Protein 5A Research Articles

Proteomic study revealed antipsychotics-induced nuclear protein regulations in B35 cells are similar to the regulations in C6 cells and rat cortex

BackgroundBased on accumulating evidence, the regulation of protein expression by antipsychotic drugs (APDs) might be closely related to the control of psychotic symptoms when these drugs are used to treat mental disorders. The low quantity of nuclear proteins in the cell hinders their detection because signal for rare proteins are masked in most proteomic detection systems.MethodsNuclear proteins fractionated from APD-treated B35 cells were labeled with iTRAQ and detected by LC/MS/MS to investigate APD-induced alterations in nuclear protein expression. Western blot, immunofluorescent cell staining, and immunohistochemical staining were applied to validate the findings.ResultsThe expression of ADP/ATP translocase 2, heat shock cognate 71 kDa protein, histone H1.2, histone H3.3, histone H4, non-POU domain-containing octamer-binding protein, nucleolin, nucleophosmin, prelamin-A/C, plectin-1, vimentin, and 40S ribosomal protein S3a was regulated by APDs in B35 cells, according to our proteomic data. According to the results of the gene ontology analysis, all these proteins played important roles in biological processes or in molecular functions in cells. Western blot results showing APD-induced alterations in nuclear protein expression in B35 cells were consistent with the LC/MS/MS results. Heat shock cognate 71 kDa protein and vimentin expression in C6 cells were not affected by the three APDs. As shown in the immunofluorescent cell staining, all the three APDs altered protein expression to similar extents. We also examined whether the expression of these proteins was affected by APDs in the prefrontal cortex of rats administered sub-chronic and chronic APD treatments by western blotting and immunohistochemical staining.ConclusionsThe findings of the proteomic analysis of APD-treated B35 cells were recapitulated in the APD-treated rat cortex. The expression of some proteins was altered by APDs in rat prefrontal cortex in a time-dependent manner.

Loss of Actin-Based Motility Impairs Ectromelia Virus Release In Vitro but Is Not Critical to Spread In Vivo.

Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus (agent of smallpox), monkeypox virus and vaccinia virus (the live vaccine for smallpox), ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed when A36R is deleted.

Overexpression of A20 Inhibits the Inflammatory Response During Dengue Fever Infection

Aim: Dengue hemorrhagic fever is a devastating disease. This study aimed to investigate the role of A20 in dengue fever infection. Materials & methods: DENV2-infected human umbilical vein endothelial cells were transfected with shRNA-A20/CD14 and A20/CD14-mimics, respectively. The expressions of inflammatory and anti-inflammatory factors, A20 and downstream proteins of the NF-κB signaling pathway were detected. Results: A20 knockdown increased the expression of IL-6, IL-10, IL-8 and CD14 during dengue virus infection, whereas overexpression of A20 had the opposite effect. FACS revealed that A20 negatively regulated the expression of CD14. Conclusion: In DENV2-infected human umbilical vein endothelial cells overexpressing A20, TNF-α stimulation inhibited NF-κB-mediated inflammatory response by negative feedback. Furthermore, A20 could affect the release of inflammatory factors via negative regulation of CD14, thus affecting the entire inflammatory response.

Proteomics analysis of intensive exercise-induced disorders of gametogenesis in the testis using isobaric tags for relative and absolute quantification (iTRAQ) analysis.

In the present study we screened a panel of regulatory proteins associated with gametogenesis disorders in the testis that are induced by intensive exercise. Four-week-old Sprague-Dawley male rats were randomly divided into three groups: a control group, a no-load exercise group and an intensive exercise group. Rats in the control group were free to move in their cage. Rats in the no-load exercise and intensive exercise groups swam for 60minday-1, six times each week, for a total 9-week exercise regimen; rats in the intensive exercise group swam with a load of 6% body mass. After the last exercise session (or at the end of the 9-week period), a sperm count, reproductive hormone assays, histological analysis of the testis and proteomics analysis were performed for each rat. Mean (±s.d.) sperm concentration was significantly lower in the intensive exercise group than in the control and no-load exercise groups (1.36±0.63 vs 2.12±0.53 and 2.57±0.48×106 spermatozoa mL-1 respectively; P<0.05). Serum testosterone concentrations were also significantly lower in the intensive exercise group (P<0.01), whereas gonadotrophin-releasing hormone, LH and FSH concentrations were slightly decreased in the intensive exercise group, but not significantly (P>0.05). Histological analysis showed that the number of spermatogenic cells in the seminiferous tubules was lower in the intensive exercise group than in the control and no-load exercise groups. Proteomics analysis identified 54 proteins that were differentially expressed between the control and intensive exercise groups (31 downregulated, 23 upregulated). Pathway enrichment analysis showed that ribosome and extracellular matrix-receptor interaction pathways play an important role in the signal transduction of testicular gametogenic disorders. Four differentially expressed proteins that were involved in the regulation of reproduction were identified by bioinformatics analysis and validated by targeted mass spectrometry analysis, namely vimentin, collagen α-1(I) chain, fatty acid-binding protein 9 and 40S ribosomal protein S3a. The data suggest that changes in the abundance of differentially expressed proteins after long-term intensive exercise affect the cycle and progression of spermatogenesis, resulting in spermatogenic disorders.

