Non-syncytium-inducing Viruses Research Articles

Longitudinal Assessment of Immune Response and Viral Characteristics in HIV-Infected Patients with Prolonged CD4+/Viral Load Discordance

Although suppression of HIV-1 RNA below the limit of detection is associated with optimal outcomes, many patients can maintain or increase their CD4(+) count for prolonged time periods in the presence of persistent low-level viremia. We followed seven patients with prolonged (>5 years) discordant CD4(+)/viral load (VL) responses on protease inhibitor (PI)-based highly active antiretroviral therapy (HAART) prospectively for 1 year to assess evolution of immune function, viral phenotype, replication capacity (RC), and resistance profile. Immune function was assessed by qualitative and quantitative measurement of cellular activation (CD38(+)HLA-DR(+) and CD38 antibodies bound per cell), and the interferon (IFN)-() ELISpot assay. Presence of syncytium-inducing (SI) or nonsyncytium-inducing (NSI) viral strains was determined by MT-2 cell culture. RC was measured by a modified rapid recombinant virus assay. The resistance profile was characterized by both genotypic and phenotypic analysis. Over the year of follow-up, IFN-() production to gag persisted, responses to other HIV antigens increased, and markers of cellular activation did not change. NSI virus predominated. The genotypic (GSS) and phenotypic (PSS) susceptibility scores remained stable. Evolution of RC was variable over the year of follow-up, but the RC of viruses remained well below that of wild-type clinical isolates. Thus, CD4(+)/VL discordance can be maintained for periods exceeding 5 years in some patients receiving PI-based HAART without significant evolution of HIV resistance.

High frequency of syncytium-inducing and CXCR4-tropic viruses among human immunodeficiency virus type 1 subtype C-infected patients receiving antiretroviral treatment.

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses have been found to almost exclusively use the chemokine receptor CCR5 as a coreceptor for entry, even in patients with advanced AIDS. We have characterized subtype C virus isolates from 28 patients from Harare, Zimbabwe, 20 of whom were receiving antiretroviral treatment. Virus from 10 of the treated patients induced syncytium formation (SI virus) when cultured with MT2 cells. Only non-syncytium-inducing (NSI) virus was cultured from the peripheral blood mononuclear cells of the eight patients who had not received treatment. The majority of these subtype C SI viruses were capable of using both CCR5 and CXCR4 as coreceptors for viral entry, and the consensus V3 loop sequences from the SI viruses displayed a high net charge compared to those of NSI viruses. While those on treatment had reverse transcriptase (RT) and protease mutations, there was no clear association between RT and protease drug resistance mutations and coreceptor tropism. These results suggest that CXCR4-tropic viruses are present within the quasispecies of patients infected with subtype C virus and that antiretroviral treatment may create an environment for the emergence of CXCR4 tropism.

Stromal-cell-derived factor 1 prevents the emergence of the syncytium-inducing phenotype of HIV-1 in vivo.

In a correlative study, the mean plasma level of the chemokine stromal-cell-derived factor 1 (SDF-1) was lower in subjects with syncytium-inducing (SI) than in subjects with non-syncytium-inducing (NSI) HIV isolates, regardless of the CD4 cell count or when compared with HIV-negative individuals. Individuals with high SDF-1 had an 81% probability of having an NSI virus phenotype compared with individuals with lower SDF-1. Increased expression of SDF-1 may help explain why the more pathogenic SI HIV-1 variants do not appear in some individuals.

Genetically and epidemiologically related "non-syncytium-inducing" isolates of HIV-1 display heterogeneous growth patterns in macrophages.

The objective of this study was to identify phenotypic parameters that could distinguish among seemingly homogeneous non-syncytium-inducing (NSI) viruses and that might provide a surrogate marker for clinical progression in pediatric human immunodeficiency virus type 1 (HIV-1) infection. We undertook a pilot analysis of 15 independent HIV-1 isolates collected prospectively from two mothers and their four children who displayed a spectrum of disease stages ranging from CDC categories A1 to C3. Viruses were evaluated for their ability to replicate in primary cells (including monocyte-derived macrophages [MDM]) and cell lines, for their co-receptor preference and for genetic features of the V3 hypervariable domain of env. Virtually all isolates displayed NSI phenotypes that were restricted in their capacity to replicate in cell lines and displayed V3 loops with uniformly low net positive charges. NSI viruses from two symptomatic children and one mother were macrophage-tropic, whereas NSI isolates from two asymptomatic children were unable to replicate in MDM and were designated primary lymphotropic viruses. Only one isolate was syncytium-inducing (SI), replicated in a variety of cell lines and in MDM, used multiple co-receptors, and was dual tropic, rather than a mixture of T-cell tropic and M-tropic viruses, as assessed by genetic analysis. Phenotypic heterogeneity among NSI viruses is revealed in the ability of isolates to replicate in MDM. This characteristic is related to disease stage and provides a potentially new in vitro criterion to distinguish among NSI isolates that is unlinked to other surrogate markers.

