Insights into the role of the cobalt(III)-thiosemicarbazone complex as a potential inhibitor of the Chikungunya virus nsP4.

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disease that can result in disability. Until now, there is no antiviral treatment against CHIKV, demonstrating that there is a need for development of new drugs. Studies have shown that thiosemicarbazones and their metal complexes possess biological activities, and their synthesis is simple, clean, versatile, and results in high yields. Here, we evaluated the mechanism of action (MOA) of a cobalt(III) thiosemicarbazone complex named [CoIII(L1)2]Cl based on its in vitro potent antiviral activity against CHIKV previously evaluated (80% of inhibition on replication). Furthermore, the complex has no toxicity in healthy cells, as confirmed by infecting BHK-21 cells with CHIKV-nanoluciferase in the presence of the compound, showing that [CoIII(L1)2]Cl inhibited CHIKV infection with the selective index of 3.26. [CoIII(L1)2]Cl presented a post-entry effect on viral replication, emphasized by the strong interaction of [CoIII(L1)2]Cl with CHIKV non-structural protein 4 (nsP4) in the microscale thermophoresis assay, suggesting a potential mode of action of this compound against CHIKV. Moreover, in silico analyses by molecular docking demonstrated potential interaction of [CoIII(L1)2]Cl with nsP4 through hydrogen bonds, hydrophobic and electrostatic interactions. The evaluation of ADME-Tox properties showed that [CoIII(L1)2]Cl presents appropriate lipophilicity, good human intestinal absorption, and has no toxicological effect as irritant, mutagenic, reproductive, and tumorigenic side effects.

Identification of NS5B resistance against SOFOSBUVIR in hepatitis C virus genotype 3a, naive and treated patients.

AIMS: Pakistan has the second highest prevalence of HCV with genotype 3a (GT-3a) being the most frequently circulating genotype. Currently, resistance-associated substitutions (RASs) are a major challenge in HCV treatment with direct-acting antivirals (DAAs). Sofosbuvir (SOF) is an FDA-approved NS5B nucleotide inhibitor. The aim of this study was to identify these RASs in the NS5B gene in naive and treated Pakistani HCV 3a isolates against SOF. METHODS AND RESULTS: Blood samples were collected from anti-HCV-positive patients, followed by HCV RNA isolation and real-time PCR quantification. HCV-positive patients were processed for HCV RNA genotyping, patients with genotype 3a were processed for NS5B gene amplification and sequencing. GT-3a was the most prevalent genotype (62.2%). S282T was identified in 2 (8.7%) patients, C316Y/G/R in 3 (13%), V321A and L320P in 1 (4.3%) each in SOF/RBV-resistant patients. Variants of S282 were detected in 3 (13%) of SOF/RBV-treated patients. While INF/RBV-associated mutations were also analysed, D244N, A333R and A334E were identified in 2 (9.5%), 3 (14.2%) and 7 (33.3%) in treatment-naive and 15 (65.2%), 7 (30.4%) and 5 (21.7%) treated patients, respectively. Q309R was observed only in one treatment-experienced patients. Some substitutions were present at higher frequency in both groups like N307G, K304R, A272D and R345H, considered that they do not have any role in sofosbuvir resistance. CONCLUSION: It was concluded that sofosbuvir RASs are present in Pakistani HCV GT-3a isolates, and they should be monitored carefully, especially in treatment-experienced patients, for further selection of treatment regimens. SIGNIFICANCE AND IMPACT OF STUDY: HCV RASs have been studied very well across the world but there is scarcity of data regarding this topic in Pakistani population, this study provides data regarding the prevalence of these RASs in Pakistani HCV isolates emphasizing the fact that these RASs must be carefully monitored before starting HCV treatment, especially in treatment failure patients.

Linkage of resistance-associated substitutions in GT1 sofosbuvir + NS5A inhibitor failures treated with glecaprevir/pibrentasvir.

