Construction and Phenotypic Characterization of HIV Type 1 Functional Envelope Clones of Subtypes G and F

Abstract

Subtype G has been estimated to represent the fourth most prevalent clade in the HIV-1 pandemic and subtype F is widely circulating in parts of South America (frequently within BF recombinant forms) and in Romania. However, functional envelope clones of these subtypes are lacking, which are needed for studies on antibody-mediated neutralization, coreceptor usage, and efficiency of viral entry inhibitor drugs. Here we report the construction, neutralization properties, and coreceptor usage of HIV-1 functional envelope clones of subtypes G (n = 15) and F (n = 7). These clones were obtained through RT-PCR amplification of HIV-1 gp160 from plasma RNA, and were used for pseudovirus production. All 15 subtype G-enveloped pseudoviruses were resistant to neutralization by gp120-targeted broadly neutralizing monoclonal antibodies (MAbs) b12 and 2G12, while a majority were neutralized by gp41-targeted MAbs 2F5 and 4E10. With regard to the subtype F envelopes, all seven pseudoviruses were resistant to 2F5 and b12, six were resistant to G12, and six were neutralized by 4E10. Coreceptor usage testing revealed that 21 of 22 envelopes were CCR5-tropic, including all 15 subtype G envelopes, seven of which were from patients with CD4(+) T cell counts <200/ml. These results confirm the broadly neutralizing activity of 4E10 on envelope clones across all tested group M clades, including subtypes G and F, reveal the resistance of most subtype F-enveloped pseudoviruses to broadly neutralizing MAbs b12, 2G12, and 2F5, and suggest that, similarly to subtype C, CXCR4 tropism is uncommon in subtype G, even at advanced stages of infection.

Independent evolution of macrophage-tropism and increased charge between HIV-1 R5 envelopes present in brain and immune tissue

“…Outer primers for episomal DNA amplification were RevenvA and LAI (5'-GCGCTTCAGCAAGCCGAGTCCT-3') [56]. The inner primers were the same for both proviral and episomal rev-env amplification as follows; RevenvBTOPO (5'-CACCTAGGCATCTCCTATGGCAGGAAGAAG-3') and Env-lo (5'-GTTTCTTCCAGTCCCCCCTTTTCTTTTAAAAAG-3'; [77]). The PCR products in positive wells at endpoint dilutions were purified from a 0.8% crystal violet stained agarose gel using a QIAquick Gel Extraction Kit (Qiagen).…”

Short Communication: Molecular Epidemiology, Phylogeny, and Phylodynamics of CRF63_02A1, a Recently Originated HIV-1 Circulating Recombinant Form Spreading in Siberia

“…This would allow for amplification of the full-length envelope sequence in nested PCR using primers Env-S and Env-A, which could be used for subsequent construction of functional envelope clones. 22 Both fragments flanking env, from vif through vpu, and nef plus a segment of the 3¢ LTR, respectively, were amplified by nested PCR from the RT-PCR product amplified with SG3-up and SG3-lo, using primers SG3-N-S and SSD2b, and NEF-S4 and 3¢ nef-3c, respectively. Gag and a fragment of the 5¢ LTR were amplified with SC-A-O-S-R and SC-A-O-A in RT-PCR and SC-A-N-S-R and SC-A-N-A in nested PCR; the 3¢ segment of pol was amplified with RTDS-O-S and SC-B-N-A in RT-PCR and RTDS-N-S and B2-N-A in nested PCR.…”

Phylogenetics of HIV-1 subtype G env: Greater complexity and older origins than previously reported

“…This included three sequences with no specified country of origin listed in the Genbank database: GenBank: HQ236615, GenBank: HQ236619 (Revilla et al, 2011) and GenBank: HQ236607. All three sequences (which we coded as being collected in Spain) were embedded in lineage G1 in both the ML tree ( Fig.…”

“…All three sequences (which we coded as being collected in Spain) were embedded in lineage G1 in both the ML tree ( Fig. 1 and in an Iberian cluster in (Revilla et al, 2011)) and the MCC tree ( Fig. 2 and Suppl Fig.…”

“…Angola (partial seq) Lineages G4 was poorly populated, but included one of the new sequences reported here. It was geographically diverse, including sequences from Cameroon, Kenya and one sequence from a Ghanaian individual in Spain, accession number EU885764 (Revilla et al, 2011). In the MCC tree, G4 contained two Angolan sequences that were not present in G4 in the ML tree, perhaps contributing to the low posterior support for the clade in the MCC tree (0.55).…”