Multifaceted Mechanisms of HIV Inhibition and Resistance to CCR5 Inhibitors PSC-RANTES and Maraviroc

Abstract

Small-molecule CCR5 antagonists, such as maraviroc (MVC), likely block HIV-1 through an allosteric, noncompetitive inhibition mechanism, whereas inhibition by agonists such as PSC-RANTES is less defined and may involve receptor removal by cell surface downregulation, competitive inhibition by occluding the HIV-1 envelope binding, and/or allosteric effects by altering CCR5 conformation. We explored the inhibitory mechanisms of maraviroc and PSC-RANTES by employing pairs of virus clones with differential sensitivities to these inhibitors. Intrinsic PSC-RANTES-resistant virus (YA versus RT) or those selected in PSC-RANTES treated macaques (M584 versus P3-4) only displayed resistance in multiple-cycle assays or with a CCR5 mutant that cannot be downregulated. In single-cycle assays, these HIV-1 clones displayed equal sensitivity to PSC-RANTES inhibition, suggesting effective receptor downregulation. Prolonged PSC-RANTES exposure resulted in desensitization of the receptor to internalization such that increasing virus concentration (substrate) could saturate the receptors and overcome PSC-RANTES inhibition. In contrast, resistance to MVC was observed with the MVC-resistant HIV-1 (R3 versus S2) in both multiple-and singlecycle assays and with altered virus concentrations, which is indicative of allosteric inhibition. MVC could also mediate inhibition and possibly resistance through competitive mechanisms.

HIV-1 Group O Genotypes and Phenotypes: Relationship to Fitness and Susceptibility to Antiretroviral Drugs

“…In the absence of coreceptor switch to X4 usage, MVC resistance is typically associated with multiple amino acid mutations in both gp120 and gp41, which result in the ability of the MVC-resistant HIV-1 to utilize the drug-bound CCR5 receptor for host cell entry. 43 Most drug-resistant HIV-1 isolates (e.g., Y181C HIV-1) replicate at higher concentrations of drug (e.g., NVP) than the wild-type virus due to reduced drug binding to viral target (e.g., RT). This drug resistance mechanism results in a shift in the drug susceptibility curve and increase in IC 50 values.…”

“…We have observed a weak correlation between replicative fitness of group M HIV-1 isolates and sensitivity to CCR5 antagonist/agonists, such as MVC, but not to NNRTIs or PIs. 43,54 For this study, the IC 50 values of group O HIV-1 isolates to each drug were compared to the mean replicative fitness values of these same group O isolates (in the absence of drug). We did not observe a significant correlation, even when comparing IC 50 values for MVC and replicative fitness of the group O isolates (data not shown).…”

“…To determine if any polymorphism in envelope could map to reduce susceptibility, we sequenced the entire envelop gp120 of these isolates and compared these sequences to any mutations or polymorphisms related to variable inhibition by MVC or other CCR5 antagonists/agonists. [42][43][44][45][46][47] Specifically six amino acid positions namely 33 and 117, 290, 315, 396, 425 that span the C1, C2, V3, V4, and C4 regions of gp120, respectively, were analyzed (Table 3). Compared to group M, the amino acid residues at these positions showed a high variation in the group O sequences with less than 5% similarity with the exception of Q290, which was 100% conserved in group O.…”

Functional bottlenecks for generation of HIV-1 intersubtype Env recombinants

“…The pREC_nfl_HIV NL4-3 vector containing a near full length (nfl) HIV-1 isolate NL4-3 backbone has previously been described [ 34 ] and routinely used to clone different HIV genes to create various chimeric HIV-1 viruses through yeast gap repair homologous recombination [ 2 , 65 , 66 ]. To clone an HIV-1 sequence into pREC_nfl_HIV NL4-3 vector, the corresponding region in the NL4-3 backbone is first replaced with the yeast orotidine 5-phosphate decarboxylase ( URA3 ) gene using specific primers tagged with 40–60 nucleotides to allow for homologous recombination at either side of the target sequence.…”

Natural Cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling

“…Our results show that CysC95-146 prevents GPR15-mediated HIV-2 and SIV infection, while GPR15L displayed little if any inhibitory activity although it induced downmodulation of GPR15 from the cell surface. It is known that both receptor removal from the cell surface as well as competitive inhibition by occupation of the interaction site(s) of the HIV envelope glycoprotein by chemokines might contribute to inhibition of CCR5-or CXCR4-dependent HIV-1 infection (Lobritz et al, 2013;Steen et al, 2009). Our data support that competition by Cterminal Cystatin C fragments is more effective than GPR15L-induced downregulation of GPR15 in inhibiting lentiviral infection in both GHOST indicator and primary CD4+ T cells suggesting that only a certain threshold is required for viral entry.…”