Coevolved Multidrug-Resistant HIV-1 Protease and Reverse Transcriptase Influences Integrase Drug Susceptibility and Replication Fitness

Integrase strand transfer inhibitors (InSTIs) are recommended agents in first-line combination antiretroviral therapy (cART). We examined the evolution of drug resistance mutations throughout HIV-1 pol and the effects on InSTI susceptibility and viral fitness. We performed single-genome sequencing of full-length HIV-1 pol in a highly treatment-experienced patient, and determined drug susceptibility of patient-derived HIV-1 genomes using a phenotypic assay encompassing full-length pol gene. We show the genetic linkage of multiple InSTI-resistant haplotypes containing major resistance mutations at Y143, Q148 and N155 to protease inhibitor (PI) and reverse transcriptase inhibitor (RTI) resistance mutations. Phenotypic analysis of viruses expressing patient-derived IN genes with eight different InSTI-resistant haplotypes alone or in combination with coevolved protease (PR) and RT genes exhibited similar levels of InSTI susceptibility, except for three haplotypes that showed up to 3-fold increases in InSTI susceptibility (p ≤ 0.032). The replicative fitness of most viruses expressing patient-derived IN only significantly decreased, ranging from 8% to 56% (p ≤ 0.01). Interestingly, the addition of coevolved PR + RT significantly increased the replicative fitness of some haplotypes by up to 73% (p ≤ 0.024). Coevolved PR + RT contributes to the susceptibility and viral fitness of patient-derived IN viruses. Maintaining patients on failing cART promotes the selection of fitter resistant strains, and thereby limits future therapy options

Manipulation of both virus- and cell-specific factors is required for robust transient replication of a hepatitis C virus genotype 3a sub-genomic replicon

Hepatitis C virus (HCV) genotype (GT) 3 is the second most prevalent of the seven HCV genotypes and exhibits the greatest resistance to the highly potent, direct-acting antivirals (DAAs) that are currently in use. Previously a stable cell line harbouring the S52 GT3 subgenomic replicon (SGR) was established, but this SGR was unable to robustly replicate transiently. As transient SGRs are a critical tool in the development of DAAs, and the study of viral resistance, we sought to establish a transient SGR system based on S52. Next generation sequencing was used to identify putative culture-adaptive substitutions that had arisen during long term selection of the S52 SGR. A subset of these substitutions were built back into the S52 SGR in the context of a CpG/UpA-low luciferase reporter, with a single point mutation in NS4A conferring the greatest replication capability upon S52. Modification of the innate immune-sensing pathways of Huh7.5 hepatoma cells by expression of the parainfluenza virus type 5 V protein and SEC14L2 resulted in a further enhancement of S52 replication. Furthermore, this transiently-replicating SGR showed genotype-specific differences in sensitivity to two clinically-relevant NS5A DAAs. In conclusion, we report that a single substitution in NS4A, coupled with host cell modifications, enabled robust levels of transient replication by the GT3 S52 SGR. This system will have beneficial uses in both basic research into the unique aspects of GT3 biology and drug discovery

Next generation sequencing of HIV-1 protease in the PIVOT trial of protease inhibitor monotherapy

BACKGROUND: The PIVOT trial examined whether patients with suppressed viral load on combination antiretroviral therapy could be safely switched long-term to ritonavir-boosted protease inhibitor (PI) monotherapy. The main trial publication reported that only one of 296 patients allocated to PI monotherapy experienced a loss of drug options due to protease mutations (identified by local Sanger sequencing resistance tests) likely selected by study drug. OBJECTIVES: To assess if we had missed low frequency mutations, using a more sensitive methodology. STUDY DESIGN: We performed next generation sequencing (NGS) on all available frozen plasma samples with VL >1000 copies/ml from patients who were randomised to PI monotherapy. Assays were performed at Public Health England laboratories using a previously described method. Median coverage depth was 76,000 and the threshold for detection of minority variants was 2%. Drug susceptibility was predicted using the Stanford HIVdb algorithm. RESULTS: 17 of 26 potential samples, all from different patients, were identified and successfully tested. The median viral load was 6780 copies/ml and the median time since randomisation was 43 weeks. NGS revealed previously unidentified minority variant protease mutations (G73D, I54T, L89V) in three samples, at frequencies ranging between 2% and 10%. None of these mutations predicted intermediate or high level resistance, the trial primary outcome. DISCUSSION: This report adds to the body of evidence that ritonavir-boosted PI monotherapy, when used as a switch strategy with prompt detection of viral load rebound and early re-introduction of combination therapy, rarely leads to the development of clinically important protease resistance mutations

