Functional complementation of the envelope hypervariable V3 loop of human immunodeficiency virus type 1 subtype B by the subtype E V3 loop.

The hypervariable V3 loop within gp120 of human immunodeficiency virus type 1 (HIV-1) is the major determinant of cell tropism and the entry coreceptor usage of the virus. However, the information obtained thus far has been from only subtype B from North America and Europe, and little is known about other subtypes whose V3 amino acids differ by as much as 50% from subtype B V3. In this study, we examined the functional potential of the V3 element of the HIV-1 subtype E, the most crucial variant causing the AIDS epidemic throughout southeast Asia. A panel of HIV-1LAI recombinants was constructed by the overlap extension method, by which the LAI V3 loop was precisely replaced by that of the subtype E nonsyncytium-inducing (NSI) or syncytium-inducing (SI) variant. All of the recombinant viruses infected peripheral blood mononuclear cells, whereas only those with SI V3 infected MT2 cells, a CD4(+) T cell line. Consistently, the SI V3 recombinants used CXCR4, while the NSI V3 recombinants used CCR5 for infection of HOS-CD4(+) cells. Finally, only the NSI V3 sequence conferred CC-chemokine sensitivity on the parental virus. The data support the notion that the HIV-1 V3 loop consists of a relatively independent domain in gp120 and suggest that the subtype E V3 loop indeed contains the functional element to dictate the cell tropism, coreceptor preference, and chemokine sensitivity of the virus. These findings are of immediate importance in understanding V3 structure-function relationship and for examining phenotypic evolution of HIV-1 subtype E.     

The HIV-2 genotype and the HIV-1 syncytium-inducing phenotype are associated with a lower virus replication in dendritic cells

During sexual transmission, HIV infects the mucosal dendritic cells and is transferred to CD4 T cells. Whether HIV variants of a particular genetic (sub)type or phenotype selectively infect dendritic cells (DC) or are preferentially transferred to T cells remains highly controversial. To avoid the cumbersome use of primary dendritic cells, in vitro dendritic cell models were generated from precursors, either hematopoietic progenitor cells (HPC) or monocytes (MO). Productive infection in the dendritic cells and transfer of the virus to T cells was assessed for a range of HIV variants. HPC-derived dendritic cells (HPC-DC) were more susceptible to HIV-1 than to HIV-2 isolates. The HIV-1 group O strains were more productive in HPC-DC than group M, but amongst the latter, no subtype-related difference was observed. Both non-syncytium-inducing (NSI) and SI HIV isolates and lab strains could productively infect HPC-DC, albeit with a different efficiency. Adding blocking antibodies confirmed that both CCR-5 and CXCR-4 co-receptors were functional. Biological HIV-1 clones of the NSI/R5 phenotype infected more readily HPC-DC than SI/X4 clones. MO-derived dendritic cells were, however, more exclusive in their preference for NSI/R5 clones. Some HIV variants, that did not grow readily in HPC-DC alone, could be rescued by adding resting or pre-activated T cells. The present data show that HIV-2 isolates and SI clones replicate less in model-DC, but no preference for a particular HIV-1 subtype was evident. Co-culture with T cells could "correct" a limited growth in dendritic cells. Clearly, both intrinsic dendritic cell susceptibility and enhancement by T cells are explained only partly by HIV genotype and phenotype. The in vitro dendritic cell models seem useful tools to further unravel interactions between HIV, DC, and T cells.     

High IL-7 plasma levels may induce and predict the emergence of HIV-1 virulent strains in pediatric infection.