BACKGROUND & AIMS: Retreatment with glecaprevir/pibrentasvir (G/P) resulted in a rate of sustained virologic response 12 weeks after treatment completion (SVR12) of >90% in HCV genotype 1 (GT1) patients who previously failed a regimen of sofosbuvir plus an NS5A inhibitor (NS5Ai). This study investigated the prevalence and impact of baseline NS3 and NS5A resistance-associated substitutions (RASs) on the efficacy of G/P in prior GT1 sofosbuvir+NS5Ai failures and the persistence of treatment-emergent RASs. METHODS: Longitudinal samples from 177 patients enrolled in a phase IIIb, randomized pragmatic clinical trial were analyzed. Patients without cirrhosis were randomized to 12 or 16 weeks of G/P, and patients with compensated cirrhosis were randomized to G/P and ribavirin for 12 weeks or G/P for 16 weeks. Linkage of RAS was identified using Primer-ID next-generation sequencing at a 15% cut-off. RESULTS: Of 177 patients, 169 (95.5%) were PI-naïve. All 33 GT1b-infected patients achieved SVR12. In GT1a-infected patients, baseline NS5A RASs were prevalent (74.5%, 105/141) but NS3 RASs were uncommon. Baseline NS3 RASs had no impact on G/P efficacy and patients with baseline NS5A RASs showed a numerically but not statistically significantly lower SVR12 rate compared to those without NS5A RASs (89% vs. 97%). SVR12 was achieved in 34 of 35 (97%) patients without NS5A baseline substitution, and 53 of 57 (93%), 35 of 40 (88%), 5 of 8 (63%) with single, double-linked, and triple-linked NS5A substitutions, respectively. Among 13 patients with virologic failure, 4 acquired treatment-emergent NS3 RASs and 10 acquired NS5A RASs. CONCLUSION: Baseline NS5A RASs were highly prevalent. The presence of an increasing number of linked NS5A RASs in GT1a showed a trend in decreasing SVR12 rates, although no specific NS5A RASs or their linkage pattern were associated with lower SVR12 rates. LAY SUMMARY: Direct-acting antivirals have revolutionized the treatment of chronic hepatitis C infection, but treatment failure occurs in some patients. Retreatment of patients who previously failed a regimen consisting of sofosbuvir and an NS5A inhibitor with a regimen of glecaprevir and pibrentasvir (G/P) is >90% effective. Herein, we analyzed samples from these patients and showed that retreatment efficacy with G/P is lower in patients with double- or triple-linked NS5A resistance mutations than in patients with single or no NS5A resistance mutations. CLINICAL TRIAL NUMBER: NCT03092375.

Preparation of a polyclonal antibody against the non-structural protein, NSs of SFTSV.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is newly discovered virus which is the member of the order Bunyavirales, family phenuiviridae, phlebovirus genus. Its genome is composed of 3 segments of negative-sense RNA L, M and S. NSs is a non structure protein encoded by S segment which is important for viral replication and virulence. NSs protein of SFTSV is only involved in the regulation of host innate immune responses and suppression of IFN-promoter activities. So, the exact functions of this protein need to be studied deeply. To understand the exact role of NSs from SFTSV in viral replication and host immune response, a qualified antibody against this protein is required. In this study, NSs gene of SFTSV, was cloned into a bacterial expression vector (pGEX-6P-1) and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells. The SFTSV NSs fusion protein was purified using Glutathione Sepharose 4B and utilized as an antigen to immunize rabbits and obtain an anti-SFTSV NSs polyclonal antibody. Proper expression of the fusion protein and polyclonal antibody specificity were confirmed by western blotting and immunofluorescence analyses. The polyclonal antibody recognized NSs from SFTSV specifically. This is the first report that NSs can form viroplasm-like structures not only in infected cells but also in transfected cells with NSs plasmids. This polyclonal antibody will be useful for future studies of NSs functions.

First report on molecular docking analysis and drug resistance substitutions to approved HCV NS5A and NS5B inhibitors amongst Iranian patients.

