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Sequences necessary for transduction of human endogenous retrovirus (HERV)-Kcon, a consensus of the HERV-K(HML-2) family, were analyzed and found to reside in the leader/gag region. They act in an orientation-dependent way and consist of at least two sites working together. Having defined these sequences, we exploited this information to produce a simple system to investigate to what extent virions of HERV-Kcon, murine leukemia virus, and HIV-1 have the ability to transduce each other's genomes, leading to potential contamination of gene therapy vectors., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Dynamic changes in Human Immunodeficiency Virus 1 (HIV-1) sequence diversity and divergence are associated with immune control during primary infection and progression to AIDS. Consensus sequencing or single genome amplification sequencing of the HIV-1 envelope (env) gene, in particular the variable (V) regions, is used as a marker for HIV-1 genome diversity, but population diversity is only minimally, or semi-quantitatively sampled using these methods., Results: Here we use second generation deep sequencing to determine inter-and intra-patient sequence heterogeneity and to quantify minor variants in a cohort of individuals either receiving or not receiving antiretroviral treatment following seroconversion; the SPARTAC trial. We show, through a cross-sectional study of sequence diversity of the env V3 in 30 antiretroviral-naive patients during primary infection that considerable population structure diversity exists, with some individuals exhibiting highly constrained plasma virus diversity. Diversity was independent of clinical markers (viral load, time from seroconversion, CD4 cell count) of infection. Serial sampling over 60 weeks of non-treated individuals that define three initially different diversity profiles showed that complex patterns of continuing HIV-1 sequence diversification and divergence could be readily detected. Evidence for minor sequence turnover, emergence of new variants and re-emergence of archived variants could be inferred from this analysis. Analysis of viral divergence over the same time period in patients who received short (12 weeks, ART12) or long course antiretroviral therapy (48 weeks, ART48) and a non-treated control group revealed that ART48 successfully suppressed viral divergence while ART12 did not have a significant effect., Conclusions: Deep sequencing is a sensitive and reliable method for investigating the diversity of the env V3 as an important component of HIV-1 genome diversity. Detailed insights into the complex early intra-patient dynamics of env V3 diversity and divergence were explored in antiretroviral-naïve recent seroconverters. Long course antiretroviral therapy, initiated soon after seroconversion and administered for 48 weeks, restricts HIV-1 divergence significantly. The effect of ART12 and ART48 on clinical markers of HIV infection and progression is currently investigated in the SPARTAC trial.

Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50-fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs) that target the membrane proximal external region (MPER) of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly discovered activity for Nef has important implications for anti-HIV-1 immunity and AIDS pathogenesis.

Objective: An epidemic of acute hepatitis C virus (HCV) infection in HIV-positive men-who-have-sex-with-men (MSM) is emerging in Europe, Australia and the USA. The aim of this study was to characterise the natural history of primary HCV in this setting and to assess host and viral factors which predict spontaneous clearance., Methods: This prospective longitudinal cohort study was carried out in 112 HIV-positive patients who were followed in a single centre (the St Mary's Acute HCV Cohort). Plasma and peripheral blood mononuclear cells (PBMCs) were obtained at monthly intervals for 3 months and at 3-monthly intervals thereafter for a median of 45 months (IQR = 29-69 months). The primary end point was spontaneous clearance of HCV. Cox regression was used to assess the impact of clinical and virological variables on outcome, including liver function, CD4 count, rate of HCV RNA decline, T cell response and clonal sequence evolution within the HCV E2 envelope gene., Results: 15% of patients cleared HCV spontaneously, while 85% progressed towards chronicity. The latter group included a significant proportion of 'fluctuating' progressors (37.5%), in whom a fall followed by a rise (>1 log₁₀) in viraemia was observed. This was associated with superinfection with new HCV strains and partially effective T cell responses. Spontaneous clearance was strongly associated with a 2.2 log₁₀ viral load drop within 100 days of infection (HR = 1.78; p < 0.0001), elevated bilirubin (≥ 40 μmol/l; HR = 5.04; p = 0.006), elevated alanine aminotransferase (ALT; ≥ 1000 IU/ml; HR = 2.62; p = 0.048) and baseline CD4 count ≥ 650 × 10⁶/l (HR = 2.66; p = 0.045), and only occurred in patients with genotype 1 infection. Evolution to spontaneous clearance occurred in patients with low viral diversity in the presence of an early multispecific T cell response., Conclusions: Spontaneous clearance of acute HCV in HIV-positive men can be predicted by a rapid decline in viral load, high CD4 count, elevated bilirubin and ALT, and is associated with low viral diversity and strong T cell responses.

