Nonstructural Protein 5B Research Articles

Genetic diversity of hepatitis C virus in Ethiopia.

Hepatitis C virus (HCV) is genetically highly divergent and classified in seven major genotypes and approximately hundred subtypes. These genotypes/subtypes have different geographic distribution and response to antiviral therapy. In Ethiopia, however, little is known about their molecular epidemiology and genetic diversity. The aim of this study was to investigate the distribution and genetic diversity of HCV genotypes/subtypes in Ethiopia, using 49 HCV RNA positive samples. HCV genotypes and subtypes were determined based on the sequences of the core and the nonstructural protein 5B (NS5B) genomic regions. Phylogenetic analysis revealed that the predominant was genotype 4 (77.6%) followed by 2 (12.2%), 1 (8.2%), and 5 (2.0%). Seven subtypes were identified (1b, 1c, 2c, 4d, 4l, 4r and 4v), with 4d (34.7%), 4r (34.7%) and 2c (12.2%) as the most frequent subtypes. Consistent with the presence of these subtypes was the identification of a potential recombinant virus. One strain was typed as genotype 2c in the NS5B region sequence and genotype 4d in the core region. In conclusion, genotype 4 HCV viruses, subtypes 4d and 4r, are most prevalent in Ethiopia. This genotype is considered to be difficult to treat, thus, our finding has an important impact on the development of treatment strategies and patient management in Ethiopia.

Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/\u03b2-catenin pathway

BackgroundHepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/β-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/β-catenin signaling in various liver cells, and explored the molecular mechanism underlying HCV-related hepatocarcinogenesis.ResultsCompared with the empty vector control, HCV core protein and NS4B demonstrated the following characteristics in the Huh7 cells: significantly enhanced β-catenin/Tcf-dependent transcriptional activity (F = 40.87, P < 0.01); increased nuclear translocation of β-catenin (F = 165.26, P < 0.01); upregulated nuclear β-catenin, cytoplasmic β-catenin, Wnt1, c-myc, and cyclin D1 protein expression (P < 0.01); and promoted proliferation of Huh7 cells (P < 0.01 or P < 0.05). Neither protein enhanced β-catenin/Tcf-dependent transcriptional activity in the LO2 cells (F = 0.65, P > 0.05), but they did significantly enhance Wnt3a-induced β-catenin/Tcf-dependent transcriptional activity (F = 64.25, P < 0.01), and promoted the nuclear translocation of β-catenin (F = 66.54, P < 0.01) and the Wnt3a-induced proliferation of LO2 cells (P < 0.01 or P < 0.05). Moreover, activation of the Wnt/β-catenin signaling pathway was greater with the core protein than with NS4B (P < 0.01 or P < 0.05).ConclusionsHCV core protein and NS4B directly activate the Wnt/β-catenin signaling pathway in Huh7 cells and LO2 cells induced by Wnt3a. These data suggest that HCV core protein and NS4B contribute to HCV-associated hepatocellular carcinogenesis.

Impact of hepatitis C virus recombinant form RF1_2k/1b on treatment outcomes within the Georgian national hepatitis C elimination program.