Characterization of porcine sapelovirus isolated from Japanese swine with PLC/PRF/5 cells.

Porcine sapelovirus (PSV) is a causative agent of neurological disorders, fertility disorders and dermal lesions of swine. In this study, we isolated two PSV strains, Jpsv477 and Jpsv1315, from swine faecal specimens using a PLC/PRF/5 cell culture system. The PSV infection of PLC/PRF/5 cells induced a cytopathic effect (CPE). Two types of virus particles with identical diameter (~35nm) but different densities (1.300 and 1.285g/cm3 ) were observed in the cell culture supernatants. Analysis of the entire genome sequence of Jpsv477 and Jpsv1315 revealed that both strains possess 7,558 nucleotides and the poly (A) tail and have a typical PSV genome organization consisting of a 5' terminal untranslated region (5'UTR), a large open reading frame (ORF), and a 3' terminal untranslated region (3'UTR). The ORF encodes a single polyprotein that is subsequently processed into a leader protein (L), four structural proteins (VP1, VP2, VP3 and VP4) and seven functional proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). The structural proteins VP1, VP2, VP3 and VP4 have molecular masses of ~35, ~26, ~25 and ~6kDa. The N-terminal amino acid sequence analysis of VP1, VP2, VP3 and VP4 confirmed that the cleavage sites between VP4 and VP2, VP2 and VP3, and VP3 and VP1 are K/A, Q/G and Q/G, respectively. We further confirmed that HepG2/C3A, Vero E6 and primary green monkey kidney cells (PGMKC) were also susceptible to PSV infection. The stability assay demonstrated that PSV was inactivated by heating at 60°C for 10min or 65°C for 5min. The virus also lost infectivity by incubation with 62.5ppm of NaClO for 30min.

Hepatitis C Virus: Viral Quasispecies and Genotypes.

Hepatitis C virus (HCV) mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs), which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

Protection of Mice from Lethal Vaccinia Virus Infection by Vaccinia Virus Protein Subunits with a CpG Adjuvant.

Smallpox vaccination carries a high risk of adverse events in recipients with a variety of contra-indications for live vaccines. Although alternative non-replicating vaccines have been described in the form of replication-deficient vaccine viruses, DNA vaccines, and subunit vaccines, these are less efficacious than replicating vaccines in animal models. DNA and subunit vaccines in particular have not been shown to give equivalent protection to the traditional replicating smallpox vaccine. We show here that combinations of the orthopoxvirus A27, A33, B5 and L1 proteins give differing levels of protection when administered in different combinations with different adjuvants. In particular, the combination of B5 and A27 proteins adjuvanted with CpG oligodeoxynucleotides (ODN) gives a level of protection in mice that is equivalent to the Lister traditional vaccine in a lethal vaccinia virus challenge model.

Esculentoside A specifically binds to ribosomal protein S3a and impairs LPS-induced signaling in macrophages

Esculentoside A (EsA), a saponin isolated from Phytolacca esculenta, is reported as a potent suppressor of pro-inflammatory functions of macrophages. However, little is known about the target proteins of EsA for its anti-inflammatory activity. In the present study, to identify the intracellular target for EsA, affinity resins bearing immobilized EsA were used to capture binding proteins of EsA from RAW264.7 cell lysates. Mass spectrography and Western blot analysis of captured proteins indicated that ribosomal protein S3a preferentially bound to EsA affinity resin. Competition experiment further demonstrated that free EsA can disturb the specific interaction between recombinant RPS3a and affinity resin. Surface Plasmon Resonance analysis confirmed that EsA directly bound to RPS3a. Lentivirus-mediated RNAi RPS3a resulted in suppression of TNF-α and IL-6 production and impediment of signal transduction in LPS-stimulated RAW264.7 cells, indicating that RPS3a is required for LPS-triggered signaling during induction of pro-inflammatory cytokines. In addition, EsA inhibited the expression of inflammatory factors more strongly in the case of RPS3a interference. These results suggest that EsA exerts its anti-inflammatory activity by targeting RPS3a and impairing its signaling function. These new findings not only extended our understanding on the intracellular mechanisms of EsA, but also indicated RPS3a as an essential component for LPS-mediated pro-inflammatory signaling, thus implying RPS3a as a novel therapeutic target for anti-inflammatory therapy.

