Quantifying the relationship between HIV-1 susceptibility to CCR5 antagonists and virus affinity for antagonist-occupied co-receptor.

Previous studies have demonstrated that HIV-1 develops resistance to CCR5 antagonists by gaining the ability to use drug-occupied co-receptor. However, the effects of CCR5 antagonists on the affinity of virus-co-receptor interactions have been difficult to quantify. We developed a pharmacological model for allosteric interaction at G-protein coupled receptors to analyze the effect of different CCR5 antagonists on infection by three laboratory adapted viruses with low, moderate and high susceptibility to the inhibitors. Infection data for these viruses fitted a model in which susceptibility to inhibition by CCR5 antagonists was directly related to fold reduction in virus affinity for CCR5. Dissociation constants for CCR5 antagonists calculated from the modeled data were consistent with values obtained by standard methods, suggesting that this approach can quantify pharmacologically relevant changes in co-receptor:ligand affinity in the context of infection of whole cells by authentic HIV-1 particles.

Structure-function analysis of human immunodeficiency virus type 1 gp120 amino acid mutations associated with resistance to the CCR5 coreceptor antagonist vicriviroc.

Vicriviroc (VCV) is a small-molecule CCR5 coreceptor antagonist currently in clinical trials for treatment of R5-tropic human immunodeficiency virus type 1 (HIV-1) infection. With this drug in development, identification of resistance mechanisms to VCV is needed to allow optimal outcomes in clinical practice. In this study we further characterized VCV resistance in a lab-adapted, VCV-resistant RU570 virus (RU570-VCV(res)). We show that K305R, R315Q, and K319T amino acid changes in the V3 loop, along with P437S in C4, completely reproduced the resistance phenotype in a chimeric ADA envelope containing the C2-V5 region from RU570 passage control gp120. The K305R amino acid change primarily impacted the degree of resistance, whereas K319T contributed to both resistance and virus infectivity. The P437S mutation in C4 had more influence on the relative degree of virus infectivity, while the R315Q mutation contributed to the virus concentration-dependent phenotypic resistance pattern observed for RU570-VCV(res). RU570-VCV(res) pseudovirus entry with VCV-bound CCR5 was dramatically reduced by Y10A, D11A, Y14A, and Y15A mutations in the N terminus of CCR5, whereas these mutations had less impact on entry in the absence of VCV. Notably, an additional Q315E/I317F substitution in the crown region of the V3 loop enhanced resistance to VCV, resulting in a stronger dependence on the N terminus for viral entry. By fitting the envelope mutations to a molecular model of a recently described docked N-terminal CCR5 peptide consisting of residues 2 to 15 in complex with HIV-1 gp120 CD4, potential new interactions in gp120 with the N terminus of CCR5 were uncovered. The cumulative results of this study suggest that as the RU570 VCV-resistant virus adapted to use the drug-bound receptor, it also developed an increased reliance on the N terminus of CCR5.

Mapping resistance to the CCR5 co-receptor antagonist vicriviroc using heterologous chimeric HIV-1 envelope genes reveals key determinants in the C2-V5 domain of gp120.

Several small molecule drugs that bind to the host CCR5 co-receptor and prevent viral entry have been developed for the treatment of HIV-1 infection. The innate variability found in HIV-1 envelope and the complex viral/cellular interactions during entry makes defining resistance to these inhibitors challenging. Here we found that mapping determinants in the gp160 gene from a primary isolate RU570-VCV(res), selected in culture for resistance to the CCR5 entry inhibitor vicriviroc, was complicated by inactivity of the cloned envelope gene in pseudovirus assays. We therefore recombined the envelope from RU570-VCV(res) into a highly active and susceptible ADA gp160 backbone. The chimeric envelopes generated robust signals in the pseudovirus assay and a 200 amino acid fragment, encompassing a C2-V5 region of the RU570-VCV(res) envelope, was required to confer resistance in both the single-cycle assay and in replicating virus. In contrast, a chimeric envelope that contained only the V3-loop region from this resistant virus was completely susceptible suggesting that the V3-loop changes acquired are context dependent.

Antiviral activity of transiently expressed IFN-kappa is cell-associated.

Most type I interferons (IFNs) are expressed by the majority of cell types in response to viral infection. In contrast, IFN-kappa has been reported to have a cellular distribution limited to keratinocytes and certain lymphoid cell populations. Recombinant expressed IFN-kappa has been shown previously to possess weak antiviral activity when directly compared with IFN-beta. In order to expand on the antiviral potential of IFN-kappa, we transiently transfected human cell lines to circumvent the need to purify recombinant proteins and to avoid the possible loss of biologic activity by the purification process. We evaluated the transcriptional signaling and antiviral activity of IFN-kappa in parallel with IFN-alpha2b with mammalian expression vectors to express each protein transiently. Both IFN-kappa and IFN-alpha2b exhibited comparable transcriptional and antiviral activities. However, in contrast to IFN-alpha2b transcriptional signaling and antiviral activity, IFN-kappa activity was not detectable in conditioned cell culture medium. Subsequent experiments revealed there was a direct relationship between IFN-kappa-expressing cells and antiviral activity. These results were confirmed in immunocytochemical studies. Furthermore, IFN-kappa exhibited cell-associated antiviral activity against a hepatitis C virus (HCV) replicon cell line. This novel IFN signaling strategy may represent an important distinct and divergent mechanism for limiting viral infections.