Nonstructural Protein 5A Research Articles

Cellular Vimentin Interacts with Foot-and-Mouth Disease Virus Nonstructural Protein 3A and Negatively Modulates Viral Replication.

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids that is in most FMDVs examined to date, and it plays important roles in virus replication, virulence, and host range. To better understand the role of 3A during FMDV infection, we used coimmunoprecipitation followed by mass spectrometry to identify host proteins that interact with 3A in FMDV-infected cells. Here, we report that cellular vimentin is a host binding partner for 3A. The 3A-vimentin interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pull down, and immunofluorescence assays. Alanine-scanning mutagenesis indicated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin. Using reverse genetics, we demonstrate that mutations in 3A that disrupt the interaction between 3A and vimentin are also critical for virus growth. Overexpression of vimentin significantly suppressed the replication of FMDV, whereas knockdown of vimentin significantly enhanced FMDV replication. However, chemical disruption of the vimentin network by acrylamide resulted in a significant decrease in viral yield, suggesting that an intact vimentin network is needed for FMDV replication. These results indicate that vimentin interacts with FMDV 3A and negatively regulates FMDV replication and that the vimentin-3A interaction is essential for FMDV replication. This study provides information that should be helpful for understanding the molecular mechanism of FMDV replication.IMPORTANCE Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, host range, and virulence. To further understand the role of 3A during FMDV infection, identification of host cell factors that interact with FMDV 3A is needed. Here, we found that vimentin is a direct binding partner of FMDV 3A, and manipulation of vimentin has a negative effect on virus replication. We also demonstrated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin and that the 3A-vimentin interaction is critical for viral replication since the full-length cDNA clone harboring mutations in 3A, which were disrupt 3A-vimentin reactivity, could not produce viable virus progeny. This study provides information that not only provides us a better understanding of the mechanism of FMDV replication but also helps in the development of novel antiviral strategies in the future.

OCIAD1 is a host mitochondrial substrate of the hepatitis C virus NS3-4A protease.

The hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) protease is a key component of the viral replication complex and the target of protease inhibitors used in current clinical practice. By cleaving and thereby inactivating selected host factors it also plays a role in the persistence and pathogenesis of hepatitis C. Here, we describe ovarian cancer immunoreactive antigen domain containing protein 1 (OCIAD1) as a novel cellular substrate of the HCV NS3-4A protease. OCIAD1 was identified by quantitative proteomics involving stable isotopic labeling using amino acids in cell culture coupled with mass spectrometry. It is a poorly characterized membrane protein believed to be involved in cancer development. OCIAD1 is cleaved by the NS3-4A protease at Cys 38, close to a predicted transmembrane segment. Cleavage was observed in heterologous expression systems, the replicon and cell culture-derived HCV systems, as well as in liver biopsies from patients with chronic hepatitis C. NS3-4A proteases from diverse hepacivirus species efficiently cleaved OCIAD1. The subcellular localization of OCIAD1 on mitochondria was not altered by NS3-4A-mediated cleavage. Interestingly, OCIAD2, a homolog of OCIAD1 with a cysteine residue in a similar position and identical subcellular localization, was not cleaved by NS3-4A. Domain swapping experiments revealed that the sequence surrounding the cleavage site as well as the predicted transmembrane segment contribute to substrate selectivity. Overexpression as well as knock down and rescue experiments did not affect the HCV life cycle in vitro, raising the possibility that OCIAD1 may be involved in the pathogenesis of hepatitis C in vivo.

The 3A protein of coxsackievirus B3 acts as a viral suppressor of RNA interference

RNA interference (RNAi) is a potent antiviral defence mechanism in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs). Coxsackievirus B3 (CVB3) belongs to the genus Enterovirus in the family Picornaviridae, and has been reported to be a major causative pathogen for viral myocarditis. Despite the importance of CVB3, it is unclear whether CVB3 can also encode proteins that suppress RNAi. Here, we showed that the CVB3 nonstructural protein 3A suppressed RNAi triggered by either small hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) in mammalian cells. We further uncovered that CVB3 3A interacted directly with double-stranded RNAs (dsRNAs) and siRNAs in vitro. Through mutational analysis, we found that the VSR activity of CVB3 3A was significantly reduced by mutations of D24A/L25A/L26A, Y37A/C38A and R60A in conserved residues. In addition, the 3A protein encoded by coxsackievirus B5 (CVB5), another member of Enterovirus, also showed VSR activity. Taken together, our findings showed that CVB3 3A has in vitro VSR activity, thereby providing insights into the pathogenesis of CVB3 and other enteroviruses.

