Differential CD4/CCR5 utilization, gp120 conformation, and neutralization sensitivity between envelopes from a microglia-adapted human immunodeficiency virus type 1 and its parental isolate

Abstract

Human immunodeficiency virus type 1 (HIV-1) infects and induces syncytium formation in microglial cells from the central nervous system (CNS). A primary isolate (HIV-1(BORI)) was sequentially passaged in cultured microglia, and the isolate recovered (HIV-1(BORI-15)) showed high levels of fusion and replicated more efficiently in microglia (J. M. Strizki, A. V. Albright, H. Sheng, M. O'Connor, L. Perrin, and F. González-Scarano, J. Virol. 70:7654-7662, 1996). The parent and adapted viruses used CCR5 as coreceptor. Recombinant viruses demonstrated that the syncytium-inducing phenotype was associated with four amino acid differences in the V1/V2 region of the viral gp120 (J. T. C. Shieh, J. Martin, G. Baltuch, M. H. Malim, and F. González-Scarano, J. Virol. 74:693-701, 2000). We produced luciferase-reporter, env-pseudotyped viruses using plasmids containing env sequences from HIV-1(BORI), HIV-1(BORI-15), and the V1/V2 region of HIV-1(BORI-15) in the context of HIV-1(BORI) env (named rBORI, rB15, and rV1V2, respectively). The pseudotypes were used to infect cells expressing various amounts of CD4 and CCR5 on the surface. In contrast to the parent recombinant, the rB15 and rV1V2 pseudotypes retained their infectability in cells expressing low levels of CD4 independent of the levels of CCR5, and they infected cells expressing CD4 with a chimeric coreceptor containing the third extracellular loop of CCR2b in the context of CCR5 or a CCR5 Delta4 amino-terminal deletion mutant. The VH-rB15 and VH-rV1V2 recombinant viruses were more sensitive to neutralization by a panel of HIV-positive sera than was VH-rBORI. Interestingly, the CD4-induced 17b epitope on gp120 was more accessible in the rB15 and rV1V2 pseudotypes than in rBORI, even before CD4 binding, and concomitantly, the rB15 and rV1V2 pseudotypes were more sensitive to neutralization with the human 17b monoclonal antibody. Adaptation to growth in microglia--cells that have reduced expression of CD4 in comparison with other cell types--appears to be associated with changes in gp120 that modify its ability to utilize CD4 and CCR5. Changes in the availability of the 17b epitope indicate that these affect conformation. These results imply that the process of adaptation to certain tissue types such as the CNS directly affects the interaction of HIV-1 envelope glycoproteins with cell surface components and with humoral immune responses.

FIG. 1

Microglial infection. Human adult microglial cultures were established as previously described (1, 2, 95, 116) and infected with 5 ng/ml p24^gag of supernatants containing env-pseudotyped, luciferase viruses. After 48 to 62 h, cells were lysed and the extent of infection was measured by the intensity of chemiluminescence when mixing equal volumes of substrate and cell lysate. A representative experiment (performed in triplicate) is shown, and the results are expressed as the mean + standard deviation (error bars).

FIG. 2

Effect of sCD4 on infection of cells expressing CCR5 alone or with CD4. (A) HOS cells stably transfected for the expression of CD4 (HOS-CD4), CCR5 (HOS-CCR5), or both (HOS-CD4-CCR5) were infected with supernatants containing env-pseudotyped, luciferase viruses (p24^gag [5 ng/ml]). The results are expressed as the mean + standard deviation (error bars) from four independent experiments each performed in triplicate. (B) Treatment with sCD4 (5 μg/ml) inhibited infection of HOS-CD4-CCR5 cells by rB15 and rV1V2 but not by rBORI pseudotypes. Results are shown as percent luciferase activity relative to the value in the absence of sCD4 treatment, in four independent experiments each performed in triplicate. The differences in values between rBORI and rB15 or rV1V2 were statistically significant (Wilcoxon's rank-sum test: P < 0.05 in both cases).