BACKGROUND: HIV-1 infection results in severe immunodeficiency when T-cell loss cannot be compensated. IL-7 is one of the main cytokines involved in the maintenance of T-cell homeostasis. However, IL-7 can also enhance HIV-1 replication in vivo and lead to an accelerated progression of AIDS. OBJECTIVE: The aim of our study was to evaluate if the increase of IL-7 levels in response to CD4+ T cell depletion could favor the emergence of HIV-1 strains with more aggressive phenotypes in pediatric infection. STUDY DESIGN: Plasma IL-7 levels were measured in 42 HIV-1 vertically infected infants at different times of infection, and HIV-1 isolates were obtained from primary cell cultures to determine replication rate and syncytium-inducing (SI) capability on MT-2 cell line. RESULTS: IL-7 levels were significantly higher in infants harboring HIV-1 SI strains compared to those with non-syncytium-inducing (NSI) viruses (p<0.0001). Likewise, IL-7 levels were higher in infants with rapid replicating viral strains versus those with slow replicating viruses (p=0.0006). Despite the strong negative correlation between IL-7 levels and CD4+ T lymphocyte counts (r=-0.55, p=0.0001), covariance analysis demonstrated that the high levels of IL-7 were associated with more virulent phenotype features (SI and rapid replicating strains) independently of CD4+ T cell depletion. In 19 of the 42 infants, longitudinal follow-up studies showed that SI to NSI phenotype switch can occur after HAART administration, with a reduction in IL-7 levels and an increase in CD4+ T cell counts. CONCLUSIONS: IL-7 response to T-cell depletion may enhance T-cell production, but at the same time may foster HIV-1 disease progression favoring the emergence of more virulent HIV-1 strains characterized by SI capability and rapid replication rate.     

Phenotypic variations and switches in HIV isolated from the blood and the gastrointestinal tissues of patients with HIV-1 infection

The objective of this study was to determine the initial and subsequent phenotypes of HIV-1 isolated from the blood, duodenal, and colonic biopsies of 51 HIV-1 positive patients followed prospectively over 2 years. Blood and tissues were cocultured with stimulated peripheral blood monocytes, and HIV was analyzed for phenotypic expression of syncytia-induction (SI). A total of 45/51 patients had HIV-1 isolated from the blood and 35/51 had HIV isolated from gastrointestinal tract. In 12/45 patients SI-HIV-1 was isolated from the blood. In 6/12 patients the blood phenotype reverted to the NSI phenotype. SI phenotypes were also isolated from the colon and duodenum of 8/35 patients and reversion from SI to NSI virus phenotype was again observed in gut tissue of 3/8 patients. These results show that gastrointestinal tissues can harbor SI HIV phenotype. Discordant phenotypes can be found in tissue and blood of late-stage patients. Reversion of phenotypic SI expression to NSI may occur in patients receiving monotherapy as antiretroviral treatment. These results suggest that gastrointestinal tissues may act as a separate and distinct site involved in HIV replication and its associated pathogenesis. J. Med. Virol. 52:31–34, 1997. © 1997 Wiley-Liss, Inc.     

Apoptosis induced in synchronized human immunodeficiency virus type 1-infected primary peripheral blood mononuclear cells is detected after the peak of CD4+ T-lymphocyte loss and is dependent on the tropism of the gp120 envelope glycoprotein

Disease progression in human immunodeficiency virus type-1 (HIV-1)-infected individuals is frequently accompanied by declining CD4 cell numbers and the acquisition of a T-tropic (X4) or dual tropic (R5X4) phenotype. Understanding the mechanism of CD4 cell loss in HIV-1 infection is essential for the development of effective therapeutic strategies. In this study, donor populations of peripheral blood mononuclear cells (PBMCs) were selected for their ability to support an equivalent acute infection by both R5 and X4 virus phenotypes. This demonstrated that CD4+ T-lymphocyte loss was due to the gp120 region of Env and was replication independent. Furthermore, apoptosis was only detected in cells infected with an X4 virus after the majority of CD4+ T-lymphocyte loss had occurred. These observations indicate that the CD4+ T-lymphocyte loss in an X4 HIV-1 infection is not directly mediated by apoptosis, although apoptosis may be induced in the remaining cell population as a consequence of this CD4+ T-lymphocyte loss.     

