2Th-CD4^lo/CCR5 cells were stained for CD4 and CCR5 expression. The Cf2Th-CCR5 cells were stained only for CCR5 expression, and the primary blood lymphocytes were stained only for CD4. Staining for CCR5 expression was performed with the 2D7 antibody, and staining for CD4 expression was performed with the Q4120 antibody. An isotype-matched antibody was used as a negative control (shaded peaks) and yielded staining similar to that observed for parental Cf2Th cells stained with the Q4120 antibody (data not shown).

FIG. 2.

Effects of R5 and R5X4 HIV-1 envelope glycoprotein expression on viability of cells expressing CCR5 and different levels of CD4. The percentage of EGFP-positive cells in Cf2Th-CD4^hi/CCR5 (upper row), Cf2Th-CD4^lo/CCR5 (middle row), and Cf2Th-CCR5 (bottom row) cultures transduced by recombinant HIV-1 vectors expressing the indicated HIV-1 envelope glycoproteins or a control Env(−) vector is shown. The HIV-1 envelope glycoproteins in the experiments shown in the left column are R5 envelope glycoproteins, and those in the experiments shown in the right column are R5X4 envelope glycoproteins. The percentage of EGFP-positive cells in each culture 48 h after incubation with recombinant viruses was designated as 100%, and subsequent percentages were normalized to this value. The F522Y variants (designated F/Y) are also shown except for YU2 F/Y in the Cf2Th-CD4^lo/CCR5 cultures, which had EGFP-positive percentages that exceeded the scale of the graph and were left out for the sake of clarity for the remaining samples.

FIG. 3.

Ability of Cf2Th-CD4^hi/CCR5 and Cf2Th-CCR5 cells to participate in syncytium formation. (A) The ability of Cf2Th-CD4^hi/CCR5 (black bars) and Cf2Th-CCR5 cells (white bars) to serve as target cells for syncytium formation was examined. 293T cells were transduced with single-round HIV-1 vectors expressing the indicated envelope glycoproteins or with an Env(−) control vector. Twenty-four hours later, the 293T cells were split into two plates, and each was cocultivated at 37°C with either Cf2Th-CD4^hi/CCR5 or Cf2Th-CCR5 cells. Twenty-four hours later, syncytia were counted by microscopy. (B) Syncytium formation resulting from HIV-1 envelope glycoprotein expression within Cf2Th-CD4^hi/CCR5 and Cf2Th-CCR5 cells. Cf2Th-CD4^hi/CCR5 and Cf2Th-CCR5 cells cultured at high density (10⁵ cells in six-well plates) were transduced with recombinant HIV-1 vectors expressing the indicated HIV-1 envelope glycoproteins. The Env(−) control vector does not express HIV-1 envelope glycoproteins. Syncytia were scored 48 h later by light microscopy.

FIG. 4.

Increase in cytotoxicity of a CD4-independent HIV-1 envelope glycoprotein in CD4-expressing cells due to a decrease in CD4-binding ability. Cf2Th-CCR5 cells (A) and Cf2Th-CD4^hi/CCR5 cells (B) were infected with recombinant HIV-1 vectors expressing either ADAΔV1/V2 or ADAΔV1/V2 D368R envelope glycoproteins or with a control vector that does not express HIV-1 envelope glycoproteins [Env(−)]. All of the vectors expressed EGFP. The percentage of EGFP-positive cells in the culture 48 h after transduction was considered 100%, and the percentages of EGFP-positive cells in each culture at subsequent time points were normalized to this value.

FIG. 5.

Decrease in HIV-1 envelope glycoprotein precursor processing by CD4 binding. Cf2Th-CD4^lo/CCR5, Cf2Th-CD4^hi/CCR5, and Cf2Th-CCR5 cells were infected with recombinant HIV-1 vectors expressing the indicated HIV-1 envelope glycoproteins. Twenty-four hours later, the cells were washed and labeled with [³⁵S]methionine. Twenty-four hours later, the cells were lysed, and the lysates were used for immunoprecipitation by pooled sera from HIV-1-infected individuals. Precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (A and B). The gp160 envelope glycoprotein precursor and the mature gp120 envelope glycoprotein are indicated. (C) The gel image in B was analyzed with ImageQuant software. The background present in each lane was determined and subtracted from the values obtained for each band of interest. Cleavage was calculated as [gp120/(gp120 + gp160)] × 100. Repetition of this experiment yielded similar results.

FIG. 6.

Generation of inactivated HIV-1 virions. (A) Virions that incorporated the HXBc2, HXBc2-P, 89.6, or KB9 envelope glycoproteins as well as the F522Y fusion-defective variants of these envelope glycoproteins were made by cotransfecting the three plasmids shown. The recombinant viruses were inactivated by UV irradiation. (B) Effect of the duration of UV treatment on the number of EGFP-positive cells that resulted from incubation of the viruses with Jurkat T lymphocytes is shown. (C) Effect of duration of UV irradiation on the reverse transcriptase (RT) activity measured in pelleted virions is shown. (D) Jurkat T lymphocytes were incubated with the recombinant HIV-1 vector containing the vesicular stomatitis virus G protein envelope glycoproteins either after no UV irradiation or after 20 min of UV irradiation. The EGFP-positive cells were visualized. Equivalent numbers of Jurkat cells were present in both fields (data not shown). Cells incubated with the recombinant HIV-1 vector expressing HXBc2 envelope glycoprotein showed similar results (data not shown).

FIG. 7.

Assay for detecting cytotoxicity. Jurkat T lymphocytes were stained with propidium iodide (PI) and by TUNEL and analyzed by fluorescence-activated cell sorting. The results with untreated Jurkat cells are shown in the upper left panel. The other panels show the results obtained with Jurkat cells treated for 16 h with the indicated concentrations of staurosporine.

FIG. 8.

Effect of recombinant HIV-1 virions lacking genomic RNA on primary CD4⁺ T cells. To produce virions lacking genomic RNA, 293T cells were transfected with the pCMVΔP1ΔenvpA plasmid and, in some cases, with the pSVIIIenv plasmid expressing the indicated HIV-1 envelope glycoproteins or a plasmid expressing the vesicular stomatitis virus G protein glycoprotein. Virions lacking envelope glycoproteins (Gag/Pol), medium from cells transfected with plasmids expressing envelope glycoproteins (HXBc2 Env and vesicular stomatitis virus G protein Env), or medium from untransfected 293T cells (medium) were used as negative controls. CD4⁺ T cells were isolated by negative selection from human peripheral blood mononuclear cells and stimulated with phytohemagglutinin and interleukin-2 for 3 days. The cells were then incubated with recombinant HIV-1 virions lacking genomic RNA at 37°C. On the indicated days, a TUNEL assay was performed, with the results shown.