HIV-1 Evolves into a Nonsyncytium-Inducing Virus upon Prolonged Culture in Vitro

HIV-1 LAI is a syncytium-inducing (SI) virus with a broad host cell range. We previously isolated a LAI variant that improved replication in the SupT1 T cell line due to mutations within the C1 and C4 constant regions of the Env protein. We now report that this variant exhibits a severely restricted host cell range, as replication in other T cell lines and primary cells was abolished. Several Env-mediated functions were analyzed to provide a mechanistic explanation for this selective adaptation. The change in host cell tropism was not caused by a switch to a SupT1-specific coreceptor. Biosynthesis of the variant Env glycoprotein was not improved in SupT1 cells, and in fact a small defect in intracellular Env processing was observed. SupT1 infection assays did not reveal an improved Env function either, and a dramatic loss of infectivity was measured with other cell types. The Env-mutated HIV-1 reached an approximately fivefold higher level of virus production in SupT1 cells at the peak of infection. Unlike the LAI virus, the variant did not trigger the formation of syncytia. Our combined results suggest that the HIV-1 variant allows the infected host cell to survive longer, thus producing more viral progeny. The intricate virus-cell interaction results in a balance between optimal virus replication and host cell survival, causing a cytopathic SI isolate to evolve toward a nonsyncytium-inducing (NSI) phenotype in cell culture. These findings may help explain the absence of SI variants in the initial phase of HIV-1 infection, and the results dispute the notion that HIV-1 evolution should always go from the NSI to SI phenotype.

Sensitivity of human immunodeficiency virus infection to various alpha, beta and gamma chemokines.

Examination of a large panel of chemokines indicates that in addition to RANTES, MIP-1alpha and MIP-1beta, the beta-chemokine MCP-2 and, to a lesser extent, the gamma-chemokine lymphotactin also show anti-human immunodeficiency virus (HIV) activity in cell culture. The amount of chemokine needed to suppress HIV replication by > or = 50% was generally greater (> or = 250 ng/ml) than that required for inhibition of virus infection by RANTES, MIP-1alpha and MIP-1beta. The beta-chemokine MCP-3 was found to enhance the replication of both non-syncytium-inducing (NSI) and syncytium-inducing (SI) viruses at high concentrations (0.5-5 microg/ml). In contrast to a previous report, macrophage-derived chemokine was not found to inhibit HIV replication of either NSI or SI viruses, but at low concentrations enhanced NSI virus replication. When small amounts of RANTES or MCP-2 were added together with high concentrations of non-inhibitory chemokines, the anti-HIV effects were countered. Information on chemokines that affect HIV infection could be useful for future therapeutic strategies.

Differential effects of human immunodeficiency virus isolates on beta-chemokine and gamma interferon production and on cell proliferation.

All human immunodeficiency virus (HIV) isolates can grow readily in primary CD4(+) T cells, but they can be distinguished by their ability to replicate in macrophages and established T-cell lines. The macrophage-tropic viruses are generally non-syncytium inducing (NSI), whereas the T-cell-line-tropic viruses are syncytium inducing (SI) in cultured cells. We now demonstrate that infection of CD4(+) T cells by NSI and SI viruses shows a differential effect on production of beta-chemokines and gamma interferon. Infection by NSI viruses increased production of MIP-1alpha, MIP-1beta, and gamma interferon, whereas infection by SI viruses had no effect or decreased production of these cytokines. Production of RANTES was slightly increased during infection by both virus phenotypes. This differential effect of NSI and SI viruses was observed at the level of beta-chemokine mRNA as well as at the level of protein expression. Infection by NSI viruses also increased CD4(+) cell proliferation. These results may have relevance for a differential role of HIV strains in AIDS pathogenesis.

A 32-bp deletion within the CCR5 locus protects against transmission of parenterally acquired human immunodeficiency virus but does not affect progression to AIDS-defining illness.