Hepatitis C virus (HCV) recombinant form RF1_2k/1b is common in ethnic Georgians. This chimera virus contains genomic fragments of genotype 2 and genotype 1 and is misclassified as genotype 2 by standard genotyping. We aimed to identify RF1_2k/1b strains among genotype 2 patients and assess its impact on treatment outcomes. The study included 148 patients with HCV genotype 2 as determined by 5-untranslated region/core genotyping assay. RF1_2k/1b was identified by sequencing the non-structural protein 5B region. Patients were treated within the national hepatitis C elimination program with sofosbuvir/ribavirin (SOF/RBV), interferon (IFN)/SOF/RBV, or ledipasvir (LDV)/SOF/RBV. Of 148 patients, 103 (69.5%) had RF1_ 2k/1b. Sustained virologic response (SVR) data was available for 136 patients (RF1_ 2k/1b, n = 103; genotype 2, n = 33). Sustained virologic response was achieved in more genotype 2 patient than in RF1_2k/1b patients (97.0% vs. 76.7%, P = 0.009). Twelve weeks of LDV/SOF/RBV treatment was highly effective (100% SVR) in both genotypes. Among RF1_2k/1b patients, LDV/SOF/RBV for 12weeks was superior (100% SVR) to SOF/RBV for 12weeks (56.4%, P <0.0001) or 20weeks (79.2%, P = 0.05). Twelve weeks of IFN/SOF/RBV also showed better response than SOF/RBV for 12weeks (88.9% vs. 56.4%, P = 0.02) in these patients. High prevalence of the RF1_2k/1b strain can significantly affect treatment outcomes. Treatment with IFN/SOF/RBV and especially LDV/SOF/RBV ensured significantly higher SVR in patients infected with RF1_2k/1b strain compared to standard HCV genotype 2 treatment with SOF/RBV. There is a need to reassess existing methods for the management of HCV genotype 2 infections, especially in areas with high prevalence of the RF1_2k/1b strain.

Nonstructural protein 5B promotes degradation of the NORE1A tumor suppressor to facilitate hepatitis C virus replication.

HCV uses NS5B to specifically suppress NORE1A, facilitating viral replication and elevated Ras signaling. (Hepatology 2017;65:1462-1477).

Morphologic and Molecular Characterization of a Strain of Zika Virus Imported into Guangdong, China.

The recent outbreaks of Zika virus (ZIKV) disease have caused worldwide concerns. Guangdong province is one of the commercial centers in China and communicates frequently with the epidemic areas. To date, 65.2% of the ZIKV infection cases in China were imported via port of entry in Guangdong. The continuous surveillance of imported cases is crucial for the prevention and control of potential ZIKV infection outbreak in China. In this study, a strain of ZIKV was isolated from the serum of a 6-year-old child returning from Venezuela. The morphology of the ZIKV was analyzed in vivo and in vitro by electron microscopy, and clusters of virus particles were found in the loose cytoplasmic membrane structures. The genomic sequence of the isolated ZIKV was determined, and the alignment and phylogenetic analysis identified one unique amino acid substitution occurring in the non-structural protein 4B (NS4B), and the isolated virus belonged to the Asian lineage.

Computer-aided analysis of phytochemicals as potential dengue virus inhibitors based on molecular docking, ADMET and DFT studies

Dengue fever, caused by dengue virus (DENV), has become a serious threat to human lives. Phytochemicals are known to have great potential to eradicate viral, bacterial and fungal-borne diseases in human beings. This study was aimed at in silico drug development against nonstructural protein 4B (NS4B) of dengue virus 4 (DENV4). A total of 2750 phytochemicals from different medicinal plants were selected for this study. These plants grow naturally in the climate of Pakistan and India and have been used for the treatment of various pathologies in human for long-time. The ADMET studies, molecular docking and density functional theory (DFT) based analysis were carried out to determine the potential inhibitory properties of these phytochemicals. The ADMET analysis and docking results revealed nine phytochemicals, i.e. Silymarin, Flavobion, Derrisin, Isosilybin, Mundulinol, Silydianin, Isopomiferin, Narlumicine and Oxysanguinarine to have potential inhibitory properties against DENV and can be considered for additional in vitro and in vivo studies to assess their inhibitory effects against DENV replication. They exhibited binding affinity ≥ -8 kcal/mol against DENV4-NS4B. Furthermore, DFT based analysis revealed high reactivity for these nine phytochemicals in the binding pocket of DENV4-NS4B, based on ELUMO, EHOMO and band energy gap. Five out of nine phytochemicals are reported for the first time as novel DENV inhibitors. These included three phytochemicals from Silybum marianum, i.e. Derrisin, Mundulinol, Isopomiferin, and two phytochemicals from Fumaria indica, i.e. Narlumicine and Oxysanguinarine. However, all the nine phytochemicals can be considered for in vitro and in vivo analysis for the development of potential DENV inhibitors.