BACKGROUND: NS5A and NS5B proteins of hepatitis C virus (HCV) are the main targets of compounds that directly inhibit HCV infections. However, the emergence of resistance-associated substitutions (RASs) may cause substantial reductions in susceptibility to inhibitors. METHODS: Viral load and genotyping were determined in eighty-seven naïve HCV-infected patients, and the amplified NS5A and NS5B regions were sequenced by Sanger sequencing. In addition, physicochemical properties, structural features, immune epitopes, and inhibitors-protein interactions of sequences were analyzed using several bioinformatics tools. RESULTS: Several amino acid residue changes were found in NS5A and NS5B proteins; however, we did not find any mutations related to resistance to the treatment in NS5B. Different phosphorylation and few glycosylation sites were assessed. Disulfide bonds were identified in both proteins that had a significant effect on the function and structure of HCV proteins. Applying reliable software to predict B-cell epitopes, 3 and 5 regions were found for NS5A and NS5B, respectively, representing a considerable potential to induce the humoral immune system. Docking analysis determined amino acids involved in the interaction of inhibitors and mentioned proteins may not decrease the drug efficiency. CONCLUSIONS: Strong interactions between inhibitors, NS5A and NS5B proteins and the lack of efficient drug resistance mutations in the analyzed sequences may confirm the remarkable ability of NS5A and NS5B inhibitors to control HCV infection amongst Iranian patients. The results of bioinformatics analysis could unveil all features of both proteins, which can be beneficial for further investigations on HCV drug resistance and designing novel vaccines.

Zika virus NS5 protein inhibits cell growth and invasion of glioma.

Glioma is the most common primary brain tumor with high mortality. Given the poor outcomes with standard-of-care treatments, novel treatment strategies are needed. Oncolytic viral therapy for glioma has developed as an exciting therapeutic method in recent years. Zika virus, a member of flavivirus family, has oncolytic activity against glioma cells but the mechanism is unknown. Here, we aimed to determine which viral protein might play a critical role in mitigating glioma cell growth. We examined the tumor suppressor function of four nonstructural proteins NS1, NS3, NS4B and NS5 in human glioma cell line U87. As a result, we found that only NS5 significantly inhibited proliferation, migration and invasion of U87 cells. Moreover, expression of NS5 suppressed tumorigenicity of mouse GL261 glioma cell in vivo. Our findings provide some clues for further exploration of oncolytic Zika virus in the treatment of glioma.

Bovine viral diarrhea virus non-structural protein NS4B induces autophagosomes in bovine kidney cells.

Bovine viral diarrhea virus (BVDV) is an important pathogen in cattle that causes economic losses in livestock industries. Autophagy is an essential cell system for the maintenance of homeostasis and is induced by various triggers, including infection by viruses. BVDV infection leads to autophagy in order to enhance its replication in cells. In this study, we investigated the effect of BVDV non-structural proteins on the induction of autophagosomes. We found that NS4B alone could induce autophagosomes, suggesting a novel and important function of NS4B in BVDV replication.

Interactome Analysis of NS1 Protein Encoded by Influenza A H7N9 Virus Reveals an Inhibitory Role of NS1 in Host mRNA Maturation.

Influenza A virus infections can result in severe respiratory diseases. The H7N9 subtype of avian influenza A virus has been transmitted to humans and caused severe disease and death. Nonstructural protein 1 (NS1) of influenza A virus is a virulence determinant during viral infection. To elucidate the functions of the NS1 encoded by influenza A H7N9 virus (H7N9 NS1), interaction partners of H7N9 NS1 in human cells were identified with immunoprecipitation followed by SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). We identified 36 cellular proteins as the interacting partners of the H7N9 NS1, and they are involved in RNA processing, mRNA splicing via spliceosome, and the mRNA surveillance pathway. Two of the interacting partners, cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, were confirmed to interact with H7N9 NS1 using coimmunoprecipitation and immunoblotting based on the previous finding that the two proteins are involved in pre-mRNA polyadenylation machinery. Furthermore, we illustrate that overexpression of H7N9 NS1, as well as infection by the influenza A H7N9 virus, interfered with pre-mRNA polyadenylation in host cells. This study comprehensively profiled the interactome of H7N9 NS1 in host cells, and the results demonstrate a novel endotype for H7N9 NS1 in inhibiting host mRNA maturation.

