Loss of N-linked glycans in the V3-loop region of gp120 is correlated to an enhanced infectivity of HIV-1.

We describe mutants of human immunodeficiency virus type-1 (HIV-1) strain NL4-3, which are lacking the thirteenth, fifteenth, or seventeenth sites for N-linked glycosylation (g13, g15, g17) of the envelope protein gp120. All three sites are located within the hypervariable V3 loop region of gp120. Those mutants lacking carbohydrates g15 or combinations of g15/g17 showed markedly higher infectivity for GHOST cells (human osteosarcoma cells) expressing CXCR4 (GHOST-X4), compared to the fully glycosylated NL4-3 wild type virus. In addition, these mutants could also infect cells which exhibits low background expression of CXCR4, corresponding to <10% of that observed for GHOST-X4 cells. In addition to the enhanced infectivity observed, mutants lacking g15 and g17 showed increased resistance to inhibition by SDF-1, the natural ligand of CXCR4. Thus, loss of the oligosaccharides g15 and g17 in the V3 region of gp120 markedly influences CXCR4-specific infection.

Membrane-anchored peptide inhibits human immunodeficiency virus entry.

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.

A recombinant virus assay using full-length envelope sequences to detect changes in HIV-1 co-receptor usage.

The clinical management of HIV-1 infection has benefited enormously from molecular characterization of drug resistance as well as determination of the viral phenotype in vitro. HIV-1 infected individuals on HAART are currently monitored for the development of drug resistance variants allowing clinicians to redesign drug regimens. An understanding of the molecular basis of the evolution of drug resistance in vivo allows the improvement of the drugs as well as in vitro evaluation of new antiviral compounds alone or in combination with those currently approved. New findings suggest that viral envelopes could be a target to inhibit infection and replication. Therefore the generation of a recombinant virus assay (RVA) to allow the phenotypic determination of drug resistance against entry inhibitors (EI) is anticipated. We constructed an env-deleted clone of HIV-1 using the molecular clone NL-4.3. PCR amplified complete envelope genes (NL-4.3, BaL, primary envelope-genes) were ligated in vitro with a deletion clone (pNL-deltaK) and PM1-cells, supporting the replication of R5- and X4-tropic viruses, were transfected. Determination of co-receptor usage of the harvested recombinant virus-swarm revealed no difference compared to the molecular clones derived individually from three different patients. These results clearly show that an envelope-based RVA is practicable to monitor HIV-co-receptor usage at a given time point. Furthermore, this assay will allow to monitor resistance development against existing and future entry inhibitors and will aid to improve the management of HIV-therapy.

Coreceptor requirements of primary HIV type 1 group O isolates from Cameroon.

HIV-1 group O has its epicenter in Cameroon and neighboring countries and is responsible for 3 to 5% of all HIV infections in this region. It is believed that HIV-1 group O was introduced into the human population by a separate cross-species transmission, occurring independently of the HIV-1 (group M and group N) and HIV-2 transmissions. We have studied the coreceptor requirements of 12 primary HIV-1 O-type isolates from individuals with different clinical symptoms. Only 2 of these 12 viruses showed a syncytium-inducing phenotype after infection of primary peripheral blood mononuclear cells (PBMCs) and were infectious for the T cell line C8166. These isolates used CXCR4 as a coreceptor for entry, whereas the remaining isolates used only CCR5 efficiently. One isolate was able to use BOB and CCR8 as coreceptors in addition to CXCR4. All group O isolates tested were efficiently inhibited by SDF-1 or RANTES, the natural ligands of CXCR4 and CCR5, respectively. These results indicate that CXCR4 and CCR5 are the principal coreceptors for HIV-1 O-type viruses. Most of the HIV-1 group O isolates studied were derived from patients at later stages of the disease. Although HIV-1 group O and group M infections do not differ in their pathogenesis, the studied isolates did not evolve to use a broad range of coreceptors as described for HIV-1 group M and HIV-2.