EFFECT OF AN ANTI-HEPATITIS C VIRAL DRUG ON RAT SUBMANDIBULAR SALIVARY GLAND

Background: In the last 3 years, hepatitis C virus (HCV) treatment has undergone significant changes. Sofosbuvir is a new drug; it is a nucleotide analogue that is a highly potent inhibitor of the nonstructural protein 5B (NS5B) polymerase in HCV.Methodology: 30 adult male albino rats weighing about ±200 gm were used. The rats were divided into three groups 10 rats each. Group I(control group) received distilled water. Group II received sofosbuvir 40 mg/kg/day dissolved in distilled water for 45 days. Group III received sofosbuvir 40 mg/kg/day dissolved in distilled water for 90 days. All animals were sacrificed by ketamine overdose. The submandibular salivary glands were dissected. Specimens were examined histologically, immunohistochemically using anti- nuclear factor κ B antibody, histomorphomotry and statistically.Results: Group I showed normal histological structure for the ducts and acini however, group II showed acinar and ductal vacuolization, discontinuity of the epithelial lining with retained secretion in the excretory duct, and chronic inflammatory cell infiltration. These changes were accentuated in group III accompanied by acinar and ductal shrinkage. Immunohistochemical results of group І showed negative immunoreactivity for NF-κB in nuclei of the acini and ducts with very weak cytoplasmic reaction. However group II showed moderate nuclear and cytoplasmic immunoreactivity for NF-κB in the acinar and ductal cells. Regarding group III, the ductal and acinar cells exhibited strong immunoreaction in their nuclei and cytoplasm. Statistical results proved that the difference in the mean values were statistically highly significant between groups II & I, groups III & I and groups II & III.Conclusion: We concluded that sofosbuvir may affect the function of submandibular salivary gland through increased ROS levels in acinar and ductal cells leading to degenerative effect.