The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis.

Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93-140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells.

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

BACKGROUND: Hepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/ß-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/ß-catenin signaling in various liver cells, and explored the molecular mechanism underlying HCV-related hepatocarcinogenesis. RESULTS: Compared with the empty vector control, HCV core protein and NS4B demonstrated the following characteristics in the Huh7 cells: significantly enhanced ß-catenin/Tcf-dependent transcriptional activity (F = 40.87, P < 0.01); increased nuclear translocation of ß-catenin (F = 165.26, P < 0.01); upregulated nuclear ß-catenin, cytoplasmic ß-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 ß-catenin/Tcf-dependent transcriptional activity in the LO2 cells (F = 0.65, P > 0.05), but they did significantly enhance Wnt3a-induced ß-catenin/Tcf-dependent transcriptional activity (F = 64.25, P < 0.01), and promoted the nuclear translocation of ß-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/ß-catenin signaling pathway was greater with the core protein than with NS4B (P < 0.01 or P < 0.05). CONCLUSIONS: HCV core protein and NS4B directly activate the Wnt/ß-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.

Structural Insight into NS5 of Zika Virus Leading to the Discovery of MTase Inhibitors.

Zika virus (ZIKV) is an emerging mosquito-borne virus recently linked to intrauterine growth restriction including abnormal fetal brain development. The recent outbreak of ZIKV reached pandemic level resulting in an alarming public health emergency. At present, there is limited understanding of the infectious mechanism and no approved therapy. Nonstructural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase (MTase) and RNA dependent RNA polymerase domain. Here we used molecular modeling to obtain the structure of ZIKV MTase and molecular docking to identify the additional hydrophobic region uniquely conserved in flavivirus MTase that can be used as a druggable site. Subsequently, a virtual screening with a library of 28 341 compounds identified 10 best hits showing decisive contacts with the MTase. In vitro efficacy analysis of these compounds against ZIKV, by plaque reduction assay, has confirmed four of the top scored ligands (Life Chemicals ID: F3043-0013, F0922-0796, F1609-0442, and F1750-0048) having EC50 (50% effective concentration) values of 4.8 ± 2.3, 12.5 ± 7.4, 17.5 ± 8.4, and 17.6 ± 3.1 µM respectively, identifying lead compounds for anti-ZIKV drug development.

Dengue NS1 antigen contributes to disease severity by inducing interleukin (IL)-10 by monocytes.

Both dengue NS1 antigen and serum interleukin (IL)-10 levels have been shown to associate with severe clinical disease in acute dengue infection, and IL-10 has also been shown to suppress dengue-specific T cell responses. Therefore, we proceeded to investigate the mechanisms by which dengue NS1 contributes to disease pathogenesis and if it is associated with altered IL-10 production. Serum IL-10 and dengue NS1 antigen levels were assessed serially in 36 adult Sri Lankan individuals with acute dengue infection. We found that the serum IL-10 levels correlated positively with dengue NS1 antigen levels (Spearman's r = 0·47, P < 0·0001), and NS1 also correlated with annexin V expression by T cells in acute dengue (Spearman's r = 0·63, P = 0·001). However, NS1 levels did not associate with the functionality of T cell responses or with expression of co-stimulatory molecules. Therefore, we further assessed the effect of dengue NS1 on monocytes and T cells by co-culturing primary monocytes and peripheral blood mononuclear cells (PBMC), with varying concentrations of NS1 for up to 96 h. Monocytes co-cultured with NS1 produced high levels of IL-10, with the highest levels seen at 24 h, and then declined gradually. Therefore, our data show that dengue NS1 appears to contribute to pathogenesis of dengue infection by inducing IL-10 production by monocytes.