The beta-chemokine receptor CCR5 is required as a coreceptor by non-syncytium-inducing (NSI) strains of human immunodeficiency virus type 1 (HIV-1). NSI viruses predominate early during an infection and are thought to be important for the transmission of HIV-1. The importance of CCR5 during parenteral transmission of HIV-1 was investigated. The distribution of the homozygous deleted CCR5 genotype among 566 exposed persons with hemophilia and 97 exposed transfusion recipients indicated that the lack of CCR5 expression protected persons from infection. This suggests that the initial predominance of NSI viruses during an infection does not result from limited availability of CXCR4-expressing cells within the mucosa but rather implies a more fundamental requisite for CCR5-expressing cells early during an infection regardless of the route of transmission. In addition, no difference in the rate of progression to AIDS (CDC 1987 definition) of infected heterozygous compared with homozygous wild type subjects was observed.

MT-2 tropism and CCR-5 genotype strongly influence disease progression in HIV-1-infected individuals.

The beta-chemokine receptor CCR-5 is the coreceptor for cellular entry by non-syncytium-inducing (NSI) HIV-1 strains that dominate early in infection. A 32 base-pair deletion (delta32) in the CCR-5 gene renders this coreceptor non-functional. Heterozygosity for this deletion [delta32/wild-type (wt)] is associated with slow disease progression. The purpose of this study was to document the combined impact on HIV-1 disease progression of the CCR-5 genotype and the biological phenotype of HIV-1. In a cross-sectional study of 258 HIV-1-infected Swedish individuals, the CCR-5 genotype (wt/wt or delta32/wt) was determined by polymerase chain reaction and the biological phenotype [NSI or syncytium-inducing (SI)] of virus isolates was determined in the MT-2 cell assay. Clinical status, HIV-1 RNA levels in plasma, CD4+ lymphocyte counts, and rate of CD4+ lymphocyte decline, based on retrospective analysis of CD4+ lymphocyte counts, were also recorded. None of the individuals were treated with protease inhibitors. The prevalence of the delta32/wt genotype was 23%. Subjects with the delta32/wt CCR-5 genotype more often carried SI virus than subjects with the wt/wt genotype (49 versus 35%; P=0.067), but there were no differences between the two groups in prevalence of AIDS, viral load, CD4+ lymphocyte count or CD4+ slope. NSI virus isolates were found in 159 (62%) out of 258 individuals. Individuals with NSI had lower prevalence of AIDS (39 versus 19%; P < 0.01), higher CD4+ lymphocyte counts (289+/-188 x 10(6)/l versus 153+/-162 x 10(6)/l; P=0.001), lower viral loads (median, 4.45 log10 versus 4.91 log10 copies/ml; P < 0.01) and a lower prevalence of the delta32/wt genotype (19 versus 29%; P=0.067) compared with individuals with SI virus. When the material was further subdivided, subjects with the delta32/wt genotype and SI virus had the highest prevalence of AIDS (P < 0.001), lowest CD4+ lymphocyte count (P=0.0001) and highest viral load (P=0.023) whereas the opposite was true for subjects with the delta32/wt genotype and NSI virus. A significantly higher proportion of subjects with NSI virus with delta32/wt and wt/wt CCR-5 genotype had been immunized with recombinant gp160. In summary, the delta32/wt CCR-5 genotype has a protective effect against HIV-1 disease progression that appears to be limited to individuals carrying HIV-1 variants with NSI phenotype. Immunization with recombinant gp160 tended to reduce the frequency of SI phenotypes.

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 overtime 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.

HIV type 1 V3 variation dynamics in vivo: long-term persistence of non-syncytium-inducing genotypes and transient presence of syncytium-inducing genotypes during the course of progressive AIDS.

We performed a population-based sequence analysis of the envelope V3 region of human immunodeficiency virus type 1 (HIV-1) in two infected hemophiliacs. The study was conducted over 6-9 years, extending from the asymptomatic phase to AIDS. In both patients, serial analysis showed that the V3 population at the initial stage consisted exclusively of putative non-syncytium-inducing (NSI) genotypes. Several of these clones continued to be present without change for many years until the terminal stage and often represented the dominant species in the population at each time interval. On the other hand, syncytium-inducing (SI) genotypes were initially absent but appeared shortly before severe depletion of CD4+ T cells and their proportion in the population appeared to correlate with the viral load. In sharp contrast to NSI genotypes, SI genotypes displayed a significantly shorter presence. Thus, rapid gross population changes were found in SI genotypes, which were particularly frequent in the asymptomatic phase and less frequent in the terminal stage. Furthermore, the ratio of nonsynonymous nucleotide substitutions per synonymous substitutions in the V3 region in SI genotypes was higher than the corresponding value of NSI genotypes and the phylogenetic tree analysis revealed that a longer branch length was observed in SI genotypes than in NSI genotypes. These results suggest that there might be a stronger pressure for selection on SI viruses than on NSI viruses during the high CD4 counts on the contrary to the fact that emergence of SI genotypes was well correlated with the rapid decline of CD4 count.