Anja Böckmann

Angewandte Chemie International EditionVolume 56, Issue 11 p. 2834-2834 Author ProfileFree Access Anja Böckmann First published: 21 December 2016 https://doi.org/10.1002/anie.201611871AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract “The biggest challenge facing scientists is to work together efficiently. My favorite musician is Eliane Elias. ...” This and more about Anja Böckmann can be found on page 2834. Anja Böckmann The author presented on this page has recently published her 10th article in Angewandte Chemie in the last 10 years: “Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy”: T. Wiegand, R. Cadalbert, C. Gardiennet, J. Timmins, L. Terradot, A. Böckmann, B. H. Meier, Angew. Chem. Int. Ed. 2016, 55, 14164; Angew. Chem. 2016, 128, 14370. Date of birth: December 27, 1966 Position: Directrice de Recherche CNRS, Head of Protein Solid-State NMR group, Institute for Molecular Microbiology and Structural Biochemistry, UMR 5086 CNRS/Université de Lyon E-mail: a.bockmann@ibcp.fr Homepage: https://www.ibcp.fr/mmsb/-RMN-et-virus-de-l-hepatite-C-A-?lang=fr ORCID: 0000-0001-8149-7941 Education: 1993 Undergraduate degree, Technische Universität Berlin 1996 PhD from Université Paris-Sud, Orsay, for work supervised by Dr. Eric Guittet, Institut de Chimie des Substances Naturelles, Gif-sur-Yvette 1996–1997 Postdoctoral work with Ann E. McDermott, Columbia University, New York Awards: 2007 Médaille de bronze, CNRS; 2014 Prix Pierre Desnuelle, Académie des Sciences Research: New approaches and applications in protein solid-state NMR Hobbies: Family, friends, traveling, reading, biking, hiking, running, playing the piano The biggest challenge facing scientists is to work together efficiently. My favorite musician is Eliane Elias. Science is fun because it is like a big puzzle—you find bits and pieces, and if you can put them all together you can create a clear image. My favorite drink is beer accompanied by good scientific discussions. My favorite quote is “The way forward is paradoxically to look not ahead, but to look around” (John Seely Brown and Paul Duguid, The Social Life of Information, 2000). If I could be any age I would be three—the age of discovery. My favorite way to spend a holiday is to exchange homes and live like a local. I am waiting for the day when someone will discover a cure for Alzheimer's disease. As our children will get even older then us, this would allow them to do so without losing their minds. My science “heroes” are women scientists who paved the way, headed forward, and believed in what they were doing against all odds. My favorite book is The Susan Effect by Peter Høeg. My 5 top papers: References 1“Atomic-resolution structure of a disease-relevant Aβ(1–42) amyloid fibril”: M. A. Wälti, F. Ravott, H. Arai, C. G. Glabe, J. S. Wall, A. Böckmann, P. Güntert, B. H. Meier, R. Riek, Proc. Natl. Acad. Sci. USA 2016, 113, E 4976. (I hope this work will help advance the design of interactant molecules that are potential drug candidates.) 2“Atomic-Resolution Three-Dimensional Structure of Amyloid β Fibrils Bearing the Osaka Mutation”: A. K. Schütz et al., Angew. Chem. Int. Ed. 2015, 54, 331; Angew. Chem. 2015, 127, 337. (Central in establishing a general, reliable, and consistent protocol for fibril structure determination.) 3“The Conformation of the Prion Domain of Sup35p in Isolation and in the Full-Length Protein”: N. Luckgei, A. K. Schütz, L. Bousset, B. Habenstein, Y. Sourigues, C. Gardiennet, B. H. Meier, R. Melki, A. Böckmann, Angew. Chem. Int. Ed. 2013, 52, 12741; Angew. Chem. 2013, 125, 12974. (Highlights the importance of studying full-length proteins.) 4“Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus”: M. L. Fogeron et al., J. Biomol. NMR 2016, 65, 87. (An approach to structurally studying challenging membrane proteins in their lipid environment.) 5“A Sedimented Sample of a 59 kDa Dodecameric Helicase Yields High–Resolution Solid-State NMR Spectra”: C. Gardiennet, A. Schütz, A. Hunkeler, B. Kunert, L. Terradot, A. Böckmann, B. H. Meier, Angew. Chem. Int. Ed. 2012, 51, 7855; Angew. Chem. 2012, 124, 7977. (Samples can be prepared by concentrating the protein directly in the NMR rotor.) Volume56, Issue11March 6, 2017Pages 2834-2834 ReferencesRelatedInformation

151 - RNA-Dependent RNA Polymerase Activity of HCV NS5B Protein Is Modulated by Glutathionylation

Hepatitis C virus (HCV) is a human blood-borne pathogen that causes chronic liver disease. Chronic hepatitis C often leads to development of liver fibrosis, cirrhosis and hepatocellular carcinoma as well as other hepatic and extrahepatic pathologies. HCV genome is presented by a short positive strand RNA encoding ten structural and non-structural proteins. Among these non-structural protein 5B (NS5B) exhibits RNA-dependent RNA-polymerase activity capable of replicating genomic RNA de novo thus acting as a key component of HCV replicase. Investigation of mechanisms of regulation of HCV replication was focused mostly on search for NS5B-interacting viral and cellular proteins. Much less attention was given to metabolic alterations in the infected cells. It was already shown that HCV triggers massive reactive oxygen species (ROS) production, and virus replication can be inhibited by extracellular H 2 O 2 . Our aim was to explore possible redox modifications of NS5B protein as a link between these events. Using antibodies against glutathionylated proteins we showed that the recombinant NS5B protein of 1b or 2a genotypes, expressed in E.coli , contain glytathione residues. Treatment of NS5B with oxidized glutathione increases degree of glutathionylation, whereas reduced glutathione causes opposite effect. Increase of NS5B glutathionylation degree was accompanied by inhibition of its enzymatic activity both in primer-dependent and primer-independent ( de novo ) systems. MALDI MS analysis revealed that glutathionylation of the recombinant protein of 2a genotype occurs at cysteine residues at positions 89, 146, 274, and 279/295, and GSSG treatment affected residues 170, 223, and 521. Construction of a panel of NS5B mutants with substitution of any single cysteine by serine demonstrated that C279S mutant lost its enzymatic activity, whereas other mutant forms retained sensitivity to glutathione and reducing agents. Currently these data are being verified in HCV cellular replicon system. Acknowledgements This study was supported by Russian Science Foundation (grant #14-14-01021).