Background: Nucleoside-sparing combination antiretroviral therapy (cART) regimens might be an attractive therapeutic option for HIV type-1 (HIV-1)-infected patients; however, the pharmacokinetic profiles of such regimens are frequently unknown., Methods: Fourteen HIV-1-infected patients (age 21-55 years, 64% male) on stable cART with plasma HIV RNA <50 copies/ml entered this Phase I pharmacokinetic study. In period 1, patients received tenofovir/emtricitabine/-darunavir/ritonavir (300/200/800/100 mg) all once daily. During period 2, raltegravir 400 mg twice daily was added to the regimen and in period 3 tenofovir/emtricitabine was discontinued. At steady state, intensive pharmacokinetic sampling was undertaken. Differences in the geometric mean ratio (GMR) for pharmacokinetic parameters between periods 2 versus 1 and period 3 versus 1 were assessed for darunavir and ritonavir (period 3 versus 2 for raltegravir)., Results: No statistically significant differences in pharmacokinetic parameters were observed between period 2 versus period 1. During period 3, darunavir GMR (95% confidence interval) values for trough and maximum plasma concentration (C(trough) and C(max)), area under the plasma concentration-time curve (AUC) and elimination half-life (t(1/2)) were 0.64 ng/ml (0.44-0.93), 1.05 ng/ml (0.90-1.24), 0.92 ng h/ml (0.78-1.08) and 0.69 h (0.46-1.05), respectively, when compared with period 1. No statistically significant changes were observed in ritonavir or raltegravir pharmacokinetic parameters. Darunavir C(trough)<550 ng/ml (the minimum effective concentration for protease-resistant HIV viral isolates) was observed in four patients during period 3 only. No clinically significant safety concerns were reported., Conclusions: Darunavir C(trough) is reduced by 36% when administered without tenofovir/emtricitabine in HIV-1-infected patients. This interaction might be of clinical significance in the management of individuals with protease-resistant HIV viral isolates.

Foamy viruses are a family of complex retroviruses that establish common, productive infections in a wide range of nonhuman primates. In contrast, humans appear nonpermissive for foamy virus replication, although zoonotic infections do occur. Here we have analyzed the ability of primate and mouse APOBEC3G proteins to inhibit the infectivity of primate foamy virus (PFV) virions produced in their presence. We demonstrate that several APOBEC3 proteins can potently inhibit the infectivity of a PFV-based viral vector. This inhibition correlated with the packaging of inhibitory APOBEC3 proteins into PFV virions, due to a specific PFV Gag/APOBEC3 interaction, and resulted in the G to A hypermutation of PFV reverse transcripts. While inhibition of PFV virion infectivity by primate APOBEC3 proteins was largely relieved by coexpression of the PFV Bet protein, a cytoplasmic auxiliary protein of previously uncertain function, Bet failed to relieve inhibition caused by murine APOBEC3. PFV Bet bound to human, but not mouse, APOBEC3 proteins in coexpressing cells, and this binding correlated with the specific inhibition of their incorporation into PFV virions. Of note, both PFV Bet and a second Bet protein, derived from an African green monkey foamy virus, rescued the infectivity of Vif-deficient human immunodeficiency virus type 1 (HIV-1) virions produced in the presence of African green monkey APOBEC3G and blocked the incorporation of this host factor into HIV-1 virion particles. However, neither foamy virus Bet protein reduced APOBEC3 protein expression levels in virion producer cells. While these data identify the foamy virus Bet protein as a functional ortholog of the HIV-1 Vif auxiliary protein, they also indicate that Vif and Bet block APOBEC3 protein function by distinct mechanisms.