Dynamics of syncytium-inducing and non-syncytium-inducing type 1 human immunodeficiency viruses during primary infection.

Syncytium-inducing (SI) type 1 human immunodeficiency viruses (HIV-1) replicate faster in vitro and are generally more cytopathic to T cells than non-syncytium-inducing (NSI) HIV-1. Early in infection, the virus population typically consists of NSI viruses, with SI viruses appearing later. This is true even when both SI and NSI viruses are transmitted, and when SI viruses dominate the virus population peak seen during primary infection. Here, Phillips's model of HIV dynamics during primary infection (Science 271:497-499) is modified to map the growth trajectories of SI and NSI subpopulations. The model predicts that with certain rate constants, SI viruses may show a more precipitous decline in their numbers during primary infection than NSI viruses, and this may account for the observed dominance of NSI viruses early in infection.

The role of viral phenotype and CCR-5 gene defects in HIV-1 transmission and disease progression.

Cellular entry of human immunodeficiency virus type 1 (HIV-1) requires binding to both CD4 (ref, 1, 2) and to one of the chemokine receptors recently discovered to act as coreceptors. Viruses that infect T-cell lines to form syncytia (syncytium-inducing, SI) are frequently found in late-stage HIV disease and utilize the chemokine receptor CXCR-4; macrophage-tropic viruses are non-syncytium-inducing (NSI), found throughout disease and utilize CCR-5 (ref. 3-11). We postulated that CCR-5 gene defects might reduce infection risk in seronegative subjects and prolong AIDS-free survival in seropositive subjects with NSI but not SI virus. Homozygous (delta ccr5/delta ccr5) and heterozygous (CCR5/delta ccr5) CCR-5 deletions (delta ccr5) were found in 7 (2.7%) and 51 (19.5%), respectively, of 261 seronegative subjects from the San Francisco Men's Health Study. CCR-5/delta ccr5 genotype was identified in 33 of 172 (19.2%) nonprogressors and 25 of 234 (10.7%) progressors from the seropositive arm of this cohort. The delta ccr5 allele conferred a significant protective effect against HIV-1 infection (P = 0.001) and a survival advantage against disease progression (P = 0.02). Although both progressing and nonprogressing CCR5/delta ccr5 subjects were identified, a distinct survival advantage was shown for those with NSI virus (P < 0.0001). Thus, the protective effect of delta ccr5 against disease progression is lost when the infecting virus uses CXCR-4 as a coreceptor.

Macrophages and CD4+ T lymphocytes from two multiply exposed, uninfected individuals resist infection with primary non-syncytium-inducing isolates of human immunodeficiency virus type 1.

Despite multiple, high-risk sexual exposures, some individuals remain uninfected with human immunodeficiency virus type 1 (HIV-1). CD4+ lymphocytes from these individuals are less susceptible to infection in vitro with some strains of HIV-1, suggesting that the phenotype of the virus may influence its ability to interact with certain CD4+ cells. In the present study, we examined the susceptibility of CD4+ T lymphocytes and macrophages from two exposed uninfected individuals (EU2 and EU3) to infection with a panel of biologically cloned isolates of HIV-1 having either a non-syncytium-inducing (NSI) or a syncytium-inducing (SI) phenotype. Our results indicate that CD4+ T lymphocytes from EU2 and EU3 are resistant to infection with NSI isolates of HIV-1 but are susceptible to infection with primary SI isolates. In addition, we found that macrophages from EU2 and EU3 are resistant to infection with both NSI and SI isolates. The latter finding was confirmed by using several uncloned NSI and SI isolates obtained from patients during acute HIV-1 infection. In further experiments, env clones encoding glycoproteins characteristic of NSI or SI viruses were used in single-cycle infectivity assays to evaluate infection of CD4+ lymphocytes and macrophages from EU2 and EU3. Consistent with our previous results, we found that macrophages from these individuals are resistant to infection with NSI and SI env-pseudotyped viruses, while CD4+ T lymphocytes are resistant to NSI, but not SI, pseudotyped viruses. Overall, our results demonstrate that CD4+ cells from two exposed uninfected individuals resist infection in vitro with primary, macrophage-tropic, NSI isolates of HIV-1, which is the predominant viral phenotype found following HIV-1 transmission. Furthermore, infection with NSI isolates was blocked in both CD4+ T lymphocytes and macrophages from these individuals, suggesting that there may be a common mechanism for resistance in both cell types.