Poly (I:C) enhances the anti-tumor activity of canine parvovirus NS1 protein by inducing a potent anti-tumor immune response.

The canine parvovirus NS1 (CPV2.NS1) protein selectively induces apoptosis in the malignant cells. However, for an effective in vivo tumor treatment strategy, an oncolytic agent also needs to induce a potent anti-tumor immune response. In the present study, we used poly (I:C), a TLR3 ligand, as an adjuvant along with CPV2.NS1 to find out if the combination can enhance the oncolytic activity by inducing a potent anti-tumor immune response. The 4T1 mammary carcinoma cells were used to induce mammary tumor in Balb/c mice. The results suggested that poly (I:C), when given along with CPV2.NS1, not only significantly reduced the tumor growth but also augmented the immune response against tumor antigen(s) as indicated by the increase in blood CD4+ and CD8+ counts and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed that Th1 cytokines (IFN-γ and IL-2) were significantly upregulated in the treatment group indicating activation of cell-mediated immune response. The present study reports the efficacy of CPV2.NS1 along with poly (I:C) not only in inhibiting the mammary tumor growth but also in generating an active anti-tumor immune response without any visible toxicity. The results of our study may help in developing CPV2.NS1 and poly (I: C) combination as a cancer therapeutic regime to treat various malignancies.

Recombinant Dengue virus protein NS2B alters membrane permeability in different membrane models.

BACKGROUND: One of the main phenomena occurring in cellular membranes during virus infection is a change in membrane permeability. It has been observed that numerous viral proteins can oligomerize and form structures known as viroporins that alter the permeability of membranes. Previous findings have identified such proteins in cells infected with Japanese encephalitis virus (JEV), a member of the same family that Dengue virus (DENV) belongs to (Flaviviridae). In the present work, we investigated whether the small hydrophobic DENV protein NS2B serves a viroporin function. METHODS: We cloned the DENV NS2B sequence and expressed it in a bacterial expression system. Subsequently, we evaluated the effect of DENV NS2B on membranes when NS2B was overexpressed, measured bacterial growth restriction, and evaluated changes of permeability to hygromycin. The NS2B protein was purified by affinity chromatography, and crosslinking assays were performed to determine the presence of oligomers. Hemolysis assays and transmission electron microscopy were performed to identify structures involved in permeability changes. RESULTS: The DENV-2 NS2B protein showed similitude with the JEV viroporin. The DENV-2 NS2B protein possessed the ability to change the membrane permeability in bacteria, to restrict bacterial cell growth, and to enable membrane permeability to hygromycin B. The NS2B protein formed trimers that could participate in cell lysis and generate organized structures on eukaryotes membranes. CONCLUSIONS: Our data suggest that the DENV-2 NS2B viral protein is capable of oligomerizing and organizing to form pore-like structures in different lipid environments, thereby modifying the permeability of cell membranes.

Conformational Heterogeneity Determined by Folding and Oligomer Assembly Routes of the Interferon Response Inhibitor NS1 Protein, Unique to Human Respiratory Syncytial Virus.