Effect of the HIV-1 syncytium-inducing phenotype on disease stage in vertically-infected children

The syncytium-inducing (SI) capability of HIV-1 isolates from 48 HIV-infected children was determined in order to examine the association of the SI phenotype with an AIDS diagnosis and/or with other clinical parameters in HIV-infected children. In a retrospective cross-sectional analysis, phenotypic data were linked to clinical and immunologic data from each patient. Multiple longitudinal samples were analyzed from 14 patients. Children with SI viruses were older than children with nonsyncytium-inducing (NSI) strains. Twelve of 13 children less than 2 years old carried NSI viruses, seven of the 12 already had a diagnosis of AIDS. Two children under 2 years of age died within 1 month of NSI virus isolation. Although plasma p24 antigen levels tended to be higher in the NSI group, the difference appeared to reflect high p24 levels in children under 2 years old with AIDS. When children under 2 were omitted, differences in age, CD4+ cell counts, p24 antigenemia, and clinical parameters were not significant. The SI phenotype of HIV-1 did not occur more frequently in children with an AIDS diagnosis. Four children remained stable with SI isolates over time periods of 16 to 31 months. Three children's isolates converted from NSI to SI and 2 converted from SI to NSI. These data indicate that SI viruses do not play a significant role in progression to AIDS during the first 2 years of life. Furthermore, for children above the age of 2, the association between advanced disease stage and the SI phenotype in adults may not apply. J. Med. Virol. 55:56–63, 1998. © 1998 Wiley-Liss, Inc.     

Five human immunodeficiency virus type 1 phenotypic variants with different MT-2 cell tropisms correlate with prognostic markers of disease

OBJECTIVES: We correlated virologic and immunologic parameters of disease progression with cytopathogenicity of HIV isolates. STUDY DESIGN/METHODS: Human immunodeficiency virus type 1 (HIV-1) isolates from 207 patients with CD4+ cell counts < 500/mm3 were examined for biologic phenotype in MT-2 cells. We used a cross-sectional study design. RESULTS: Three subtypes of syncytium-inducing (SI) strains with different times of appearance of syncytia formation in cell culture and two subtypes of non-syncytium-inducing (NSI) isolates, with (NSI/MT2+) or without (NSI/MT2-) replicative capacity in MT-2 cells, were identified. Early SI strains were associated with the lowest CD4+ cell counts and the highest levels of viral load, and NSI/MT2- isolates correlated with the highest CD4+ cell counts and the lowest viral loads. Patients with late SI and NSI/MT2+ strains showed minimal differences in immunologic and virologic markers. CONCLUSIONS: Five HIV phenotypic variants that correlate significantly (P < 0.001) with markers of disease progression were identified.     

Coreceptor usage and RANTES sensitivity of non-syncytium-inducing HIV-1 isolates obtained from patients with AIDS

OBJECTIVES: The biologic phenotype of HIV-1 primary isolates obtained from approximately 50% of patients who progress to AIDS switches from non-syncytium-inducing (NSI) to syncytium-inducing (SI). We evaluated possible associations between virus coreceptor usage, sensitivity to inhibition by beta-chemokines, and disease progression of patients who continue to yield NSI isolates after developing AIDS. STUDY DESIGN/METHODS: Sequential virus isolates were analyzed for biologic phenotype using the MT-2 cell assay, for sensitivity to beta-chemokines using RANTES inhibition, and for coreceptor usage using U87.CD4 and GHOST.CD4 cells expressing different chemokine/orphan receptors or donor peripheral blood mononuclear cells (PBMC) defective in CCR5 expression. In addition, the env V3 region was sequenced and the length of the V2 region determined. RESULTS: All NSI isolates, regardless of patient status at time of isolation, were dependent on CCR5 expression for cell entry. Furthermore, there was no indication of broadened coreceptor usage of NSI isolates obtained from persons with late-stage AIDS. A majority of NSI isolates remained RANTES sensitive; however, virus variants with reduced sensitivity were observed. The V2 lengths and the V3 sequences exhibited no or minor changes at analysis of sequential NSI isolates. CONCLUSIONS: Our data suggest that NSI isolates obtained from AIDS patients remain CCR5 dependent (ie, R5) and, in many cases, also remain sensitive to RANTES inhibition. However, virus variants with decreased sensitivity to RANTES inhibition may evolve during disease progression, not only as a result of a switch from NSI to SI but also in patients who develop AIDS while continuing to maintain R5 isolates.     