FIG. 3

Infection of cells expressing decreasing amounts of CD4 and/or CCR5. U87 cells were transiently transfected with plasmids expressing CD4 and CCR5 and subsequently infected with supernatants containing env-pseudotyped, luciferase viruses (p24^gag [5 ng/ml]). (A) rBORI pseudotype did not infect cells expressing low CD4 and high CCR5 or low CD4/CCR5, while rB15 and rV1V2 did. Results are expressed as the mean + standard deviation (error bars) from seven independent experiments each performed in triplicate. (B) The luciferase activity measured following infection of low-CD4 and high-CCR5 or low-CD4/CCR5 cells was expressed as a percentage of the activity induced in the high-CD4/CCR5 cells. We performed seven independent experiments with the rBORI, rB15, and rV1V2 pseudotypes. The differences in the values between rBORI and rB15 or rV1V2 were statistically significant (Wilcoxon's rank-sum test: P < 0.01 in both comparisons for low CD4 and high CCR5 and for low CD4/CCR5).

FIG. 3

Infection of cells expressing decreasing amounts of CD4 and/or CCR5. U87 cells were transiently transfected with plasmids expressing CD4 and CCR5 and subsequently infected with supernatants containing env-pseudotyped, luciferase viruses (p24^gag [5 ng/ml]). (A) rBORI pseudotype did not infect cells expressing low CD4 and high CCR5 or low CD4/CCR5, while rB15 and rV1V2 did. Results are expressed as the mean + standard deviation (error bars) from seven independent experiments each performed in triplicate. (B) The luciferase activity measured following infection of low-CD4 and high-CCR5 or low-CD4/CCR5 cells was expressed as a percentage of the activity induced in the high-CD4/CCR5 cells. We performed seven independent experiments with the rBORI, rB15, and rV1V2 pseudotypes. The differences in the values between rBORI and rB15 or rV1V2 were statistically significant (Wilcoxon's rank-sum test: P < 0.01 in both comparisons for low CD4 and high CCR5 and for low CD4/CCR5).

FIG. 4

Infection of cells expressing CD4 and the 5552 or Δ4 chimeric coreceptors. U87 cells were transiently transfected with plasmids expressing CD4 and the chimeric coreceptors 5552 (amino terminus, ECL1 and ECL2 of CCR5, and ECL3 of CCR2b) or Δ4 (deletion of the first four amino acids in the amino terminus of CCR5), and subsequently infected with supernatants containing env-pseudotyped, luciferase viruses (p24^gag [5 ng/ml]). Luciferase activity is shown as the percentage with respect to wild-type CCR5 for each virus and experiment. Horizontal lines represent the median of each group of data. For the 5552 chimera, rBORI infections were significantly less efficient than rB15 and rV1V2 infections (Wilcoxon's rank-sum test: P < 0.001 in both cases). For the Δ4 mutant, rBORI infected significantly less than rB15 and rV1V2 (P < 0.01 and P < 0.02, respectively).

FIG. 5

Neutralization with HIV-1-positive human sera. A MAGI-CCR5 cell-based, single-cycle infection assay was performed as described in Materials and Methods, to test the neutralization sensitivity of VH-rBORI, VH-rB15, and VH-rV1V2 recombinant viruses (93) either in the presence of a 1/10 dilution of NHS or in the presence of increasing dilutions of serum from four HIV-1-infected individuals. Results were calculated as percentage of infection with respect to the extent observed with virus plus NHS, and the values shown represent the average of two to three independent experiments.

FIG. 6

17b ELISA and neutralization assays. (A) An ELISA was performed using the 17b MAb with supernatants containing env pseudotypes, either with or without sCD4. Results are shown as mean optical density at 405 nm + standard error of the mean (error bars) from four independent experiments using different pseudotype stocks. Absent sCD4, reactivity with the 17b antibody was greater in rB15 and rV1V2 than in rBORI pseudotypes (Student's t test: P < 0.02 and P < 0.05, respectively). (B) 17b MAb neutralization was performed in U87 cells transiently transfected with CD4-CCR5 (left; four independent experiments) and in microglial cultures (right; three independent experiments with two microglial preparations). The pseudotypes were incubated with 17b MAb (20 μg/ml) at 37°C for 1 h prior to inoculation. Data are shown as percentage of luciferase activity with respect to untreated controls for each pseudotype. In both cell types, 17b inhibited infection by rB15 and rV1V2 pseudotypes but not by rBORI.