Regulation of hepatitis C virus replication via threonine phosphorylation of the NS5A protein.

The hepatitis C virus non-structural 5A (NS5A) protein is highly phosphorylated and plays roles in both virus genome replication and assembly of infectious virus particles. NS5A comprises three domains separated by low complexity sequences (LCS). Mass spectrometry analysis of NS5A revealed the existence of a singly phosphorylated tryptic peptide corresponding to the end of LCS I and the beginning of domain II that contained a number of potential phosphorylatable residues (serines and threonines). Here we use a mutagenic approach to investigate the potential role of three of these threonine residues. Phosphomimetic mutations of two of these (T242E and T244E) resulted in significant reductions in virus genome replication and the production of infectious virus, suggesting that the phosphorylation of these residues negatively regulated virus RNA synthesis. Mutation of T245 had no effect, however when T245E was combined with the other two phosphomimetic mutations (TripleE) the inhibitory effect on replication was less pronounced. Effects of the mutations on the ratio of basally/hyperphosphorylated NS5A, together with the apparent molecular weight of the basally phosphorylated species were also observed. Lastly, two of the mutations (T245A and TripleE) resulted in a perinuclear restricted localization of NS5A. These data add further complexity to NS5A phosphorylation and suggest that this analysis be extended outwith the serine-rich cluster within LCS I.

Prevalence of naturally occurring NS5A resistance-associated substitutions in patients infected with hepatitis C virus subtype 1a, 1b, and 3a, co-infected or not with HIV in Brazil

BackgroundNon-structural 5A protein (NS5A) resistance-associated substitutions (RASs) have been identified in patients infected with hepatitis C virus (HCV), even prior to exposure to direct-acting antiviral agents (DAAs). Selection for these variants occurs rapidly during treatment and, in some cases, leads to antiviral treatment failure. DAAs are currently the standard of care for hepatitis C treatment in many parts of the world. Nevertheless, in Brazil, the prevalence of pre-existing NS5A RASs is largely unknown. In this study, we evaluated the frequency of naturally occurring NS5A RASs in Brazilian patients infected with HCV as either a monoinfection or coinfection with human immunodeficiency virus (HIV).MethodsDirect Sanger sequencing of the NS5A region was performed in 257 DAA-naïve patients chronically infected with HCV (156 monoinfected with HCV and 101 coinfected with HIV/HCV).ResultsThe frequencies of specific RASs in monoinfected patients were 14.6% for HCV GT-1a (M28 V and Q30H/R), 6.0% for GT-1b (L31F/V and Y93H), and 22.6% for GT-3a (A30K and Y93H). For HIV/HCV-coinfected patients, the frequencies of RAS were 3.9% for GT-1a (M28 T and Q30H/R), and 11.1% for GT-1b (Y93H); no RASs were found in GT-3a sequences.ConclusionsSubstitutions that may confer resistance to NS5A inhibitors exist at baseline in Brazilian DAA-naïve patients infected with HCV GT-1a, −1b, and -3a. Standardization of RAS definitions is needed to improve resistance analyses and to facilitate comparisons of substitutions reported across studies worldwide. Therapeutic strategies should be optimized to efficiently prevent DAA treatment failure due to selection for RASs, especially in difficult-to-cure patients.