Viral Interference Between Dengue Virus and Hepatitis C Virus Infections.

Both dengue virus (DENV) and hepatitis C virus (HCV) belong to the Flaviviridae family and could induce hepatitis. We aimed to investigate the interference between them. In total, 515 patients confirmed with dengue fever (DF) were enrolled. Thirty-two patients (6.21%) were seropositive for anti-HCV; 12 of 32 anti-HCV-positive patients had detectable HCV-RNA at presentation of DF. The proportion of dengue hemorrhagic fever was comparable between patients with or without anti-HCV and between those with or without HCV-RNA. Eleven of 32 patients received HCV-RNA testing during a median interval of 23 months after DF, which revealed significantly increased HCV-RNA levels (5.43 ± 0.77 vs 3.09 ± 1.24 log IU/mL, follow-up vs acute-DF phase; P = .003). Four of 11 patients with baseline HCV-RNA values before DF demonstrated a nadir viremia during acute DF. We also included age-, sex-, and follow-up duration–matched HCV-monoinfected patients as controls; higher delta HCV-RNA changes were demonstrated in patients with DF than in controls during the follow-up period (2.34 ± 1.15 vs –0.27 ± 0.76 log IU/mL; P < .001). Further in vitro experiments showed that HCV nonstructural protein 5A was downregulated in Con1 HCV replicon cells infected by DENV1. These clinical and experimental findings suggested possible viral interference in DENV/HCV. However, HCV viremia did not affect the disease outcomes of DF.

Foot-and-mouth disease virus non-structural protein 3A modulates cellular innate immune responses to influence host tropism

The picornavirus foot-and-mouth disease virus (FMDV) is responsible for one of the most significant diseases of livestock, leading to large economic losses due to reduced productivity and trade embargoes for areas not certified as disease-free. The picornavirus non-structural protein 3A is involved in replication of the viral RNA genome and is implicated in host tropism of several picornaviruses. Deletions in the C-terminus of 3A have been observed in FMDV outbreaks specific for swine and such viruses are non-pathogenic in cattle. The mechanism for species specific attenuation of FMDV is unknown. We have shown that FMDV containing a C-terminal deletion in 3A is attenuated in bovine cell culture and that the attenuated phenotype can be reversed by the JAK1/2 inhibitor Ruxolitinib (Rux), identifying a role for the induction of interferon stimulated genes (ISGs) in the restricted bovine tropism of the 3A-deleted virus.

Orally bioavailable HCV NS5A inhibitors of unsymmetrical structural class

A novel unsymmetrical structural class of orally bioavailable hepatitis C virus (HCV) nonstructural 5A protein (NS5A) inhibitors has been generated by improving both the solubility and membrane permeability of the lead compound found in our previous work. The representative compound 14, with a 5-hydroxymethylpyrazine group and a 3-t-butylpropargyl group on each side of the molecule, exhibited the best oral bioavailability in this study, inhibiting not only the HCV genotype 1a, 1b, 2a, and 3a replicons with EC50 values in the picomolar range, but also inhibited 1a Q30 mutants induced by launched symmetrical inhibitors with EC50 values in the low nanomolar range.

Utilization and effectiveness of elbasvir/grazoprevir and adoption of resistance-associated substitutions testing in real-world treatment of hepatitis C virus genotype 1A infection: results from the German Hepatitis C-Registry.