HIV-1 drug resistance mutations emerging on darunavir therapy in PI-naive and -experienced patients in the UK

BACKGROUND: Darunavir is considered to have a high genetic barrier to resistance. Most darunavir-associated drug resistance mutations (DRMs) have been identified through correlation of baseline genotype with virological response in clinical trials. However, there is little information on DRMs that are directly selected by darunavir in clinical settings. OBJECTIVES: We examined darunavir DRMs emerging in clinical practice in the UK. PATIENTS AND METHODS: Baseline and post-exposure protease genotypes were compared for individuals in the UK Collaborative HIV Cohort Study who had received darunavir; analyses were stratified for PI history. A selection analysis was used to compare the evolution of subtype B proteases in darunavir recipients and matched PI-naive controls. RESULTS: Of 6918 people who had received darunavir, 386 had resistance tests pre- and post-exposure. Overall, 2.8% (11/386) of these participants developed emergent darunavir DRMs. The prevalence of baseline DRMs was 1.0% (2/198) among PI-naive participants and 13.8% (26/188) among PI-experienced participants. Emergent DRMs developed in 2.0% of the PI-naive group (4 mutations) and 3.7% of the PI-experienced group (12 mutations). Codon 77 was positively selected in the PI-naive darunavir cases, but not in the control group. CONCLUSIONS: Our findings suggest that although emergent darunavir resistance is rare, it may be more common among PI-experienced patients than those who are PI-naive. Further investigation is required to explore whether codon 77 is a novel site involved in darunavir susceptibility

Enhanced surveillance of HIV-1 drug resistance in recently infected MSM in the UK

OBJECTIVES: To determine the prevalence of inferred low-frequency HIV-1 transmitted drug resistance (TDR) in MSM in the UK and its predicted effect on first-line therapy. METHODS: The HIV-1 pol gene was amplified from 442 newly diagnosed MSM identified as likely recently infected by serological avidity testing in 2011-13. The PCR products were sequenced by next-generation sequencing with a mutation frequency threshold of >2% and TDR mutations defined according to the 2009 WHO surveillance drug resistance mutations list. RESULTS: The majority (75.6%) were infected with subtype B and 6.6% with rare complex or unique recombinant forms. At a mutation frequency threshold of >20%, 7.2% (95% CI 5.0%-10.1%) of the sequences had TDR and this doubled to 15.8% (95% CI 12.6%-19.6%) at >2% mutation frequency (P 20% and 2%-20% mutation frequency differed for each drug class, these respectively being: L90M (n = 7) and M46IL (n = 10) for PIs; T215rev (n = 9) and D67GN (n = 4) for NRTIs; and K103N (n = 5) and G190E (n = 2) for NNRTIs. Combined TDR was more frequent in subtype B than non-B (OR = 0.38; 95% CI = 0.17-0.88; P = 0.024) and had minimal predicted effect on recommended first-line therapies. CONCLUSIONS: The data suggest differences in the types of low-frequency compared with majority TDR variants that require a better understanding of the origins and clinical significance of low-frequency variants. This will better inform diagnostic and treatment strategies