Complete genome sequence of a novel bromovirus infecting elderberry (Sambucus nigra L.) in the Czech Republic

The genus Bromovirus currently contains six species whose members have relatively narrow host ranges. In the present work, a new bromovirus infecting elderberry (Sambucus nigra L.) is reported. dsRNA was purified and sequenced by next-generation sequencing, and with minimal additional completion by Sanger sequencing, the full tripartite genome was obtained. RNA1 is 3241 nt long and contains ORF1 (1a protein), RNA2 is 2810 nt long and contains ORF2 (2a protein), and RNA3 is 2244 nt long and contains ORF3a (movement protein) and ORF3b (coat protein, CP), separated by an intercistronic poly(A) stretch. Proteins 1a and 2a showed highest sequence identity (69.9% and 69.4%) to the corresponding proteins of melandrium yellow fleck virus. The coat protein showed highest sequence identity (67.9%) to that of brome mosaic virus. The genome shows a typical bromovirus organisation comprising of all the conserved protein domains within the genus. Phylogenetic analysis supports the assignment of this virus as a new member of the genus Bromovirus, for which the name "sambucus virus S" (SVS) is proposed.

Tagging of the vaccinia virus protein F13 with mCherry causes aberrant virion morphogenesis.

Vaccinia virus produces two distinct infectious virions; the single-enveloped intracellular mature virus (IMV), which remains in the cell until cell lysis, and the double-enveloped extracellular enveloped virus (EEV), which mediates virus spread. The latter is derived from a triple-enveloped intracellular enveloped virus (IEV) precursor, which is transported to the cell periphery by the kinesin-1 motor complex. This transport involves the viral protein A36 as well as F12 and E2. A36 is an integral membrane protein associated with the outer virus envelope and is the only known direct link between virion and kinesin-1 complex. Yet in the absence of A36 virion egress still occurs on microtubules, albeit at reduced efficiency. In this paper double-fluorescent labelling of the capsid protein A5 and outer-envelope protein F13 was exploited to visualize IEV transport by live-cell imaging in the absence of either A36 or F12. During the generation of recombinant viruses expressing both A5-GFP and F13-mCherry a plaque size defect was identified that was particularly severe in viruses lacking A36. Electron microscopy showed that this phenotype was caused by abnormal wrapping of IMV to form IEV, and this resulted in reduced virus egress to the cell surface. The aberrant wrapping phenotype suggests that the fluorescent fusion protein interferes with an interaction of F13 with the IMV surface that is required for tight association between IMVs and wrapping membranes. The severity of this defect suggests that these viruses are imperfect tools for characterizing virus egress.

Differential Innate Immune Signaling in Macrophages by Wild-Type Vaccinia Mature Virus and a Mutant Virus with a Deletion of the A26 Protein.

The Western Reserve (WR) strain of mature vaccinia virus contains an A26 envelope protein that mediates virus binding to cell surface laminin and subsequent endocytic entry into HeLa cells. Removal of the A26 protein from the WR strain mature virus generates a mutant, WRΔA26, that enters HeLa cells through plasma membrane fusion. Here, we infected murine bone marrow-derived macrophages (BMDM) with wild-type strain WR and the WRΔA26 mutant and analyzed viral gene expression and cellular innate immune signaling. In contrast to previous studies, in which both HeLa cells infected with WR and HeLa cells infected with WRΔA26 expressed abundant viral late proteins, we found that WR expressed much less viral late protein than WRΔA26 in BMDM. Microarray analysis of the cellular transcripts in BMDM induced by virus infection revealed that WR preferentially activated type 1 interferon receptor (IFNAR)-dependent signaling but WRΔA26 did not. We consistently detected a higher level of soluble beta interferon secretion and phosphorylation of the STAT1 protein in BMDM infected with WR than in BMDM infected with WRΔA26. When IFNAR-knockout BMDM were infected with WR, late viral protein expression increased, confirming that IFNAR-dependent signaling was differentially induced by WR and, in turn, restricted viral late gene expression. Finally, wild-type C57BL/6 mice were more susceptible to mortality from WRΔA26 infection than to that from WR infection, whereas IFNAR-knockout mice were equally susceptible to WR and WRΔA26 infection, demonstrating that the ability of WRΔA26 to evade IFNAR signaling has an important influence on viral pathogenesis in vivoIMPORTANCE The vaccinia virus A26 protein was previously shown to mediate virus attachment and to regulate viral endocytosis. Here, we show that infection with strain WR induces a robust innate immune response that activates type 1 interferon receptor (IFNAR)-dependent cellular genes in BMDM, whereas infection with the WRΔA26 mutant does not. We further demonstrated that the differential activation of IFNAR-dependent cellular signaling between WR and WRΔA26 not only is important for differential host restriction in BMDM but also is important for viral virulence in vivo Our study reveals a new property of WRΔA26, which is in regulating host antiviral innate immunity in vitro and in vivo.