A REVIEW ON ANTI-HCV AGENTS TARGETING ACTIVE SITE AND ALLOSTERIC SITES OF NON-STRUCTURAL PROTEIN 5B [NS5B

Hepatitis C, a chronic disease affecting the global population significantly is caused majorly by Hepatitis C virus [HCV]. Among the several druggable targets explored for Hepatitis C, the viral protein, non-structural protein 5B [NS5B] is the target of choice for researchers as it is the key enzyme in the HCV replication and its active site is conserved among all genotypes. In the recent years the landscape of Hepatitis C therapies, have evolved from Peg-Interferon [PEG-INF]/Ribavirin, to directly acting anti-virus along with PEG-INF and finally, INF free regimens with greater than 90% sustained virological response [SVR]. The launch of Sofosbuvir, a nucleotide inhibitor of NS5B marks the major paradigm in hepatitis C research. Sofosbuvir exhibits, pan-genotypic activity, low barrier to resistance, highly effective and safe. However, the high prices of these medications limit their universal access. This review will focus on progress towards the discovery and development of NS5B inhibitors targeting allosteric sites and active site, covering the chemical class and structure-activity relationships.

MicroRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure.

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein.

Although a highly effective vaccine is available, the number of yellow fever cases has increased over the past 2 decades, which highlights the pressing need for antiviral therapeutics. In a high-throughput screening campaign, we identified an acetic acid benzodiazepine (BDAA) compound which potently inhibits yellow fever virus (YFV). Interestingly, while treatment of YFV-infected cultures with 2 μM BDAA reduced the virion production by greater than 2 logs, the compound was not active against 21 other viruses from 14 different viral families. Selection and genetic analysis of drug-resistant viruses revealed that replacement of the proline at amino acid 219 (P219) of the nonstructural protein 4B (NS4B) with serine, threonine, or alanine conferred YFV with resistance to BDAA without apparent loss of replication fitness in cultured mammalian cells. However, replacement of P219 with glycine conferred BDAA resistance with significant loss of replication ability. Bioinformatics analysis predicts that the P219 amino acid is localized at the endoplasmic reticulum lumen side of the fifth putative transmembrane domain of NS4B, and the mutation may render the viral protein incapable of interacting with BDAA. Our studies thus revealed an important role and the structural basis for the NS4B protein in supporting YFV replication. Moreover, in YFV-infected hamsters, oral administration of BDAA protected 90% of the animals from death, significantly reduced viral load by greater than 2 logs, and attenuated virus infection-induced liver injury and body weight loss. The encouraging preclinical results thus warrant further development of BDAA or its derivatives as antiviral agents to treat yellow fever. IMPORTANCE Yellow fever is an acute viral hemorrhagic disease which threatens approximately 1 billion people living in tropical areas of Africa and Latin America. Although a highly effective yellow fever vaccine has been available for more than 7 decades, the low vaccination rate fails to prevent outbreaks in at-risk regions. It has been estimated that up to 1.7 million YFV infections occur in Africa each year, resulting in 29,000 to 60,000 deaths. Thus far, there is no specific antiviral treatment for yellow fever. To cope with this medical challenge, we identified a benzodiazepine compound that selectively inhibits YFV by targeting the viral NS4B protein. To our knowledge, this is the first report demonstrating in vivo safety and antiviral efficacy of a YFV NS4B inhibitor in an animal model. We have thus reached a critical milestone toward the development of specific antiviral therapeutics for clinical management of yellow fever.

An outbreak of HCV genotype 6a and 2a infection in South China: Confirmation of iatrogenic transmission by phylogenetic analysis of the NS5B region.

