Emergence of X4 usage among HIV-1 subtype C: evidence for an evolving epidemic in South Africa
Connell, Bridgette Ja; Michler, Katherinea; Capovilla, Alexioa; Venter, Willem DFb; Stevens, Wendy Sa; Papathanasopoulos, Maria Aa
aHIV Pathogenesis Research Laboratory, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Johannesburg, South Africa
bReproductive Health and HIV Research Unit, University of the Witwatersrand Medical School, Johannesburg, South Africa.
Received 5 December, 2007
Accepted 14 December, 2007
This study investigated the genotype and phenotype of HIV-1 isolates of 20 South African AIDS patients. We found the highest percentage of CXCR4 usage among primary isolates, in which 30% efficiently utilized CXCR4 and exhibited the syncytium-inducing phenotype. Phylogenetic analysis of env confirmed that 19 of the 20 were subtype C, and syncytium-inducing viruses had genetic changes in the V3 loop, characteristic of CXCR4 usage. Results imply that the frequency of CXCR4-utilizing subtype C is increasing with time.
The HIV-1 envelope glycoprotein (Env) plays a pivotal role in the viral life cycle, and is responsible for viral transmission, host cell entry and tropism. HIV-1 enters host cells through a complex and dynamic process whereby Env binds sequentially to CD4 and a co-receptor, either CCR5 or CXCR4 . HIV-1 tropism can largely be linked to these two co-receptors.
Co-receptor usage and switching has been extensively studied in HIV-1 subtype B isolates. During the early stages of infection non-syncytium inducing, macrophage-tropic, CCR5-utilizing (R5) HIV-1 strains predominate, whereas T-cell-tropic, syncytium-inducing, CXCR4-utilizing (X4) isolates may emerge during the later phases of infection . Some viruses can use both CCR5 and CXCR4 (dual tropic). Changes in cellular tropism by HIV-1 in vivo seems to be a key event in disease pathogenesis, and broadening of the co-receptor usage profile of HIV-1 may be associated with accelerated CD4 T-cell loss, disease progression to AIDS , and more recently has clinical implications for the use of CCR5 inhibitors in antiretroviral therapy. Approximately 50–90% of isolates from AIDS patients infected with subtype B show multiple co-receptor usage . Biological and genotypic factors contributing to the R5 to X4 variant ‘switching’ during the classic course of HIV-1 infection are not completely understood. It is known, however, that the overall positive amino acid charge and specific conserved amino acids within the V3 loop of Env play a critical role in co-receptor phenotype and binding .
HIV-1 subtype C viruses now cause the vast majority (> 56%) of new HIV-1 infections worldwide, and predominate in India, Brazil, Botswana, Malawi, Ethiopia and South Africa (http://www.unaids.org). Published research up to 2007 has suggested that HIV-1 subtype C isolates in different regions of the world isolated from various cohorts during different disease stages utilize CCR5 almost exclusively and show minimal conversion to CXCR4 tropism [6–13]. To date, less than 30 CXCR4-utilizing subtype C primary isolates have been described worldwide [6–14]. This cross-sectional study investigated the genetic and phenotypic properties of 20 HIV-1 primary isolates from randomly selected antiretroviral drug-naive South African AIDS patients.
Whole blood was collected by convenience sampling from advanced AIDS patients attending a Johannesburg clinic between January and July, 2005. All patients provided informed consent. Ethical clearance was obtained for research on human subjects (medical) at the University of the Witwatersrand. Epidemiological and clinical data were obtained from patient hospital files at the time of sample collection (Table 1). Twenty primary virus isolates were obtained by standard peripheral blood mononuclear cell co-culture techniques, and their biotype and phenotype was established by growth in U87.CD4.CCR5/CXCR4 or MT-2 cell lines, respectively . Proviral DNA was extracted, and the full-length env and partial pol genes were polymerase chain reaction amplified, sequenced, and analysed.
