CCR5, GPR15, and CXCR6 are major coreceptors of human immunodeficiency virus type 2 variants isolated from individuals with and without plasma viremia.

Human immunodeficiency virus type 2 (HIV-2) is generally considered capable of using a broad range of coreceptors. Since HIV-2 variants from individuals with nonprogressive infection were not studied previously, the possibility that broad coreceptor usage is a property of variants associated with progressive infection could not be excluded. To test this, we determined the coreceptor usage of 43 HIV-2 variants isolated from six long-term-infected individuals with undetectable plasma viremia. Using GHOST indicator cells, we showed for the first time that the only coreceptors efficiently used by low-pathogenic HIV-2 variants are CCR5, GPR15 (BOB), and CXCR6 (BONZO). Surprisingly, control HIV-2 variants (n = 45) isolated from seven viremic individuals also mainly used these three coreceptors, whereas use of CCR1, CCR2b, or CCR3 was rare. Nearly a quarter of all HIV-2 variants tested could infect the parental GHOST cells, which could be partially explained by CXCR4 usage. Use of CXCR4 was observed only for HIV-2 variants from viremic individuals. Thirty-eight variants from aviremic and viremic HIV-2-infected individuals were additionally tested in U87 cells. All except one were capable of infecting the parental U87 cells, often with high efficiency. When virus production in parental cells was regarded as background in the coreceptor-transduced cell lines, the results in U87 cells were largely in agreement with the findings in GHOST cells. HIV-2 isolates from aviremic individuals commonly use as coreceptors CCR5, GPR15, and CXCR6, as well as an unidentified receptor expressed by U87 cells. Broad coreceptor usage, therefore, does not appear to be associated with pathogenicity of HIV-2.

Macrophage tropism of human immunodeficiency virus type 1 facilitates in vivo escape from cytotoxic T-lymphocyte pressure.

Early after seroconversion, macrophage-tropic human immunodeficiency virus type 1 (HIV-1) variants are predominantly found, even when a mixture of macrophage-tropic and non-macrophage-tropic variants was transmitted. For virus contracted by sexual transmission, this is presently explained by selection at the port of entry, where macrophages are infected and T cells are relatively rare. Here we explore an additional mechanism to explain the selection of macrophage-tropic variants in cases where the mucosa is bypassed during transmission, such as blood transfusion, needle-stick accidents, or intravenous drug abuse. With molecularly cloned primary isolates of HIV-1 in irradiated mice that had been reconstituted with a high dose of human peripheral blood mononuclear cells, we found that a macrophage-tropic HIV-1 clone escaped more efficiently from specific cytotoxic T-lymphocyte (CTL) pressure than its non-macrophage-tropic counterpart. We propose that CTLs favor the selective outgrowth of macrophage-tropic HIV-1 variants because infected macrophages are less susceptible to CTL activity than infected T cells.

Antiviral resistance of biologic HIV-2 clones obtained from individuals on nucleoside reverse transcriptase inhibitor therapy.

OBJECTIVE: To study phenotypic and genotypic resistance of HIV-2 against nucleoside reverse transcriptase inhibitors (NRTI). METHODS: Biologic HIV-2 clones were generated from 3 patients before and after initiation of antiretroviral therapy with zidovudine (AZT) in patient RH2-7, AZT and didanosine (ddI) in patient PH2-1, and after addition of lamivudine (3TC) to AZT monotherapy in patient RH2-5. The sensitivity to NRTI of the virus clones, as defined by the 50% inhibitory concentration (IC(50)), was determined in vitro. The predicted amino acid sequences of the reverse transcriptase proteins from these clones were determined. RESULTS: Comparing the sensitivity of the biologic HIV-2 clones obtained after start of therapy with those from antiviral naive patients, resistance had developed to AZT (patients RH2-7 and RH2-5) and 3TC (patient PH2-1 and RH2-5). No resistance to AZT was observed in the biologic clone from PH2-1 obtained after start of therapy. The resistant clones from RH2-5 and PH2-1, but not RH2-7, contained amino acid mutations at positions where HIV-1 has been shown to mutate after AZT and 3TC treatment. CONCLUSIONS: Phenotypic resistance of HIV-2 to nucleoside analogues, which developed in HIV-2-infected patients treated with NRTIs, was associated with genotypic changes. Some of the mutations at amino acid positions in the HIV-2 reverse transcriptase gene corresponded with those involved in HIV-1 resistance, although no conventional mutations associated with resistance to AZT were observed.

Broadening of coreceptor usage by human immunodeficiency virus type 2 does not correlate with increased pathogenicity in an in vivo model.