The nonstructural NS1 protein is an essential virulence factor of the human respiratory syncytial virus, with a predominant role in the inhibition of the host antiviral innate immune response. This inhibition is mediated by multiple protein-protein interactions and involves the formation of large oligomeric complexes. There is neither a structure nor sequence or functional homologues of this protein, which points to a distinctive mechanism for blocking the interferon response among viruses. The NS1 native monomer follows a simple unfolding kinetics via a nativelike transition state ensemble, with a half-life of 45 min, in agreement with a highly stable core structure at equilibrium. Refolding is a complex process that involves several slowly interconverting species compatible with proline isomerization. However, an ultrafast folding event with a half-life of 0.2 ms is indicative of a highly folding compatible species within the unfolded state ensemble. On the other hand, the oligomeric assembly route from the native monomer, which does not involve unfolding, shows a monodisperse and irreversible end-point species triggered by a mild temperature change, with half-lives of 160 and 26 min at 37 and 47 °C, respectively, and at a low protein concentration (10 µM). A large secondary structure change into ß-sheet structure and the formation of a dimeric nucleus precede polymerization by the sequential addition of monomers at the surprisingly low rate of one monomer every 34 s. The polymerization phase is followed by the binding to thioflavin-T indicative of amyloid-like, albeit soluble, repetitive ß-sheet quaternary structure. The overall process is reversible only up until ~8 min, a time window in which most of the secondary structure change takes place. NS1's multiple binding activities must be accommodated in a few binding interfaces at most, something to be considered remarkable given its small size (15 kDa). Thus, conformational heterogeneity, and in particular oligomer formation, may provide a means of expand its binding repertoire. These equilibria will be determined by variables such as macromolecular crowding, protein-protein interactions, expression levels, turnover, or specific subcellular localization. The irreversible and quasi-spontaneous nature of the oligomer assembly, together with the fact that NS1 is the most abundant viral protein in infected cells, makes its accumulation highly conceivable under conditions compatible with the cellular milieu. The implications of NS1 oligomers in the viral life cycle and the inhibition of host innate immune response remain to be determined.

Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors.

UNLABELLED: Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high-titer HCV cultures mimicking the complete viral life cycle, but such systems only exist for genotypes 1a and 2a. We developed efficient culture systems for the epidemiologically important genotype 2b. Full-length molecular clones of patient strains DH8 and DH10 were adapted to efficient growth in Huh7.5 cells by using F1468L/A1676S/D3001G (LSG) mutations. The previously developed J8cc prototype 2b recombinant was further adapted. DH8 and J8 achieved infectivity titers >4.5 log10 Focus-Forming Units/mL. A defined set of DH8 mutations had cross-isolate adapting potential. A chimeric genome with the DH10 polyprotein coding sequence inserted into a vector with J8 untranslated regions was viable. Importantly, we succeeded in generating DH8, J8, and DH10 viruses with authentic sequences in the regions targeted by lead direct-acting antivirals. Nonstructural protein (NS)5B inhibitors sofosbuvir, mericitabine, and BI207127 had activity against 1a (strain TN), 2a (strains JFH1 and J6), and the 2b strains, whereas VX-222 and filibuvir only inhibited 1a. Genotype 2b strains were least sensitive to seven lead protease inhibitors, including MK-5172 with high overall potency. NS5A inhibitor daclatasvir was exceptionally potent, but efficacy was affected by the HCV strain. CONCLUSION: Highly efficient HCV full-length 2b culture systems can be established by using consensus clones with defined mutations. Lead protease and NS5A inhibitors, as well as polymerase inhibitors sofosbuvir, mericitabine, and BI207127, show cross-activity against full-length 1a, 2a, and 2b viruses, but important sensitivity differences exist at the isolate level. Infectious cultures for different HCV strains will advance studies on viral biology and pathogenesis and promote individualized patient treatment.

NS3 of bluetongue virus interferes with the induction of type I interferon.

Upon infection with Bluetongue virus (BTV), an arthropod-borne virus, type I interferon (IFN-I) is produced in vivo and in vitro. IFN-I is essential for the establishment of an antiviral cellular response, and most if not all viruses have elaborated strategies to counteract its action. In this study, we assessed the ability of BTV to interfere with IFN-I synthesis and identified the nonstructural viral protein NS3 as an antagonist of the IFN-I system.

Screening and rational design of hepatitis C virus entry inhibitory peptides derived from GB virus A NS5A.