Changes in cellular virus load and zidovudine resistance of syncytium-inducing and non-syncytium-inducing human immunodeficiency virus populations under zidovudine pressure: a clonal analysis.

Zidovudine treatment preferentially benefits persons with only non-syncytium-inducing (NSI) human immunodeficiency virus type 1 (HIV-1) variants. To understand this differential efficacy, changes in cellular virus load, clonal composition of HIV-1 populations, and development of resistance-conferring reverse transcriptase mutations were studied in 17 persons initiating zidovudine therapy. Zidovudine treatment resulted in larger and more sustained decreases in cellular virus load in persons with NSI variants only compared with persons also carrying syncytium-inducing (SI) variants. Although the former group had a delayed emergence of resistance mutations, differences in initial responses between the 2 groups were independent of the emergence of resistance mutations. Changes in virus load in subjects also carrying SI variants were due mainly to loss of coexisting NSI virus. Resistance mutations emerged at similar rates in both coexisting variants. Data suggest that mechanisms other than drug resistance are necessary to completely explain the phenotype-dependent benefit of zidovudine.

Genetic variability and function of the long terminal repeat from syncytium-inducing and non-syncytium-inducing human immunodeficiency virus type 1.

We analyzed sequence variability and function of the long terminal repeat (LTR) from syncytium-inducing (SI) and non-syncytium-inducing (NSI) HIV-1. Twenty LTR DNA clones were obtained by polymerase chain reaction amplification and molecular cloning from short-term cultures of SI and NSI viruses from an AIDS patient and two asymptomatic individuals, respectively. All the LTR clones tested contained multiple nucleotide changes (mostly G-to-A transitions), compared to the subtype B consensus sequence, which were clustered within the negative regulatory element, including NF-AT, USF, and TCF-1 alpha binding sites. The core promoter/TAR region sequences were highly conserved. The basal and Tat-mediated transcriptional activities of selected LTR clones tested were 0.1 to 1 and 0.2 to 0.5 times that of the control, respectively, regardless of the SI or NSI origin of the clones. Phylogenetic analysis revealed interi-solate sequence divergence in the LTR that was similar but not identical to previously analyzed vif sequences from the same samples. In particular, the inter-isolate distances from reference sequences differed for the LTR and vif. This raises the possibility that recombination occurred between corresponding LTR and vif loci of the quasi-species present in the isolates described here.

Syncytium-inducing (SI) phenotype suppression at seroconversion after intramuscular inoculation of a non-syncytium-inducing/SI phenotypically mixed human immunodeficiency virus population

Two distinct biological phenotypes of human immunodeficiency virus (HIV) have been described: the non-syncytium-inducing (NSI) phenotype, best characterized by the inability to infect MT-2 cells, and the syncytium-inducing (SI) phenotype, with the ability to infect MT-2 cells. The earliest virus population observed following HIV transmission is generally of the NSI phenotype, even after exposure to inocula of mixed NSI/SI phenotype. In this study, the issue of intrapatient selection of virus phenotype following transmission was addressed by studying two cases of accidental transmission. A comparison of the sequences of the V1-V2 and the V3 coding regions of the envelope gene and the p17 region of the gag gene showed that the donor-recipient pairs were tightly clustered in all gene segments, but away from local and published transmission controls. The intrasample variation of the p17 sequence was greater in the recipients and smaller in the donors than that of the V3 region sequence, indicating selection of V3 at transmission. In these transmission cases, the effects of an intravenous inoculation of a small quantity of blood containing predominantly SI V3 sequences (6 of 8 clonal sequences) were compared with those of an intramuscular inoculation of a large quantity of blood containing predominantly NSI viruses (14 of 16 clonal sequences). Both SI and NSI V3 regions were demonstrated to be phenotypic expressions of genetically related viral strains. The inoculation of the predominantly SI virus population resulted in the persistence of an SI virus population in the recipient and a rapid CD4+ T-cell decline. The inoculation of the predominantly NSI population resulted in a selective amplification of SI viruses before seroconversion, followed by a suppression of SI viruses at seroconversion and a rapid decline of CD4+ T-cell numbers. These data suggest that the suppression of SI viruses can be accomplished following the development of HIV-specific immunity and that the ability to suppress SI viruses does not prevent the development of immunodeficiency.