The pathogenic properties of four primary human immunodeficiency virus type 2 (HIV-2) isolates and two primary HIV-2 biological clones were studied in an in vivo human-to-mouse chimeric model. The cell-associated viral load and the ability to reduce the severity of the induced graft-versus-host disease symptoms, the CD4/CD8 ratio and the level of repopulation of the mouse tissues by the graft, were determined. All HIV-2 strains, irrespective of their in vitro biological phenotype, replicated to high titres and significantly reduced graft-versus-host disease symptoms as well as the CD4/CD8 ratios. Reduction of graft repopulation caused by infection with the respective HIV-2 strains showed that the in vitro replication rate, syncytium-inducing capacity and ability to infect human macrophages did influence the in vivo pathogenic potential whereas broadening of coreceptor usage did not.

Kinetics of antiviral activity by human immunodeficiency virus type 1-specific cytotoxic T lymphocytes (CTL) and rapid selection of CTL escape virus in vitro.

The antiviral activity of a CD8(+) cytotoxic T-lymphocyte (CTL) clone (TCC108) directed against a newly identified HLA-B14-restricted epitope, human immunodeficiency virus type 1 (HIV-1) Rev(67-75) SAEPVPLQL, was analyzed with respect to its kinetics of target cell lysis and inhibition of HIV-1 production. Addition of TCC108 cells or CD8(+) reverse transcriptase-specific CTLs to HLA-matched CD4(+) T cells at different times after infection with HIV-1 IIIB showed that infected cells became susceptible to CTL-mediated lysis before peak virus production but after the onset of progeny virus release. When either of these CTLs were added to part of the infected cells immediately after infection, p55 expression and virus production were significantly suppressed. These data support a model in which CTLs, apart from exerting cytolytic activity which may prevent continued virus release, can interfere with viral protein expression during the eclipse phase via noncytolytic mechanisms. TCC108-mediated inhibition of virus replication in peripheral blood mononuclear cells caused rapid selection of a virus with a mutation (69E-->K) in the Rev(67-75) CTL epitope which abolished recognition by TCC108 cells. Taken together, these data suggest that both cytolytic and noncytolytic antiviral mechanisms of CTLs can be specifically targeted to HIV-1-infected cells.

Coreceptor usage of human immunodeficiency virus type 2 primary isolates and biological clones is broad and does not correlate with their syncytium-inducing capacities.

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by binding of the surface envelope glycoprotein to the CD4 molecule. Interaction of the resulting CD4-glycoprotein complex with alpha- or beta-chemokine receptors, depending on the biological phenotype of the virus, then initiates the fusion process. Here, we show that primary HIV-2 isolates and biological clones, in contrast to those of HIV-1, may use a broad range of coreceptors, including CCR-1, CCR-3, CCR-5, and CXCR-4. The syncytium-inducing capacity of these viruses did not correlate with the ability to infect via CXCR-4 or any other coreceptor. One cell-free passage of the intermediate isolates in mitogen-stimulated, CD8+ cell-depleted peripheral blood mononuclear cells resulted in the outgrowth of variants with CCR-5 only, whereas the coreceptor usage of late and early isolates did not change. Since HIV-2 is less pathogenic in vivo than HIV-1, these data suggest that HIV pathogenicity in vivo is not directly related to the spectrum of coreceptors used in in vitro systems.

Impact of natural sequence variation in the V2 region of the envelope protein of human immunodeficiency virus type 1 on syncytium induction: a mutational analysis.

Several studies have demonstrated a functional role for the V1-V2 region of the human immunodeficiency virus type 1 (HIV-1) envelope surface glycoprotein gp120 in the membrane fusion processes underlying viral entry and syncytium induction. In a study with chimeric primary envelope genes, we have previously demonstrated that the exchange of V2 regions was sufficient to transfer syncytium-inducing capacity to a non-syncytium-inducing envelope protein. The exchanged V2 regions, comprising a number of variable amino acids, conferred changes to both the predicted secondary structure and to the net positive charge of the V2 loops. In a syncytium-forming assay based on transient envelope protein expression in CD4+ SupT1 cells, we have extended this observation by mutating the variable positions of the V2 region to determine the relative contribution of individual amino acids to syncytium formation. It can be shown that simultaneous mutation of multiple amino acids is needed to interfere with the V2 region-determined syncytium-inducing phenotype. Single amino acid changes either influencing charge of predicted secondary structure of the V2 loop proved to be insufficient to abolish V2 region-controlled syncytium formation. This robust V2 organization may allow the virus to accumulate mutations, while retaining its biological phenotype.