Ljungan/Sebokele-like picornavirus in birds of prey, common kestrel (Falco tinnunculus) and red-footed falcon (F. vespertinus)

Ljungan and Sebokele viruses are thought to be rodent-borne (picorna)viruses in the genus Parechovirus. Using random amplification and next generation sequencing method a novel Ljungan/Sebokele-like picornavirus was identified in birds of prey. Viral RNA was detected in total of 1 (9%) of the 11 and 2 (28.6%) of the 7 faecal samples from common kestrels and red-footed falcons in Hungary, respectively. High faecal viral RNA load (4.77×106 genomic copies/ml) measured by qPCR. The complete genome of picornavirus strain falcon/HA18_080/2014/HUN (KY645497) is 7964-nucleotide (nt) long including a 867-nt 5'end and a 101-nt 3'end (excluding the poly(A)-tail). Falcon/HA18_080/2014/HUN has type-II IRES related to hunnivirus IRES, encodes a polyprotein lacking a leader protein, a VP0 maturation cleavage site and it predicted to encode three 2A proteins (2A1NPG↓P, 2A2NPG↓P and 2A3H-Box/NC), two of them end with ‘ribosome-skipping’ sites (DxExNPG↓P). Sequence analyses indicated that the ORF1 (6996nt) polyprotein (2331 amino acid - aa) of falcon/HA18_080/2014/HUN shares the highest aa identity, 59% and 57%, to the corresponding polyproteins of Ljungan and Sebokele viruses. This study reports the identification and complete genome characterization of a novel Ljungan/Sebokele-like picornavirus in faeces of birds of prey which suggests that the genetic diversity and the potential host species spectrum of Ljungan/Sebokele-like viruses in genus Parechovirus are wider than previously thought.

Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions.

ABSTRACTHepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123–3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes.IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

Characterization of Naturally Occurring NS5A and NS5B Polymorphisms in Patients Infected with HCV Genotype 3a Treated with Direct-Acting Antiviral Agents.

Hepatitis C virus (HCV) genotype (GT)3 is associated with increased risk of steatosis, development of cirrhosis and hepatocellular carcinoma. Limited data are available regarding genetic variability and use of direct-acting antiviral agents in these patients. non-structural protein 5A (NS5A) and non-structural protein 5B (NS5B) sequencing was performed on 45 HCV GT3-infected Italian patients subsequently treated with sofosbuvir ± daclatasvir (SOF ± DCV). Novel GT3a polymorphisms were observed by Sanger sequencing in three NS5A (T79S, T107K, and T107S) and three NS5B (G166R, Q180K, and C274W) baseline sequences in patients who achieved sustained virological response (SVR). Baseline NS5A resistance-associated substitutions (RASs) A30K and Y93H were detected in 9.5% of patients; one patient with A30K did not achieve SVR. Phylogenetic analyses of sequences showed no distinct clustering. Genetic heterogeneity of NS5A and NS5B was evaluated using ultra-deep pyrosequencing (UDPS) in samples longitudinally collected in patients not achieving SVR. Some novel NS5A and NS5B polymorphisms detected at baseline may not impact treatment outcome, as they were not enriched in post-failure samples. In contrast, the clinically novel GT3 NS5A-L31F RAS emerged in one treatment failure, and I184T, G188D and N310S, located on the same NS5B haplotype, became predominant after failure. These findings suggest a potential impact of these novel substitutions on the treatment outcome; however, their significance requires further investigation.

Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins.

Among the few non-structural proteins encoded by the picornaviral genome, the 2A protein is particularly special, irrespective of structure or function. During the evolution of the Picornaviridae family, the 2A protein has been highly non-conserved. We believe that the 2A protein in this family can be classified into at least five distinct types according to previous studies. These five types are (A) chymotrypsin-like 2A, (B) Parechovirus-like 2A, (C) hepatitis-A-virus-like 2A, (D) Aphthovirus-like 2A, and (E) 2A sequence of the genus Cardiovirus. We carried out a phylogenetic analysis and found that there was almost no homology between each type. Subsequently, we aligned the sequences within each type and found that the functional motifs in each type are highly conserved. These different motifs perform different functions. Therefore, in this review, we introduce the structures and functions of these five types of 2As separately. Based on the structures and functions, we provide suggestions to combat picornaviruses. The complexity and diversity of the 2A protein has caused great difficulties in functional and antiviral research. In this review, researchers can find useful information on the 2A protein and thus conduct improved antiviral research.