CXCR4 mediates entry and productive infection of syncytia-inducing (X4) HIV-1 strains in primary macrophages

CCR5 and CXCR4 are the main coreceptors for non-syncytia-inducing (NSI) and syncytia-inducing (SI) HIV-1 strains, respectively. NSI HIV-1 isolates do not infect either human lymphoid or monocytoid cell lines, and this inability correlates with the absence of CCR5 expression in these cell types. The ability of SI HIV-1 isolates to infect human primary macrophages has been disputed. Here, we report that CXCR4 is expressed in primary blood-derived human mononuclear phagocytes at all stages of differentiation, although the maturation process correlates with downregulation of CXCR4 mRNA. Infection experiments with the SI molecular clone NL4-3 tagged with a mutant of the green fluorescent protein established that both monocytes and attached macrophages are susceptible to infection with CXCR4-restricted HIV-1 strains. NL4-3 entry into primary macrophages could be blocked by SDF-1alpha in a dose-dependent manner, or by the anti-CXCR4 monoclonal antibody 12G5. HIV-1 entry led to productive infection. No evidence of postentry defects or nuclear import delay for CXCR4-restricted HIV-1 strains was detected using a quantitative real-time PCR assay measuring HIV-1 DNA entry into the nucleus. Macrophages infected by HIV-1 and expressing virus were maintained in culture for long periods of time (up to 5 months). These results demonstrate that CXCR4 is the main HIV-1 SI coreceptor in human primary macrophages and underline the importance of the macrophage as a long-living viral reservoir for HIV-1.     

Relationship between V3 genotype, biologic phenotype, tropism, and coreceptor use for primary isolates of human immunodeficiency virus type 1.

OBJECTIVES: The predictive value of positively charged amino acids at positions 11 and 25 within the V3 loop region of the human immunodeficiency virus type 1 (HIV-1) envelope gene for the syncytium-inducing (SI) phenotype was assessed. STUDY DESIGN/METHODS: Sequencing was performed on DNA extracted from primary peripheral blood mononuclear cells (PBMCs) and complementary DNA (cDNA) prepared from serial viral isolates from 10 HIV-1-seropositive subjects. Proviral DNA sequencing was also performed on biologic clones from most of these subjects. RESULTS: Positive charge at position 11 and/or 25 in 257 isolate cDNA, PBMC DNA, and biologic clone PBMC DNA sequences was compared with 69 phenotypic determinations, of which 62.3% were SI. V3 genotype was 51.2% sensitive and 85.8% specific for the SI phenotype, with positive and negative predictive values of 62.8% and 79.0%, respectively. Cellular tropism failed to correlate with V3 genotype, coreceptor use, or biologic phenotype. Exclusive use of CCR5 was associated with the nonsyncytium-inducing (NSI) phenotype. Overall, V3 loop charge was higher in SI than in NSI isolates (5.01 and 3.78, respectively; p = 0.0211). CONCLUSIONS: The predictive power of SI phenotype from V3 genotype is relatively weak, especially in a low SI prevalence population. The direct measurement of viral phenotype, cellular tropism, and coreceptor use in HIV-1 isolates is essential for accurate biologic characterization.     