An outbreak of hepatitis C virus (HCV) infections, for which the risk factor was unknown, was previously identified in North Guangdong, China. In the present study, a total of 736 local residents were surveyed regarding their lifetime risk factors for HCV infection. Serum anti‑HCV antibodies and HCV RNA were examined to confirm infection. In the HCV‑positive samples, the core and nonstructural protein5B sequences were amplified, and phylogenetic analysis was performed to determine the association between HCV subtypes and transmission routes. A total of 374 individuals were positive for anti‑HCV antibodies. Blood transfusion, blood product transfusion, people who inject drugs and intravenous injection at a local clinic were identified as independent risk factors for HCV infection. Phylogenetic analysis revealed that the two predominant subtypes of HCV, 2a and 6a, were primarily focused in four homologous clusters. Patients with a history of intravenous injection at a local clinic were more likely to be found in the four clusters, compared with patients exposed to other risk factors. The present emergency retrospective survey showed a specific epidemiological feature of HCV infection in Zijin County and found genetic homology among individuals exposed to intravenous injection at a local clinic. Further evidence is required to confirm the causal association between the outbreak of HCV infection and intravenous injection.

Secondary Structure and Membrane Topology of the Full‐Length Dengue Virus NS4B in Micelles

Dengue virus nonstructural protein 4B (NS4B) is a membrane protein consisting of 248 residues with a crucial role in virus replication and interference with the host innate immunity. The dengue virus serotype 3 NS4B was reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Backbone resonance assignment of NS4B was obtained using conventional solution NMR experiments. Further studies suggested that NS4B contained eleven helices and six of them form five potential transmembrane regions. This study provides atomic level information for an important drug target to control flavivirus infections.

Secondary Structure and Membrane Topology of the Full‐Length Dengue Virus NS4B in Micelles

AbstractDengue virus nonstructural protein 4B (NS4B) is a membrane protein consisting of 248 residues with a crucial role in virus replication and interference with the host innate immunity. The dengue virus serotype 3 NS4B was reconstituted into lyso‐myristoyl phosphatidylglycerol (LMPG) micelles. Backbone resonance assignment of NS4B was obtained using conventional solution NMR experiments. Further studies suggested that NS4B contained eleven helices and six of them form five potential transmembrane regions. This study provides atomic level information for an important drug target to control flavivirus infections.

Heads or Tails: Genotyping of Hepatitis C Virus Concerning the 2k/1b Circulating Recombinant Form.

As different hepatitis C virus (HCV) genotypes respond differently to initiated therapy, correct HCV genotyping is essential. A potential risk for misclassification of the intergenotypic HCV circulating recombinant form (CRF) 2k/1b strains exists, depending on the genotyping method used. The aim was to investigate the differences in HCV genotyping methods with regard to CRF 2k/1b and to gain insight in the prevalence of the CRF 2k/1b. Genotyping results by Versant HCV Genotype Assay were compared with nonstructural protein 5B (NS5B) sequencing. In total, from November 2001 until March 2015, 3296 serum samples were analyzed by Versant HCV Genotype Assay. As misclassified CRF is harbored among HCV genotype 2, we further focused our search on 142 (4.3%) samples positive for HCV genotype 2. On 116 (81.7%) retrieved samples, the NS5B sequencing was performed. Twelve out of the 116 retrieved samples (10.3%) were classified as CRF 2k/1b by sequencing of the NS5B region. Ten of these 12 samples were originally misclassified as genotype 2a or 2c, while 2 of them were misclassified as genotype 2. Our results show that the current prevalence of CRF 2k/1b is underestimated. The importance of correct HCV genotyping is emphasized, considering the tailored choice of treatment regimen and overall prognosis.

Assessment of the clinical cardiac drug-drug interaction associated with the combination of hepatitis C virus nucleotide inhibitors and amiodarone in guinea pigs and rhesus monkeys.

