Impact of the HIV-1 env Genetic Context outside HR1–HR2 on Resistance to the Fusion Inhibitor Enfuvirtide and Viral Infectivity in Clinical Isolates

Abstract

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1–HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1–HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1–HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1–HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy.

Coreceptor usage of Chinese HIV-1 and impact of X4/DM transmission clusters among recently infected men who have sex with men

“…[6] Geno2pheno and WebPSSM are the most widely used among genotypic assays. Although clinical evidences provide support for the validity of using an FPR between 5% and 10% when applying Geno2pheno to predict subtype B tropism, [33–35] several studies indicated that both Geno2pheno (FPR = 10%) and WebPSSM overestimates the presence of X4 viruses for CRF01_AE. [17,18] In this study, we adopted the recently published algorithm that uses combination of Geno2pheno (FPR = 10%) and WebPSSM to predict HIV-1 tropism.…”

HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG

“…Viral cDNA was synthesized in a one-step RT-PCR reaction using forward primer KVL008 and reverse primer KVL009 [48] in 50 µl mix containing 5 µl viral RNA, 20 µM of each primer, 1 µl SuperScript III One-Step RT-PCR with Platinum Taq High Fidelity mix and 8 units RNAseOUT (all from Invitrogen, Merelbeke, Belgium) under the following conditions: initial denaturation at 94°C for 2 mins and 40 amplification cycles (94°C for 15 s, 60°C for 30 s, 68°C for 4 mins) followed by a final 10 mins extension step at 68°C. 2 µl of the amplified cDNA was further amplified using forward primer MM1 FP (5′-GCCTTAGGCATCTCTTATGGCAGGAAGAAG-3′) and reverse primer rec HR1-2_RP (5′-CTCTCTCTfCCACCTTCTTCTTC-3′) [27] in a 50 µl reaction mix containing 2 mM MgSO 4 , 0.2 mM of each dNTP, 20 µM of each primer, 2.5 Units Platinum Taq High Fidelity DNA polymerase. The amplification conditions were: initial denaturation step at 95°C for 3 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 48°C for 30 s, extension at 68°C for 3 min, and a final extension step at 68°C for 10 min.…”

Isolation of an HIV-1 neutralizing peptide mimicking the CXCR4 and CCR5 surface from the heavy-chain complementary determining region 3 repertoire of a viremic controller

“…LTS-2 is a viremic controller [6][7][8] infected in 1992 and harboring an R5 provirus at the time of sampling (2006). Plasma neutralizing properties were confirmed in five longitudinal samples from 2006 to 2013 using a single cycle infection assay based on luciferase-tagged recombinant viral particles (RVA) [20,21]. LTS-2 genotyping revealed the absence of the CCR5 delta 32 deletion, the CCR2 V64I and the SDF1 G801A polymorphisms and combination of HLA A Ã 02/ Ã 11, B Ã 27/ Ã 35, and C Ã 02/ Ã 04 alleles.…”