The U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 do not confer acute pathogenicity upon SIVagm.

Two chimeric proviruses comprising the U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 in addition to other genomic regions of SIVagm3mc from African green monkeys (Cercopithecus aethiops) were constructed. The derived chimeric viruses (SIVagm3mc/SIVsmmPBj1.9) were both able to replicate in nonstimulated peripheral blood leukocytes from pig-tailed macaques (Macaca nemestrina), a biological property often correlated with acute pathogenicity. However, only one of the chimeric viruses was acutely pathogenic, inducing a rapid depletion of the peripheral CD4+ T cells in two infected pig-tailed macaques within 10 days after infection in a manner similar to infection with SIVsmmPBj1.9 itself. The other chimeric virus actively replicated during the first 8 weeks after experimental infection of two pig-tailed macaques but induced neither acute disease nor CD4+ T-cell depletion for 113 weeks after infection. Thus, the U3 promoter and the nef gene of SIVsmmPBj1.9 alone appear to be insufficient to confer acute pathogenicity to SIVagm3mc.

A broad range of chemokine receptors are used by primary isolates of human immunodeficiency virus type 2 as coreceptors with CD4.

Like human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), HIV-2 requires a coreceptor in addition to CD4 for entry into cells. HIV and SIV coreceptor molecules belong to a family of seven-transmembrane-domain G-protein-coupled receptors. Here we show that primary HIV-2 isolates can use a broad range of coreceptor molecules, including CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4. Despite broad coreceptor use, the chemokine ligand SDF-1 substantially blocked HIV-2 infectivity of peripheral blood mononuclear cells, indicating that its receptor, CXCR4, was the predominant coreceptor for infection of these cells. However, expression of CXCR4 together with CD4 on some cell types did not confer susceptibility to infection by all CXCR4-using virus isolates. These data therefore indicate that another factor(s) influences the ability of HIV-2 to replicate in human cell types that express the appropriate receptors for virus entry.

Langerhans cell tropism of human immunodeficiency virus type 1 subtype A through F isolates derived from different transmission groups.

To test the hypothesis that some subtypes of human immunodeficiency virus type 1 (HIV-1), especially subtype E, are more likely to infect mature Langerhans cells (mLC), we titrated a panel of 26 primary HIV-1 isolates of subtypes A through F on peripheral blood mononuclear cells (PBMC) and mLC. The majority of HIV-1 isolates from heterosexually infected patients did not show a preferred tropism for mLC compared to homosexually transmitted HIV-1 isolates. Only 6 of 26 isolates, 2 from patients infected by homosexual contact and 4 from patients infected by heterosexual contact, showed a higher infectivity for mLC than for PBMC. Both syncytium-inducing and non-syncytium-inducing isolates were able to infect mLC which express mRNA for the chemokine receptors CCR3, CCR5, and CXCR4.

Biological characterization of human immunodeficiency virus type 1 clones derived from different organs of an AIDS patient by long-range PCR.

In order to characterize the biological properties of human immunodeficiency virus type 1 (HIV-1) variants from different tissues (peripheral blood mononuclear cells [PBMC], lymph node, spleen, brain, and lung) of one patient, we have chosen long-range PCR to amplify virtually full-length HIV proviruses and to construct replication-competent viruses by adding a patient-specific 5' long terminal repeat. To avoid selection during propagation in CD4+ target cells, we transfected 293 cells and used the supernatants from these cells as challenge viruses for tropism studies after titration on human PBMC. Despite differences in the V3 loop of the major variants found in brain and lung compared to lymphoid tissues all recombinant HIV clones obtained showed identical cell tropism and replicative kinetics. After infection of human PBMC these viruses replicated with similar kinetics, with a slow/low-titer, non-syncytium-inducing phenotype. In contrast to the prediction of macrophage tropism, drawn from the V3 loop sequence, none of these viruses infected monocyte-derived macrophages. The challenge of blood dendritic cells by these recombinant viruses in the presence of tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-4 resulted in a productive infection only after adding stimulated CD4+ T lymphocytes. Therefore, the biological properties of the HIV-1 variants derived from nonlymphoid tissue of this patient did not differ from those of HIV-1 variants from lymphoid tissue with respect to tropism for primary cells such as PBMC, macrophages, and blood dendritic cells.

Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry.

A panel of primary syncytium-inducing (SI) human immunodeficiency virus type 1 isolates that infected several CD4+ T-cell lines, including MT-2 and C8166, were tested for infection of blood-derived macrophages. Infectivity titers for C8166 cells and macrophages demonstrated that primary SI strains infected macrophages much more efficiently than T-cell line-adapted HIV-1 strains such as LAI and RF. These primary SI strains were therefore dual-tropic. Nine biological clones of two SI strains, prepared by limiting dilution, had macrophage/C8166 infectivity ratios similar to those of their parental viruses, indicating that the dual-tropic phenotype was not due to a mixture of non-SI/macrophage-tropic and SI/T-cell tropic viruses. We tested whether the primary SI strains used either Lestr (fusin) or CCR5 as coreceptors. Infection of cat CCC/CD4 cells transiently expressing Lestr supported infection by T-cell line-adapted strains including LAI, whereas CCC/CD4 cells expressing CCR5 were sensitive to primary non-SI strains as well as to the molecularly cloned strains SF-162 and JR-CSF. Several primary SI strains, as well as the molecularly cloned dual-tropic viruses 89.6 and GUN-1, infected both Lestr+ and CCR5+ CCC/CD4 cells. Thus, these viruses can choose between Lestr and CCR5 for entry into cells. Interestingly, some dual-tropic primary SI strains that infected Lestr+ cells failed to infect CCR5+ cells, suggesting that these viruses may use an alternative coreceptor for infection of macrophages. Alternatively, CCR5 may be processed or presented differently on cat cells so that entry of some primary SI strains but not others is affected.

An in vitro assay for acute pathogenicity of immunodeficiency viruses.

As a model for AIDS, experimental infections of old-world monkeys with various simian immunodeficiency viruses (SIV) are frequently carried out to study mechanisms of pathogenicity. For example, SIVsmmPBj14 was isolated from a pig-tailed macaque (Macaca nemestrina) suffering from acute viral disease. The molecular virus clone SIVsmmPBj1.9, which displays close genetic homology to other related SIVs, was shown to induce an acute viral disease in vivo after infection of pig-tailed and rhesus macaques. The acute pathogenicity of SIVsmmPBj1.9 was correlated with its unique ability to replicate in non-stimulated peripheral blood mononuclear cells from pig-tailed macaques. We have exploited this in vitro assay to resolve putative pathogenic genetic determinants of another SIV, namelySIVagm3, isolated from African green monkeys (Cercopithecus aethiops). Hybrid viruses encompassing subgenomic regions of SVsmmPBj1.9 in place of comparable regions of molecular virus clone SIVagm3mc were constructed and tested for their ability to replicate in non-stimulated PBMC from pig-tailed macaques and African green monkeys. Only those hybrid viruses comprising the U3 region of the viral LTR of SIVsmmPBj1.9 replicated in non-stimulated peripheral blood mononuclear cells. This in vitro assay will be used to determine the potential of SIV and of hybrid viruses between different SIVs to induce acute viral disease in vivo. It will help to avoid excessive experimental infections of monkeys with respective hybrid viruses for determining genetic determinants of acute pathogenicity of immunodeficiency viruses.

The U3 promoter region of the acutely lethal simian immunodeficiency virus clone smmPBj1.9 confers related biological activity on the apathogenic clone agm3mc.