Influence of ABCB11 and HNF4α genes on daclatasvir plasma concentration: preliminary pharmacogenetic data from the Kineti-C study.

Daclatasvir is an inhibitor of HCV non-structural 5A protein and is a P-glycoprotein substrate. Pharmacogenetics has had a great impact on previous anti-HCV therapies, particularly considering the association of IL-28B polymorphisms with dual therapy outcome. We investigated the association between daclatasvir plasma concentrations at 2 weeks and 1 month of therapy and genetic variants (SNPs) in genes encoding transporters and nuclear factors (ABCB1, ABCB11 and HNF4α). Allelic discrimination was achieved through real-time PCR, whereas plasma concentrations were evaluated through LC-MS/MS. Fifty-two patients were analysed, all enrolled in the Kineti-C study. HNF4α 975 C > G polymorphism was found to be associated with the daclatasvir plasma concentrations at 2 weeks (P = 0.009) and 1 month of therapy (P = 0.006). Linear regression analysis suggested that, at 2 weeks of therapy, age, baseline BMI and haematocrit were significant predictors of daclatasvir concentrations, whereas at 1 month of therapy ABCB111131 CC and HNF4α CG/GG genotypes were significant predictors of daclatasvir concentrations. These are the first and preliminary results from our clinical study focusing on daclatasvir pharmacogenetics, showing that this approach could have a role in the era of new anti-HCV therapies.

Uncleaved 2A-peptide of foot-and-mouth disease virus can display foreign epitope-tag at the virion surface

Foot-and-mouth disease virus (FMDV) capsid precursor protein P1-2A is cleaved by viral-encoded 3C protease (3Cpro) to generate VP0, VP3, VP1 and 2A proteins. It was reported earlier that substitution of a single amino acid residue within the 2A peptide sequence (L2P) blocked the 3Cpro mediated VP1/2A cleavage and produced ‘self-tagged’ FMDV particles containing uncleaved 2A-peptide. To determine whether the uncleaved 2A-peptide can function as a target structure to harbour and display exogenous epitope on FMDV particles, a full-length FMDV cDNA clone containing a HA-tag within the uncleaved 2A-peptide sequence was constructed. Subsequently, chimeric marker FMDV, displaying a HA-tag on the viral surface was rescued through reverse genetics approach. The 2A-HA epitope tag-inserted recombinant chimeric FMDV serotype O was genetically stable through up to ten serial passages in cell culture and exhibited growth properties similar to the parental virus. Furthermore the surface displayed HA-epitope tag was able to react with anti-HA antibodies as determined by various immuno-assays. The results from our study suggest that the uncleaved 2A-peptide of FMDV is suitable to present foreign antigenic epitopes on the surface of FMD virion.

Sofosbuvir–velpatasvir: A single-tablet treatment for hepatitis C infection of all genotypes

The pharmacology, pharmacokinetics, interaction potential, efficacy, and safety of the newest direct-acting antiviral (DAA) medication for the treatment of chronic hepatitis C are reviewed. Nonstructural proteins 5A (NS5A) and 5B (NS5B) are key drivers of hepatitis C virus (HCV) replication. Velpatasvir, an inhibitor of NS5A, was coformulated with the NS5B inhibitor sofosbuvir to provide a single-tablet combination DAA (Epclusa, Gilead Sciences). Sofosbuvir-velpatasvir was shown to have excellent activity against the 6 most prevalent HCV genotypes in the United States, with reported rates of sustained virological response at 12 weeks after treatment completion ranging from 95% to 100% in various HCV-infected populations, including patients with compensated cirrhosis and prior treatment failures. In patients with decompensated cirrhosis or HIV coinfection, reported cure rates are 85-100% and 92-100%, respectively. The duration of treatment with sofosbuvir-velpatasvir is 12 weeks regardless of the HCV genotype involved, previous treatment, and the presence of cirrhosis or baseline resistance-associated NS5A mutations. In patients with decompensated cirrhosis, sofosbuvir-velpatasvir must be used in combination with ribavirin. Sofosbuvir-velpatasvir was well tolerated in clinical trials; adverse effects reported at a rate of ≥10% were fatigue, headache, nausea, and nasopharyngitis. Sofosbuvir-velpatasvir is a DAA and the first single-tablet regimen to treat HCV infection caused by all genotypes. The efficacy and tolerability of sofosbuvir-velpatasvir have been observed in patients with types of HCV infection that traditionally have been difficult to treat.