In this study we compare the ability of self-inactivating Human Immunodeficiency Virus 1 (HIV-1) and Equine Infectious Anaemia Virus (EIAV)-based vectors to mediate gene transfer to rabbit and human corneas and to a murine corneal endothelial cell line. Both vectors were pseudotyped with vesicular stomatitis virus-G (VSV-G) envelope and contained marker transgenes under the control of an internal CMV promoter. For specificity of action, the heterologous promoter in the EIAV-vector was exchanged for an inducible E-Selectin promoter, previously shown to regulate gene-expression in a plasmid system. We show that EIAV is more efficient than HIV in transducing human and rabbit corneal endothelial cells. Rabbit corneal endothelial cells are transduced in higher quantity than human corneal endothelial cells. In the inducible system, however, we detected impairment between the vector and its internal E-Selectin promoter. Instead of controlled transgene expression or silencing of promoter activity, the U3-modified long-terminal-repeats (LTR) impaired the conditional activity of the E-Selectin promoter. Significant transgene expression was seen without stimulation of the inducible promoter. We show efficient transduction by lentiviruses of a corneal endothelial cell line and of full thickness corneas from different species, confirming that those vectors would be appropriate tools for gene therapy of selected corneal diseases. However, the modification within the U3-LTR did not adequately allow regulated transgene expression. These findings have important implications for vector design for diagnostic or therapeutic opportunities.

We modified the Abbott diagnostics HIV-1 Viroseq version 2 assay trade mark in order to detect the presence of HIV-1 drug resistance mutations in patients with viraemia below 1000 copies/ml of plasma. One hundred and forty-four patients with a detectable HIV-1 plasma viral load below 1000 copies/ml were selected and HIV-1 genetic analysis carried out using a modification of the Abbott Diagnostics Viroseq 2.0 assay trade mark. The procedure differs from the standard protocol in that a nested PCR amplification step was introduced. The oligonucleotide primers for the first round of PCR were those supplied in the RT-PCR module of the kit. The nested PCR primers were primers A and H taken from the sequencing module. One hundred and twenty-eight out of 144 (89%) plasma samples with an HIV-1 viral load of less than 1000 copies/ml (ranging from 54 to 992 copies) were successfully sequenced. HIV-1 genotypes were obtained from 68 out of 81 (84%) samples with a viral load of greater than 50 but less than 300 copies/ml and 60/63 (95%) of samples with a viral load of greater than 300 but less than 1000 copies/ml. Serial dilution of a sample with a high viral load did not affect the detection of resistance mutations. Multiple sequencing of samples with low viral load did not result in detection of additional mutations, although, in one sample the K103N mutation was detected in 3/6 replicates while wild-type was detected in 2/6 and a mixture of wild-type/mutant in 1/6. Samples from patients infected with both clade B and non-B clades of HIV-1 could be genotyped at low copy number. Modification of the Abbott Viroseq assay allows reproducible sequencing of the HIV-1 genome from patients with low, but detectable, plasma virus burden.

Although several groups have demonstrated that RNA interference, induced by transfection of small interfering RNA (siRNA) duplexes, can protect cells against a viral challenge in culture, this protection is transient. Here, we describe lentivirus expression vectors that can stably express siRNAs at levels sufficient to block virus replication. We have used these vectors to stably express siRNAs specific for the essential human immunodeficiency virus type 1 (HIV-1) Tat transcription factor or specific for a cellular coreceptor, CCR5, that is required for infection by the majority of primary HIV-1 isolates. These lentivirus vectors are shown to protect cells, including primary macrophages, against HIV-1 infection in culture by inducing selective degradation of their target mRNA species. These data suggest that it should be possible to block the expression of specific viral or cellular genes in vivo by using viral vectors to stably express the appropriate siRNAs.

Objective: Few data exist on the virological response to antiretroviral therapy of individuals infected with African HIV-1 subtypes. Our objective was to compare the response, in our clinic, of African HIV-1-infected patients with their British and European contemporaries treated with the same regimes., Design: The St Mary's Hospital HIV database was used to identify drug-naive African and European patients starting a highly active antiretroviral therapy (HAART) regimen., Methods: HIV-1 subtype was determined by phylogenetic analysis of pol sequences. Kaplan-Meier survival analysis was used to estimate the proportion of patients achieving undetectable viral loads (< 500 copies/ml). The longer-term response to therapy was assessed by changes in CD4 cell counts and viral loads from baseline., Results: A total of 265 patients were classified as 'European' and 97 as 'African', confirmed by sequence. The time to first undetectable viral load was similar for the two groups (P = 0.9). Although there were no statistically significant differences in the CD4 cell count responses (P = 0.11), there was evidence of an increase in viral load after 9 months for the African group, resulting in a widening viral load gap between the two cohorts; the effect of ethnic group was statistically significant (P < 0.001)., Conclusion: The initial virological and immunological responses of the African and European cohorts to HAART were similar; although the longer-term virological response was poorer in the African cohort, which may be related to adherence. On the basis of these findings, there is no justification for withholding HAART from Africa on virological grounds.