Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance–associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1‐4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156‐RASs were not maintained. For genotypes 1 and 2, persistence of 156‐RASs depended on genome‐wide substitution networks, co‐selected under continued PI treatment and identified by next‐generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156‐RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre‐existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156‐RASs, we observed genome‐wide selection of substitutions under treatment. Conclusion : Comprehensive PI resistance profiling for HCV genotypes 1‐6 revealed 156‐RASs as key determinants of high‐level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1‐3 156‐variants, which might pose a threat to clinically relevant combination treatments

Adaptation of HIV-1 Envelope to Macaque Cells and Implications for the Design of Improved Models of HIV/AIDS

Thesis (Ph.D.)--University of Washington, 2012Research in the area of Human Immunodeficiency Virus type 1 (HIV-1) vaccine development is hindered by the limitations of the Simian/Human Immunodeficiency Viruses (SHIVs) used in macaque studies. The relevance of SHIV infection of macaques to HIV-1 infection in humans depends on how closely SHIVs mimic aspects of HIV-1 transmission, pathogenesis, and diversity - properties partly determined by the HIV-1 envelope (Env). The inclusion of relevant Envs in SHIVs has proven to be difficult as many HIV-1 Envs are unable to mediate efficient infection of macaque cells. To better understand barriers to HIV-1 Env in macaque cells, we adapted a subtype A based SHIV (SHIV-A) for replication in pig-tailed macaque (Pt) lymphocytes, and identified two mutations in gp120, A204E and G312V, that increased replication by >100-fold. Introduction of these changes into multiple subtype A Envs also greatly increased entry into Pt cells. A204E and G312V Env variants continued to require CCR5 for entry, displayed sensitivity to neutralization by soluble CD4 (sCD4), and all showed a greatly increased ability to use Pt CD4. These findings hinted at the inefficient use of CD4 as an unappreciated restriction to HIV-1 replication in macaque cells and identified changes to subtype A Envs that allowed for increased infection of macaque cells, which has direct implications for the development of a successful SHIV-A. In an effort to develop a SHIV-A for in vivo use, the A204E and G312V Env variants were introduced to SHIVs, which were further assessed for their ability to mediate spreading infection in macaque cells, as well as their neutralization properties. Two SHIV-As were identified that warrant further evaluation in vivo. To establish whether CD4 is a determinant for limited infection of macaque cells by other globally relevant HIV-1 subtypes, HIV-1 Envs representative of diverse circulating strains from subtypes A to D were assayed for their ability to infect cells expressing Pt CD4 or Rhesus (Rh) CD4. Most of the 39 Envs tested (>74%) showed a >10-fold decrease in their ability to mediate infection using macaque CD4 as compared to human CD4. Infectivity using macaque CD4 was highly associated with sensitivity to sCD4 and the ability to mediate infection utilizing low levels of CD4 (p < 0.0001), thus identifying properties of HIV-1 Envs that allow for the increased ability to use macaque CD4. Additionally, the determinants of CD4 sufficient for increased infection by HIV-1 were mapped to a single amino acid difference in the D1 domain. This demonstrates that the inefficient use of macaque CD4 acts as a potent barrier to replication mediated by Envs from circulating strains of HIV-1 and identifies characteristics of HIV-1 Envs that may allow for the successful creation of relevant SHIV models

Several cell-intrinsic effectors drive type I interferon-mediated restriction of HIV-1 in primary CD4+ T cells

Summary: Type I interferon (IFN) upregulates proteins that inhibit HIV within infected cells. Prior studies have identified IFN-stimulated genes (ISGs) that impede lab-adapted HIV in cell lines, yet the ISG(s) that mediate IFN restriction in HIV target cells, primary CD4+ T cells, are unknown. Here, we interrogate ISG restriction of primary HIV in CD4+ T cells by performing CRISPR-knockout screens with a custom library that specifically targets ISGs expressed in CD4+ T cells. Our investigation identifies previously undescribed HIV-restricting ISGs (HM13, IGFBP2, LAP3) and finds that two factors characterized in other HIV infection models (IFI16 and UBE2L6) mediate IFN restriction in T cells. Inactivation of these five ISGs in combination further diminishes IFN’s protective effect against diverse HIV strains. This work demonstrates that IFN restriction of HIV is multifaceted, resulting from several effectors functioning collectively, and establishes a primary cell ISG screening model to identify both single and combinations of HIV-restricting ISGs

Analysis of the Percentage of Human Immunodeficiency Virus Type 1 Sequences That Are Hypermutated and Markers of Disease Progression in a Longitudinal Cohort, Including One Individual with a Partially Defective Vif▿ †

Hypermutation, the introduction of excessive G-to-A substitutions by host proteins in the APOBEC family, can impair replication of the human immunodeficiency virus (HIV). Because hypermutation represents a potential antiviral strategy, it is important to determine whether greater hypermutation is associated with slower disease progression in natural infection. We examined the level of HIV-1 hypermutation among 28 antiretroviral-naive Kenyan women at two times during infection. By examining single-copy gag sequences from proviral DNA, hypermutation was detected in 16 of 28 individuals. Among individuals with any hypermutation, a median of 15% of gag sequences were hypermutated (range, 5 to 43%). However, there was no association between the level of gag hypermutation and the viral load or CD4 count. Thus, we observed no overall relationship between hypermutation and markers of disease progression among individuals with low to moderate levels of hypermutation. In addition, one individual sustained a typical viral load despite having a high level of hypermutation. This individual had 43% of gag sequences hypermutated and harbored a partially defective Vif, which was found to permit hypermutation in a peripheral blood mononuclear cell culture. Overall, our results suggest that a potential antiviral therapy based on hypermutation may need to achieve a substantially higher level of hypermutation than is naturally seen in most individuals to impair virus replication and subsequent disease progression