BackgroundThe genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate.Methods and FindingsTo assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates.ConclusionGlobal surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

SAMHD1 has recently been identified as an HIV-1 restriction factor operating in myeloid cells. As a countermeasure, the Vpx accessory protein from HIV-2 and certain lineages of SIV have evolved to antagonize SAMHD1 by inducing its ubiquitin-proteasome-dependent degradation. Here, we show that SAMHD1 experienced strong positive selection episodes during primate evolution that occurred in the Catarrhini ancestral branch prior to the separation between hominoids (gibbons and great apes) and Old World monkeys. The identification of SAMHD1 residues under positive selection led to mapping the Vpx-interaction domain of SAMHD1 to its C-terminal region. Importantly, we found that while SAMHD1 restriction activity toward HIV-1 is evolutionarily maintained, antagonism of SAMHD1 by Vpx is species-specific. The distinct evolutionary signature of SAMHD1 sheds light on the development of its antiviral specificity.

BackgroundThe HIV-1 genome is subject to pressures that target the virus resulting in escape and adaptation. On the other hand, there is a requirement for sequence conservation because of functional and structural constraints. Mapping the sites of selective pressure and conservation on the viral genome generates a reference for understanding the limits to viral escape, and can serve as a template for the discovery of sites of genetic conflict with known or unknown host proteins.ResultsTo build a thorough evolutionary, functional and structural map of the HIV-1 genome, complete subtype B sequences were obtained from the Los Alamos database. We mapped sites under positive selective pressure, amino acid conservation, protein and RNA structure, overlapping coding frames, CD8 T cell, CD4 T cell and antibody epitopes, and sites enriched in AG and AA dinucleotide motives. Globally, 33% of amino acid positions were found to be variable and 12% of the genome was under positive selection. Because interrelated constraining and diversifying forces shape the viral genome, we included the variables from both classes of pressure in a multivariate model to predict conservation or positive selection: structured RNA and α-helix domains independently predicted conservation while CD4 T cell and antibody epitopes were associated with positive selection.ConclusionsThe global map of the viral genome contains positive selected sites that are not in canonical CD8 T cell, CD4 T cell or antibody epitopes; thus, it identifies a class of residues that may be targeted by other host selective pressures. Overall, RNA structure represents the strongest determinant of HIV-1 conservation. These data can inform the combined analysis of host and viral genetic information.

The results of the present study show that M89I/V is associated with PI experience in subtypes C, F and G but not in subtype B. M89I/V should be considered a secondary PI mutation with an important effect on nelfinavir susceptibility in the presence of L90M.

Primary liver cancer comprises a diverse group of liver tumors. The heterogeneity of these tumors is seen as one of the obstacles to finding an effective therapy. The Hippo pathway, with its downstream transcriptional co-activator Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), has a decisive role in the carcinogenesis of primary liver cancer. Therefore, we examined the expression pattern of YAP and TAZ in 141 patients with hepatocellular carcinoma keratin 19 positive (HCC K19+), hepatocellular carcinoma keratin 19 negative (HCC K19−), combined hepatocellular–cholangiocarcinoma carcinoma (cHCC-CCA), or cholangiocarcinoma (CCA). All cHCC-CCA and CCA patients showed high expression levels for YAP and TAZ, while only some patients of the HCC group were positive. Notably, we found that a histoscore of both markers is useful in the challenging diagnosis of cHCC-CCA. In addition, positivity for YAP and TAZ was observed in the hepatocellular and cholangiocellular components of cHCC-CCA, which suggests a single cell origin in cHCC-CCA. Within the K19− HCC group, our results demonstrate that the expression of YAP is a statistically significant predictor of poor prognosis when observed in the cytoplasm. Nuclear expression of TAZ is an even more specific and independent predictor of poor disease-free survival and overall survival of K19− HCC patients. Our results thus identify different levels of YAP/TAZ expression in various liver cancers that can be used for diagnostics.

The major limitation of drug resistance genotyping for human immunodeficiency virus remains the interpretation of the results. We evaluated the concordance in predicting therapy response between four different interpretation algorithms (Rega 6.3, HIVDB-08/04, ANRS [07/04], and VGI 8.0). Sequences were gathered through a worldwide effort to establish a database of non-B subtype sequences, and demographic and clinical information about the patients was gathered. The most concordant results were found for nonnucleoside reverse transcriptase (RT) inhibitors (93%), followed by protease inhibitors (84%) and nucleoside RT inhibitor (NRTIs) (76%). For therapy-naive patients, for nelfinavir, especially for subtypes C and G, the discordances were driven mainly by the protease (PRO) mutational pattern 82I/V ؉ 63P ؉ 36I/V for subtype C and 82I ؉ 63P ؉ 36I ؉ 20I for subtype G. Subtype F displayed more discordances for ritonavir in untreated patients due to the combined presence of PRO 20R and 10I/V. In therapy-experienced patients, subtype G displayed a lot of discordances for saquinavir and indinavir due to mutational patterns involving PRO 90 M and 82I. Subtype F had more discordance for nelfinavir attributable to the presence of PRO 88S and 82A ؉ 54V. For the NRTIs lamivudine and emtricitabine, CRF01_AE had more discordances than subtype B due to the presence of RT mutational patterns 65R ؉ 115 M and 118I ؉ 215Y, respectively. Overall, the different algorithms agreed well on the level of resistance scored, but some of the discordances could be attributed to specific (subtypedependent) combinations of mutations. It is not yet known whether therapy response is subtype dependent, but the advice given to clinicians based on a genotypic interpretation algorithm differs according to the subtype.Genotyping for the assessment of anti-human immunodeficiency virus (HIV) drug resistance is often used in the management of individual patient therapy. Currently, it is recommended in European as well as American guidelines (17,38). In several retrospective and prospective studies, genotyping proved beneficial in optimizing treatment for individual patients (5,10,16,23,25,31,37).Although genotyping is commonly used, there are still many uncertainties with respect to the value of genotype in the assignment of a new regimen. The current genotypic assays are not always able to report all drug resistance mutations among non-B subtypes (11,18,19,24). Regardless of subtype, genotyping is not sensitive to mutations that are present as a minor variant in the population (22,40). Genotyping results also differ depending on the laboratory where they are performed. Quality control studies indicate that mutations, even present as a pure variant, are often underestimated (32).However, separate from the quality and sensitivity issues, the interpretation of genotypic results is still not standardized.

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