Sensitivity Changes over the Course of Infection Increases the Likelihood of Resistance Against Fusion but Not CCR5 Receptor Blockers

Abstract

As HIV-1 evolves over the course of infection, resistance against antiretrovirals may arise in the absence of drug pressure, especially against receptor and fusion blockers because of the extensive changes observed in the envelope glycoprotein. Here we show that viruses from the chronic phase of disease are significantly less sensitive to CCR5 receptor and fusion blockers compared to early infection variants. Differences in susceptibility to CCR5 antagonists were observed in spite of no demonstrable CXCR4 receptor utilization. No significant sensitivity differences were observed to another entry blocker, soluble CD4, or to reverse transcriptase, protease, or integrase inhibitors. Chronic as compared to early phase variants demonstrated greater replication when passaged in the presence of subinhibitory concentrations of fusion but not CCR5 receptor inhibitors. Fusion antagonist resistance, however, emerged from only one chronic phase virus culture. Because sensitivity to receptor and fusion antagonists is correlated with receptor affinity and fusion capacity, respectively, changes that occur in the envelope glycoprotein over the course of infection confer greater ability to use the CCR5 receptor and increased fusion ability. Our in vitro passage studies suggest that these evolving phenotypes increase the likelihood of resistance against fusion but not CCR5 receptor blockers.

HIV-1 envelope replication and α4β7 utilization among newly infected subjects and their corresponding heterosexual partners

“…Pooled envelope products were cloned into a HIV-1 NL4-3 backbone using yeast gap-repair homologous recombination [36]. From each subject, full-length envelope sequences were examined from 8 to 12 different clones.…”

Single genome amplification and standard bulk PCR yield HIV-1 envelope products with similar genotypic and phenotypic characteristics

“…All SGA and 4 independent bulk PCRs were combined to generate a SGA and bulk PCR amplified envelope library respectively. SGA and bulk PCR envelope pools were inserted into linearized pCMV-NL4-3-PBS→LTRΔGp160 plasmid using yeast gap-repair homologous recombination as previously described (Chatziandreou et al, 2012; Dudley et al, 2009). Clone pools were transfected into HEK293T cells to yield 293T transfection virus stocks.…”

Early Infection HIV-1 Envelope V1-V2 Genotypes Do Not Enhance Binding or Replication in Cells Expressing High Levels of α4β7 Integrin

“…6, 15 The chimeric envelopes were incorporated into a plasmid containing Q23-17 HIV sequences from the primer binding site (PBS) to the 3’ long terminal repeat (LTR), pCMV-Q23-17-PBS?LTR, using yeast gap-repair homologous recombination as previously described. 16-19 Replication competent viruses were generated by co-transfecting HEK293T cells with a plasmid containing the subject V1-V2 envelope within pCMV-Q23-17-PBS→LTR and another plasmid with Q23-17 sequences from 5’ LTR to early portion of gag, pCMV-Q23-17-LTR→Gag4. 16 The 293T transfection supernatants were passaged on activated CD4+ T cells for a maximum of 7 days to generate high titer peripheral blood mononuclear cell (PBMC) derived viruses.…”

“…16-19 Replication competent viruses were generated by co-transfecting HEK293T cells with a plasmid containing the subject V1-V2 envelope within pCMV-Q23-17-PBS→LTR and another plasmid with Q23-17 sequences from 5’ LTR to early portion of gag, pCMV-Q23-17-LTR→Gag4. 16 The 293T transfection supernatants were passaged on activated CD4+ T cells for a maximum of 7 days to generate high titer peripheral blood mononuclear cell (PBMC) derived viruses. Virus stocks were titered on TZM-bl cells as previously described.…”