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Entry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experienced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinant's phenotype, indicating its primary importance in conferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determinants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 binding sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context dependent and thus inherently unpredictable. IMPORTANCE The entry inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination antiretroviral therapy. Treatment failure with drug-resistant virus is particularly interesting because it tends to be rare, with lack of sensitivity usually associated with the presence of CXCR4-using virus (CXCR4 is the main alternative coreceptor HIV-1 uses, in addition to CD4). We analyzed envelope sequences from HIV-1, obtained from 20 patients who enrolled in maraviroc clinical trials and experienced treatment failure, without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We found that in these individuals, resistant viruses form a distinct population that evolved once and was successful as a result of drug pressure. Further evolutionary analysis placed the complex network of interdependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance.

Filibuvir (PF-00868554) is an investigational nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural 5B (NS5B) RNA-dependent RNA polymerase currently in development for treating chronic HCV infection. The aim of this study was to characterize the selection of filibuvir-resistant variants in HCV-infected individuals receiving filibuvir as short (3- to 10-day) monotherapy. We identified amino acid M423 as the primary site of mutation arising upon filibuvir dosing. Through bulk cloning of clinical NS5B sequences into a transient-replicon system, and supported by site-directed mutagenesis of the Con1 replicon, we confirmed that mutations M423I/T/V mediate phenotypic resistance. Selection in patients of an NS5B mutation at M423 was associated with a reduced replicative capacity in vitro relative to the pretherapy sequence; consistent with this, reversion to wild-type M423 was observed in the majority of patients following therapy cessation. Mutations at NS5B residues R422 and M426 were detected in a small number of patients at baseline or the end of therapy and also mediate reductions in filibuvir susceptibility, suggesting these are rare but clinically relevant alternative resistance pathways. Amino acid variants at position M423 in HCV NS5B polymerase are the preferred pathway for selection of viral resistance to filibuvir in vivo .

The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1). A major problem with the first approved NNRTIs was the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular K103N and Y181C, which led to resistance to the entire class. We adopted an iterative strategy to synthesize and test small molecule inhibitors from a chemical series of pyrazoles against wild-type (wt) RT and the most prevalent NNRTI-resistant mutants. The emerging candidate, lersivirine (UK-453,061), binds the RT enzyme in a novel way (resulting in a unique resistance profile), inhibits over 60% of viruses bearing key RT mutations, with 50% effective concentrations (EC50s) within 10-fold of those for wt viruses, and has excellent selectivity against a range of human targets. Altogether lersivirine is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses.

Maraviroc is a CCR5 antagonist in clinical development as one of a new class of antiretrovirals targeting human immunodeficiency virus type 1 (HIV-1) coreceptor binding. We investigated the mechanism of HIV resistance to maraviroc by using in vitro sequential passage and site-directed mutagenesis. Serial passage through increasing maraviroc concentrations failed to select maraviroc-resistant variants from some laboratory-adapted and clinical isolates of HIV-1. However, high-level resistance to maraviroc was selected from three of six primary isolates passaged in peripheral blood lymphocytes (PBL). The SF162 strain acquired resistance to maraviroc in both treated and control cultures; all resistant variants were able to use CXCR4 as a coreceptor. In contrast, maraviroc-resistant virus derived from isolates CC1/85 and RU570 remained CCR5 tropic, as evidenced by susceptibility to the CCR5 antagonist SCH-C, resistance to the CXCR4 antagonist AMD3100, and an inability to replicate in CCR5 Δ32/Δ32 PBL. Strain-specific mutations were identified in the V3 loop of maraviroc-resistant CC1/85 and RU570. The envelope-encoding region of maraviroc-resistant CC1/85 was inserted into an NL4-3 background. This recombinant virus was completely resistant to maraviroc but retained susceptibility to aplaviroc. Reverse mutation of gp120 residues 316 and 323 in the V3 loop (numbering from HXB2) to their original sequence restored wild-type susceptibility to maraviroc, while reversion of either mutation resulted in a partially sensitive virus with reduced maximal inhibition (plateau). The plateaus are consistent with the virus having acquired the ability to utilize maraviroc-bound receptor for entry. This hypothesis was further corroborated by the observation that a high concentration of maraviroc blocks the activity of aplaviroc against maraviroc-resistant virus.

Antagonists of the human immunodeficiency virus type 1 (HIV-1) coreceptor, CCR5, are being developed as the first anti-HIV agents acting on a host cell target. We monitored the coreceptor tropism of circulating virus, screened at baseline for coreceptor tropism, in 64 HIV-1-infected patients who received maraviroc (MVC, UK-427,857) as monotherapy for 10 days. Sixty-two patients harbored CCR5-tropic virus at baseline and had a posttreatment phenotype result. Circulating virus remained CCR5 tropic in 60/62 patients, 51 of whom experienced an HIV RNA reduction from baseline of >1 log 10 copies/ml, indicating that CXCR4-using variants were not rapidly selected despite CCR5-specific drug pressure. In two patients, viral load declined during treatment and CXCR4-using virus was detected at day 11. No pretreatment factor predicted the emergence of CXCR4-tropic virus during maraviroc therapy in these two patients. Phylogenetic analysis of envelope ( Env ) clones from pre- and posttreatment time points indicated that the CXCR4-using variants probably emerged by outgrowth of a pretreatment CXCR4-using reservoir, rather than via coreceptor switch of a CCR5-tropic clone under selection pressure from maraviroc. Phylogenetic analysis was also performed on Env clones from a third patient harboring CXCR4-using virus prior to treatment. This patient was enrolled due to a sample labeling error. Although this patient experienced no overall reduction in viral load in response to treatment, the CCR5-tropic components of the circulating virus did appear to be suppressed while receiving maraviroc as monotherapy. Importantly, in all three patients, circulating virus reverted to predominantly CCR5 tropic following cessation of maraviroc.