Studies on the Restriction of Murine Leukemia Viruses by Mouse APOBEC3

APOBEC3 proteins function to restrict the replication of retroviruses. One mechanism of this restriction is deamination of cytidines to uridines in (−) strand DNA, resulting in hypermutation of guanosines to adenosines in viral (+) strands. However, Moloney murine leukemia virus (MoMLV) is partially resistant to restriction by mouse APOBEC3 (mA3) and virtually completely resistant to mA3-induced hypermutation. In contrast, the sequences of MLV genomes that are in mouse DNA suggest that they were susceptible to mA3-induced deamination when they infected the mouse germline. We tested the possibility that sensitivity to mA3 restriction and to deamination resides in the viral gag gene. We generated a chimeric MLV in which the gag gene was from an endogenous MLV in the mouse germline, while the remainder of the viral genome was from MoMLV. This chimera was fully infectious but its response to mA3 was indistinguishable from that of MoMLV. Thus, the Gag protein does not seem to control the sensitivity of MLVs to mA3. We also found that MLVs inactivated by mA3 do not synthesize viral DNA upon infection; thus mA3 restriction of MLV occurs before or at reverse transcription. In contrast, HIV-1 restricted by mA3 and MLVs restricted by human APOBEC3G do synthesize DNA; these DNAs exhibit APOBEC3-induced hypermutation

High expression of antiviral proteins in mucosa from individuals exhibiting resistance to human immunodeficiency virus

ABSTARCT: Several soluble factors have been reported to have the capacity of inhibiting HIV replication at different steps of the virus life cycle, without eliminating infected cells and through enhancement of specific cellular mechanisms. Yet, it is unclear if these antiviral factors play a role in the protection from HIV infection or in the control of viral replication. Here we evaluated two cohorts: i) one of 58 HIV-exposed seronegative individuals (HESNs) who were compared with 59 healthy controls (HCs), and ii) another of 13 HIV-controllers who were compared with 20 HIV-progressors. Peripheral blood, oral and genital mucosa and gut-associated lymphoid tissue (GALT) samples were obtained to analyze the mRNA expression of ELAFIN, APOBEC3G, SAMHD1, TRIM5α, RNase 7 and SerpinA1 using real-time PCR. RESULTS: HESNs exhibited higher expression of all antiviral factors in peripheral blood mononuclear cells (PBMCs), oral or genital mucosa when compared with HCs. Furthermore, HIV-controllers exhibited higher levels of SerpinA1 in GALT. CONCLUSIONS: These findings suggest that the activity of these factors is compartmentalized and that these proteins have a predominant role depending on the tissue to avoid the infection, reduce the viral load and modulate the susceptibility to HIV infection

Parasite infection is associated with Kaposi's sarcoma associated herpesvirus (KSHV) in Ugandan women

Background: Immune modulation by parasites may influence susceptibility to bacteria and viruses. We examined the association between current parasite infections, HIV and syphilis (measured in blood or stool samples using standard methods) and antibodies against Kaposi's sarcoma herpesvirus (KSHV), measured by ELISA, in 1915 stored plasma samples from pregnant women in Entebbe, Uganda.<p></p> Results: Seroprevalence of KSHV was higher in women with malaria parasitaemia (73% vs 60% p = 0.01), hookworm (67% vs 56% p = 0.001) and Mansonella perstans (69% vs 59% p = 0.05); seroprevalence increased with increasing intensity of hookworm infection (p < 0.001[trend]). No associations were found for HIV, five other parasites or active syphilis. These effects were not explained by socioeconomic status or education.<p></p> Conclusions: Specific parasite infections are associated with presence of antibodies against KSHV, perhaps mediated via their effect on immune function.<p></p&gt

The role of unintegrated DNA in HIV infection

Integration of the reverse transcribed viral genome into host chromatin is the hallmark of retroviral replication. Yet, during natural HIV infection, various unintegrated viral DNA forms exist in abundance. Though linear viral cDNA is the precursor to an integrated provirus, increasing evidence suggests that transcription and translation of unintegrated DNAs prior to integration may aid productive infection through the expression of early viral genes. Additionally, unintegrated DNA has the capacity to result in preintegration latency, or to be rescued and yield productive infection and so unintegrated DNA, in some circumstances, may be considered to be a viral reservoir. Recently, there has been interest in further defining the role and function of unintegrated viral DNAs, in part because the use of anti-HIV integrase inhibitors leads to an abundance of unintegrated DNA, but also because of the potential use of non-integrating lentiviral vectors in gene therapy and vaccines. There is now increased understanding that unintegrated viral DNA can either arise from, or be degraded through, interactions with host DNA repair enzymes that may represent a form of host antiviral defence. This review focuses on the role of unintegrated DNA in HIV infection and additionally considers the potential implications for antiviral therapy