This study reports a high percentage (30%) of HIV-1 subtype C primary isolates (and a unique CD recombinant) able to grow in MT-2 cells and use CXCR4 for cell entry (Table 1). The earliest characterized subtype C viruses from Malawi (collected from 1981 to 1989) all exhibited the non-syncytium-inducing phenotype (152/152) . In South Africa, only five out of 29 (17%) late-stage AIDS patients (samples collected from 1998 to 2000) and four out of 40 (10%) perinatally infected children (collected from 1998 to 2002) yielded viruses with syncytium-inducing phenotypes [11,12]. A study from Botswana showed that only three out of 29 subtype C isolates (one from 1996, two from 2003) used CXCR4 more efficiently than CCR5 . It thus appears that South African HIV-1 subtype C isolates are capable of evolving to use CXCR4 for cell entry. As the primary isolates in this study were obtained from patient samples collected over a relatively short time period (6 months), these data may suggest an ongoing dynamic evolution of env and the maturing of the subtype C epidemic in South Africa.
Most of the six patients with syncytium-inducing viruses had relatively low CD4 T-cell counts. These data are concurrent with previously published data on subtype B-infected patients. The higher detection of CXCR4 usage by subtype C in this study is not likely to be an artifact of the low CD4 T-cell counts in these patients, because their clinical profiles are similar to other patients from which X4 variants were isolated in the above-mentioned studies.
Phylogenetic analysis of V3 loop sequences revealed that 19 out of 20 clustered within subtype C, and 05ZAFV10 clustered within subtype D. Partial pol sequence analysis confirmed the patients were drug naive because no isolates harboured primary resistance mutations. Comparison of env and pol sequences revealed that 05ZAFV10 is a unique CD recombinant, highlighting the importance of ongoing subtype surveillance in South Africa. Signature sequence motifs within V3 such as the tetramer crown motif, overall charge and positions 11 and 25, were strong predictors of co-receptor usage for each isolate (Table 1). The C-PSSM algorithm predicted all six syncytium-inducing isolates with 100% accuracy; however, only 12 out of 14 non-syncytium-inducing isolates were scored correctly . Overall, the subtype C Env can accommodate amino acid changes needed to use CXCR4 as a co-receptor.
This is the highest reported X4 usage in a drug-naive subtype C AIDS cohort worldwide. In addition, we have observed syncytium-inducing CXCR4-utilizing HIV-1 subtype C isolates in treatment-experienced patients (results not shown), confirming previous findings by Johnston et al. . Although the percentage CXCR4 usage reported in this study is not as high as subtypes B and D (50–90%), it has steadily increased from 0% in the 1980s , to up to 17% in 2000 . Overall, these results imply that the frequency of subtype C CXCR4-utilizing viruses may be increasing with time, as was previously documented in the CRF01_AE epidemic in Thailand. These findings need to be confirmed in a larger cohort, and future work should focus on whether env is capable of mutating/evolving from CCR5 usage to CXCR4 usage within the same patient, or whether CXCR4-utilizing viruses are inherently present from early infection. The emergence/evolution of CXCR4 usage among HIV-1 subtype C may have profound implications for viral transmission, pathogenesis, disease progression and emphasize the importance for the ongoing investigation of co-receptor usage of subtype C viruses, particularly because the CCR5 antagonist maraviroc has received US Food and Drug Administration approval.
The full-length envelope glycoprotein genes, incorporating the V3 loop gene regions, have been submitted to GenBank under the following accession numbers DQ382361–DQ382380.
The authors are grateful for the kind donations of blood specimens from HIV-positive patients in difficult circumstances. Research funding from the South African National Research Foundation, Poliomyelitis Research Foundation, USAID, PEPFAR, University of the Witwatersrand and Elevation Biotech is gratefully acknowledged.
The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: MT-2 cells from Dr Douglas Richman; U87.CD4.CCR5 and U87.CD4.CXCR4 cells from Dr HongKui Deng and Dr Dan R. Littman.
Sponsorship: Research funding came from the South African National Research Foundation, Poliomyelitis Research Foundation, USAID, PEPFAR, University of the Witwatersrand and Elevation Biotech.
Conflicts of interest: None.
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