Functional and genetic analysis of coreceptor usage by dualtropic HIV-1 subtype C isolates

Abstract

It is widely documented that a complete switch from the predominant CCR5 (R5) to CXCR4 (X4) phenotype is less common for HIV-1 subtype C (HIV-1C) compared to other major subtypes. We investigated whether dualtropic HIV-1C isolates represented dualtropic, mixed R5 and X4 clones or both. Thirty of 35 functional HIV-1 env clones generated by bulk PCR amplification from peripheral blood mononuclear cells (PBMCs) infected with seven dualtropic HIV-1C isolates utilized CXCR4 exclusively. Five of 35 clones displayed dualtropism. Endpoint dilution of one isolate did not yield a substantial proportion of R5-monotropic env clones. Sequence-based predictive algorithms showed that env sequences from PBMCs, CXCR4 or CCR5-expressing cell lines were indistinguishable and all possessed X4/dualtropic characteristics. We describe HIV-1C CXCR4-tropic env sequence features. Our results suggest a dramatic loss of CCR5 monotropism as dualtropism emerges in HIV-1C which has important implications for the use of coreceptor antagonists in therapeutic strategies for this subtype.

Drug Resistance and Coreceptor Usage in HIV Type 1 Subtype C-Infected Children Initiating or Failing Highly Active Antiretroviral Therapy in South Africa

“…V3 loop characterization confirmed previous findings in which X4 viruses had a more variable crown motif, longer length, and higher V3 net charges than R5 viruses. 11,18 In contrast to other studies, 11,17,18 these characteristics were not found in the majority of D/M viruses except for a V3 net charge above + 4.5 in 58.8%. The efficiency of genotypic predictions based on V3 loop sequences was also investigated with C-PSSM sinsi having the highest sensitivity and relatively high specificity for predicting CXCR4 usage (75.0 and 87.5%, respectively).…”

“…Second round PCR was carried out using Phusion Hot Start High-Fidelity DNA Polymerase (Finnzymes, Finland) with primers ENV1Adir and ENVM. 17 Second round cycling conditions were at 98°C for 30 s, followed by 35 cycles at 98°C for 10 s, 65°C for 30 s, and 72°C for 4 min, followed by a final extension at 72°C for 10 min. Amplified products were gel purified using the QIAquick Gel Extraction kit (Qiagen) and cloned and sequenced as previously described with the ABI Prism 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA).…”

“…Amplified products were gel purified using the QIAquick Gel Extraction kit (Qiagen) and cloned and sequenced as previously described with the ABI Prism 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). 17 Sequences were assessed for typical features of X4 viruses, including a V3 net charge above + 4.5 and basic amino acids at positions 11 and/or 25 (11/25 rule), both of which are predictive of CXCR4 usage. 18,19 Other features include an increased V3 length > 35 amino acids and a more variable crown motif.…”

Drug Resistance and Viral Tropism in HIV-1 Subtype C-Infected Patients in KwaZulu-Natal, South Africa

“…cDNA synthesis, envelope amplification and cloning were done as previously described 25 . Full-length env from 20 ARV-failing patients and 20 ARV-naïve patients was cloned into the pcDNA3.1D/V5-His-TOPO vector (Invitrogen).…”

Mutations in variable domains of the HIV-1 envelope gene can have a significant impact on maraviroc and vicriviroc resistance

“…Pseudoviruses were produced by co-transfection of Env expression plasmid and backbone pNL4-3. Luc.E-R-, as described previously [41,42]. Pseudovirus stocks were normalized with p24 ELISA prior to testing infectivity.…”