In vitro exposure to highly cytopathic HIV-1 X4 strains increases expression of mucosa-associated integrins on CD4(+) T cells

To determine whether infection with HIV-1 strains of different tropisms would influence expression of the mucosa-associated integrins alpha 4 beta 7 and alpha E beta 7 or the lymph node homing receptor L-selectin on peripheral T lymphocytes, cells were infected with the CXCR4-tropic (X4)/syncytium-inducing (SI) HIV-1(IIIB) strain or with X4/SI or CCR5-tropic (R5)/non-SI (NSI) primary human isolates. Flow cytometric analyses of CD4(+) T cells from cultures infected with HIV-1(IIIB) and one X4/SI primary HIV-1 isolate revealed a significant increase in surface expression of alpha 4 beta 7 and alpha E beta 7 12 days after infection. L-selectin expression was not significantly affected on CD4(+) T cells. However, infection with another X4/SI and two R5/NSI primary HIV-1 isolates did not significantly alter homing receptor expression on CD4(+) T cells. Since a higher degree of CD4 cytopathicity occurred in those cultures having increased integrin expression, these data suggest that significantly altered mucosal homing receptor expression on CD4(+) T cells may result as a "bystander" effect after infection with some cytopathic isolates of HIV-1.     

Human cord blood mononuclear cells are preferentially infected by non-syncytium-inducing, macrophage-tropic human immunodeficiency virus type 1 isolates.

Identification of the factors which impact on the transmission of human immunodeficiency virus type 1 (HIV-1) from an infected mother to her infant is essential for the development of effective strategies to prevent perinatal HIV-1 infection. The current study was designed to determine if unstimulated human neonatal cord blood mononuclear cells (CBMC) differ from adult peripheral blood mononuclear cells (PBMC) in susceptibility to HIV-1 infection. Both cell populations were challenged with two laboratory and two clinical HIV-1 isolates with different phenotypic properties. Infection was evaluated by quantitation of p24 antigen production and p24 antigen expression by an enzyme immunoassay and immunofluorescence, respectively. T-cell markers were determined by flow cytometry. Unstimulated CBMC were preferentially infected by macrophage-tropic, non-syncytium-inducing (non-SI) laboratory and clinical isolates, whereas PBMC were more susceptible to T-lymphotropic, SI HIV-1 strains. The macrophage-tropic strain HIV-1Ba-L replicated to 100-fold higher titers in CBMC than a similar inoculum of the SI isolate HIV-1LAI. The opposite occurred in unstimulated PBMC, which replicated HIVLAI to eightfold higher titers than the macrophage-tropic isolate. These findings indicate that a selection of viral phenotype may occur with unstimulated CBMC displaying a predominant susceptibility to infection by macrophage-tropic, non-SI HIV-1 strains and that this selection may influence mother-infant transmission of HIV-1.     

Role of HIV-1 phenotype in viral pathogenesis and its relation to viral load and CD4+ T-cell count

The predictive value of HIV-1 phenotype in peripheral blood mononuclear cell (PBMC) coculture and the relation among viral phenotype, viral load, and CD4+ T-cell count were examined in two studies. In study A, 132 HIV-1–infected individuals were examined retrospectively for the relation between the result of their initial HIV cultivation in PBMC coculture and survival rate 6 years later. In study B, 176 patients were examined since 1994 for markers of HIV disease progression. HIV-1 phenotype was determined by PBMC cocultivation, viral load by NASBA HIV RNA QT System, and CD4+ T-cell count by flow cytometry. In study A, the percentage of survival for patients with initial negative virus culture was significantly higher (95%) than in patients with nonsyncytia-inducing (NSI) isolates (78%) and syncytia-inducing (SI) isolates (21%) (P < 0.05 and P < 0.0001, respectively). When SI phenotype was subdivided into moderately cytopathogenic and highly cytopathogenic, significant differences in the rate of survival between these subgroups could be observed (45% vs. 14%; P < 0.05). In study B, progression from negative virus culture to the isolation of NSI variants was associated with increasing viral load (P < 0.0001) but did not affect CD4+ T-cell count significantly (P > 0.07), whereas the switch from NSI to SI virus was accompanied by significant decline of CD4+ T-cells (P < 0.0001) but no change in viral load (P > 0.21). Thus, isolation and phenotyping of HIV represents an additional striking predictive marker for progression of HIV infection. J. Med. Virol. 56:259–263, 1998. © 1998 Wiley-Liss, Inc.