These are the first preclinical in vivo experiments reported to replicate the severe clinical SOF+AMIO cardiac DDI and provide potential in vivo mechanism of action. As such, these data provide a preclinical risk assessment paradigm, including a clinically relevant nonhuman primate model, with which to better understand cardiovascular DDI risk for this therapeutic class. Furthermore, these studies suggest that not all HCV DAAs and, in particular, not all HCV nonstructural protein 5B inhibitors may exhibit this cardiac DDI with amiodarone. Given the selective in vivo cardiac electrophysiological effect, these data enable targeted cellular/molecular mechanistic studies to more precisely identify cell types, receptors, and/or ion channels responsible for the clinical DDI. (Hepatology 2016;64:1430-1441).

Dengue Virus Perturbs Mitochondrial Morphodynamics to Dampen Innate Immune Responses.

SummaryWith no antiviral drugs or widely available vaccines, Dengue virus (DENV) constitutes a public health concern. DENV replicates at ER-derived cytoplasmic structures that include substructures called convoluted membranes (CMs); however, the purpose of these membrane alterations remains unclear. We determine that DENV nonstructural protein (NS)4B, a promising drug target with unknown function, associates with mitochondrial proteins and alters mitochondria morphology to promote infection. During infection, NS4B induces elongation of mitochondria, which physically contact CMs. This restructuring compromises the integrity of mitochondria-associated membranes, sites of ER-mitochondria interface critical for innate immune signaling. The spatio-temporal parameters of CM biogenesis and mitochondria elongation are linked to loss of activation of the fission factor Dynamin-Related Protein-1. Mitochondria elongation promotes DENV replication and alleviates RIG-I-dependent activation of interferon responses. As Zika virus infection induces similar mitochondria elongation, this perturbation may protect DENV and related viruses from innate immunity and create a favorable replicative environment.

Daclatasvir plasma level and resistance selection in HIV patients with hepatitis C virus cirrhosis treated with daclatasvir, sofosbuvir, and ribavirin.

Effective treatment with direct-acting antiviral drugs against hepatitis C virus (HCV) is a medical need in cirrhotic HIV-HCV co-infected patients. This study investigated the plasma levels of daclatasvir (DCV) and ribavirin (RBV) in HIV-HCV co-infected subjects treated with DCV, sofosbuvir, and RBV. Drug concentrations were quantified using validated high-performance liquid chromatography methods with ultraviolet detection. The HCV non-structural protein 5A and non-structural protein 5B coding regions were analyzed by population-based sequencing. DCV was dosed at week 4 and at week 8 of treatment, and RBV at week 8. One patient had the lowest DCV level, corresponding to 32.7% of the overall median value of the other patients at week 4 and about 40% at week 8. The Y93H variant was detected in this subject at weeks 8, 16, and 20 of treatment, but not before treatment or at day 2, and the patient experienced virological failure. Another subject with the Y93H variant at baseline and appropriate DCV levels had HCV RNA <12 IU/ml at week 12 and undetectable at week 16. Sub-optimal DCV drug levels allow the selection of resistance-associated variants and fail to contribute to antiviral activity. No definite reason for the low DCV level was found. Quantifying the drug is suggested in difficult-to-treat patients.

HCV NS4B targets Scribble for proteasome-mediated degradation to facilitate cell transformation.

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is a multi-transmembrane protein, but little is known about how NS4B contributes to HCV replication and tumorigenesis. Its C-terminal domain (CTD) has been shown to associate with intracellular membrane, and we have previously shown that NS4B CTD contains a class I PDZ-binding motif (PBM). Here, we demonstrated that NS4B PBM interacts with the PDZ-containing tumor suppressor protein, Scribble, using immunofluorescence and co-immunoprecipitation assays, and this interaction requires at least three contiguous PDZ domains of Scribble. In addition, NS4B PBM specifically induced Scribble degradation by activating the proteasome-ubiquitin pathway. Similar Scribble degradation was also observed in HCV-infected cells, suggesting NS4B could work in the context of HCV. Finally, NS4B PBM mutants showed reduced colony formation capacity compared with its wild-type counterpart, indicating that NS4B PBM plays important roles in NS4B-mediated cell transformation. Altogether, we provide a mechanism by which NS4B induces cell transformation through its PBM, which specifically interacts with the PDZ domains of Scribble and targets Scribble for degradation.