Chronic infection by hepatitis C virus (HCV) is a cause of the global burden of liver diseases. HCV entry into hepatocytes is a complicated and multistep process that represents a promising target for antiviral intervention. The recently reported amphipathic α-helical virucidal peptide (C5A) from the HCV NS5A protein suggests a new category of antiviral drug candidates. In this study, to identify C5A-like HCV inhibitors, synthetic peptides derived from the C5A-corresponding NS5 protein region of selected Flaviviridae viruses were evaluated for their anti-HCV activities. A peptide from GB virus A (GBV-A), but not other flaviviruses, demonstrated an inhibitory effect on HCV infection. Through a series of sequence optimizations and modifications of the peptide helicity and hydrophobicity, we obtained a peptide designated GBVA10-9 with highly potent anti-HCV activity. GBVA10-9 suppressed infection with both cell culture-derived and pseudotyped HCV in vitro, and the 50% cell culture inhibitory concentration ranged from 20 nM to 160 nM, depending on the genotypic origin of the envelope proteins. GBVA10-9 had no detectable effects on either HCV attachment to Huh7.5.1 cells or viral RNA replication. No virucidal activity was found with GBVA10-9, suggesting an action mechanism distinct from that of C5A. The inhibitory effect of GBVA10-9 appeared to occur at the postbinding step during viral entry. Taken together, the results with GBVA10-9 demonstrated a potent activity for blocking HCV entry that might be used in combination with other antivirals directly targeting virus-encoded enzymes. Furthermore, GBVA10-9 also provides a novel tool to dissect the detailed mechanisms of HCV entry.

Rotavirus-encoded nonstructural protein 1 modulates cellular apoptotic machinery by targeting tumor suppressor protein p53.

p53, a member of the innate immune system, is triggered under stress to induce cell growth arrest and apoptosis. Thus, p53 is an important target for viruses, as efficient infection depends on modulation of the host apoptotic machinery. This study focuses on how rotaviruses manipulate intricate p53 signaling for their advantage. Analysis of p53 expression revealed degradation of p53 during initial stages of rotavirus infection. However, in nonstructural protein-1 (NSP1) mutant strain A5-16, p53 degradation was not observed, suggesting a role of NSP1 in this process. This function of NSP1 was independent of its interferon or phosphatidylinositol 3-kinase (PI3K)/AKT modulation activity since p53 degradation was observed in Vero cells as well as in the presence of PI3K inhibitor. p53 transcript levels remained the same in SA11-infected cells (at 2 to 14 h postinfection), but p53 protein was stabilized only in the presence of MG132, suggesting a posttranslational process. NSP1 interacted with the DNA binding domain of p53, resulting in ubiquitination and proteasomal degradation of p53. Degradation of p53 during initial stages of infection inhibited apoptosis, as the proapoptotic genes PUMA and Bax were downregulated. During late viral infection, when progeny dissemination is the main objective, the NSP1-p53 interaction was diminished, resulting in restoration of the p53 level, with initiation of proapoptotic signaling ensuing. Overall results highlight the multiple strategies evolved by NSP1 to combat the host immune response.

HCV core and NS3 proteins mediate toll like receptor induced innate immune response in corneal epithelium.

Direct association of dry eye syndrome and hepatitis C virus (HCV) infection is a well established fact. In this context, the current study examines the in vitro corneal inflammatory response with respect to HCV core and NS3 antigens. Toll like receptors (TLRs) are pattern recognition receptors which can mediate innate immune response. In the present study, corneal epithelial cells responded to HCV core and NS3 proteins by secreting pro-inflammatory cytokines IL-8, IL-6 and TNF-α via TLR1, TLR2 and TLR6 mediated innate immune response. MyD88/NF-kB signalling was involved in pro-inflammatory cytokine production. Corneal epithelium synthesised nitric oxide (NO) via iNOS during HCV core and NS3 exposure. On later stages of inflammation, cells underwent apoptosis which lead to cell death. SiRNA mediated silencing of TLR1, TLR2 and TLR6 resulted in a significant down regulation of IL-8 and NO. In conclusion, this study indicates that HCV core and NS3 proteins are capable of inducing immune response in corneal epithelium which can potentiate the pathology of HCV associated dry eye condition. Blocking specific TLR response can have therapeutic application in controlling the inflammatory response associated with this dry eye condition.