Infection with the acutely pathogenic molecular virus clone SIVsmmPBj1.9, cloned from isolate PBj14 of simian immunodeficiency virus (SIV) from sooty mangabey monkeys (Cercocebus atys), leads to acute viral and often lethal disease within days or weeks. SIVsmmPBj1.9 has the unique property of replicating in nonstimulated peripheral blood mononuclear cells from pig-tailed macaques. In contrast, molecular virus clone SIVagm3mc of SIV from African green monkeys (Cercopithecus aethiops), which is apathogenic in its natural host and in pig-tailed macaques, is unable to grow in nonstimulated peripheral blood cells. Chimeric proviruses were constructed by exchanging defined regions of SIVagm3mc against comparable regions of SIVsmmPBj1.9. Four of five hybrid viruses generated by transfection into the CD4-positive T-cell line C8166 replicated in T-cell lines permissive for SIVagm3mc replication and in stimulated peripheral blood cells from pig-tailed macaques and from African green monkeys. Three hybrid viruses displayed the distinct biological property of SIVsmmPBj14 to replicate in nonstimulated peripheral blood cells from pig-tailed macaques and from African green monkeys. Replication in nonstimulated peripheral blood cells was dependent on the presence of the U3 promoter region of SIVsmmPBj1.9 within the viral long terminal repeat.

CD8+ T lymphocytes of African green monkeys secrete an immunodeficiency virus-suppressing lymphokine.

Following natural and experimental infection by simian immunodeficiency virus SIVagm of African green monkeys (AGMs), the natural host, there is no evidence for the development of an immunodeficiency. Within the framework of our studies on human immunodeficiency virus (HIV)/SIV pathogenesis, we investigated the influence of CD8 T lymphocytes on SIVagm replication in AGM CD4 T lymphocytes in vitro. The following observations were made: (i) Peripheral blood mononuclear cells from both seronegative and seropositive AGMs contained only a low proportion (i.e., 10%) of CD4+ lymphocytes, whereas a high proportion (80%) of CD8+ cells was detected. Even after persistent SIVagm infection, CD4 T lymphocytes do not decrease in number. (ii) The target of in vitro infection of peripheral blood cells is the CD4+ mononuclear cell (T lymphocytes, monocytes) and SIVagm infects by binding to the CD4 molecule. (iii) In both naturally and experimentally SIVagm-infected AGMs the CD4+ T cells and monocytes, but not the CD8+ T cells, harbor DNA provirus. (iv) Virus reisolation and virus replication of SIVagm in CD4 T lymphocytes from seropositive AGMs is suppressed in the presence of autologous CD8 T lymphocytes or a soluble factor produced by these cells. Taken together, one possible reason for the apathogenicity of the SIVagm infection in AGMs may be the suppression of virus replication by a soluble, yet unidentified factor secreted by CD8 lymphocytes quantitatively dominating among peripheral blood cell populations. We have tentatively termed this factor "immunodeficiency virus-suppressing lymphokine." In addition, we show that immunodeficiency virus-suppressing lymphokine from AGMs is able to suppress HIV-1 replication in human CD4+ T cells.

Development of vivo of genetic variability of simian immunodeficiency virus.

Rapid development of genetic variability may contribute to the pathogenicity of lentiviruses as it may allow escape from immune surveillance and/or from suppression of virus replication. Although apathogenic in African green monkeys, simian immunodeficiency virus isolated from African green monkeys is shown to display extensive genetic variability and defectiveness in the V1- and V2-like variable domains of the external envelope protein comparable to that known for human immunodeficiency virus. However, in contrast to the situation in human immunodeficiency virus-infected individuals, a predominant major virus variant was detected neither in a monkey naturally infected for more than 10 years nor in two monkeys infected with a molecular virus clone for 15-20 months. Extensive variability evolves from a single genotype with a maximal rate of 7.7 mutations per 1000 nucleotides per year. A remarkable selection for nonsynonymous mutations that accounts for 92% of all changes indicates continuous selection of variants.