BackgroundFor treatment of genotype 1a (GT1a) infection with elbasvir/grazoprevir, the German guidelines recommend a differentiated approach depending on baseline viral load (BVL). For low BVL ≤800 000 IU/mL, treatment with 12 weeks elbasvir/grazoprevir should be considered, whereas for high BVL >800 000 IU/mL, this regimen is only recommended in nonstructural protein 5A (NS5A) resistance-associated substitutions (RAS) absence. With present NS5A RAS or when RAS-testing is not available, 16 weeks elbasvir/grazoprevir + ribavirin is preferred. Here, we investigated the adherence to these recommendations and the effectiveness of elbasvir/grazoprevir in a large German Hepatitis C-Registry GT1a cohort.MethodsFrom September 2016 until July 2018, 195 GT1a-infected patients were treated with elbasvir/grazoprevir ± ribavirin for 12–16 weeks. The primary outcome was per protocol SVR12 or SVR24.ResultsMean age was 50 years, 89% were male, 19% had cirrhosis, 72% were treatment-naïve. Forty-five percent had low BVL ≤800 000 IU/mL, 55% high BVL >800 000 IU/mL, of whom 49 vs. 42% were baseline RAS-tested. Four patients with high (7.7%) and two with low BVL (5%) had NS5A RAS of whom 50% received elbasvir/grazoprevir+ribavirin, respectively. Ninety-four percent of patients with low and 65% with high BVL received elbasvir/grazoprevir without ribavirin. Thirty-five percent of patients with high BVL received ribavirin, mostly without prior RAS-testing. Per protocol sustained virologic response (SVR) by low vs. high BVL was 98.8 and 95.1%. All patients with NS5A RAS achieved SVR.ConclusionsIn German, real-world most patients received elbasvir/grazoprevir without ribavirin. Ribavirin was mainly added in GT1a patients >800 000 IU/mL, who were not NS5A RAS tested. SVR rates were consistently high and comparable to clinical trial results.

Binding of Duck Tembusu Virus Nonstructural Protein 2A to Duck STING Disrupts Induction of Its Signal Transduction Cascade To Inhibit Beta Interferon Induction.

Duck Tembusu virus (DTMUV), which is similar to other mosquito-borne flaviviruses that replicate well in most mammalian cells, is an emerging pathogenic flavivirus that has caused epidemics in egg-laying and breeding waterfowl. Immune organ defects and neurological dysfunction are the main clinical symptoms of DTMUV infection. Preinfection with DTMUV makes the virus impervious to later interferon (IFN) treatment, revealing that DTMUV has evolved some strategies to defend against host IFN-dependent antiviral responses. Immune inhibition was further confirmed by screening for DTMUV-encoded proteins, which suggested that NS2A significantly inhibited IFN-β and IFN-stimulated response element (ISRE) promoter activity in a dose-dependent manner and facilitated reinfection with duck plague virus (DPV). DTMUV NS2A was able to inhibit duck retinoic acid-inducible gene-I (RIG-I)-, and melanoma differentiation-associated gene 5 (MDA5)-, mitochondrial-localized adaptor molecules (MAVS)-, stimulator of interferon genes (STING)-, and TANK-binding kinase 1 (TBK1)-induced IFN-β transcription, but not duck TBK1- and interferon regulatory factor 7 (IRF7)-mediated effective phases of IFN response. Furthermore, we found that NS2A competed with duTBK1 in binding to duck STING (duSTING), impaired duSTING-duSTING binding, and reduced duTBK1 phosphorylation, leading to the subsequent inhibition of IFN production. Importantly, we first identified that the W164A, Y167A, and S361A mutations in duSTING significantly impaired the NS2A-duSTING interaction, which is important for NS2A-induced IFN-β inhibition. Hence, our data demonstrated that DTMUV NS2A disrupts duSTING-dependent antiviral cellular defenses by binding with duSTING, which provides a novel mechanism by which DTMUV subverts host innate immune responses. The potential interaction sites between NS2A and duSTING may be the targets of future novel antiviral therapies and vaccine development.IMPORTANCE Flavivirus infections are transmitted through mosquitos or ticks and lead to significant morbidity and mortality worldwide with a spectrum of manifestations. Infection with an emerging flavivirus, DTMUV, manifests with clinical symptoms that include lesions of the immune organs and neurological dysfunction, leading to heavy egg drop and causing serious harm to the duck industry in China, Thailand, Malaysia, and other Southeast Asian countries. Mosquito cells, bird cells, and mammalian cell lines are all susceptible to DTMUV infection. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and may pose a threat to mammalian health. However, the pathogenesis of DTMUV is largely unclear. Our results show that NS2A strongly blocks the STING-induced signal transduction cascade by binding with STING, which subsequently blocks STING-STING binding and TBK1 phosphorylation. More importantly, the W164, Y167, or S361 residues in duSTING were identified as important interaction sites between STING and NS2A that are vital for NS2A-induced IFN production and effective phases of IFN response. Uncovering the mechanism by which DTMUV NS2A inhibits IFN in the cells of its natural hosts, ducks, will help us understand the role of NS2A in DTMUV pathogenicity.

Discovery of a Silicon-Containing Pan-Genotype Hepatitis C Virus NS5A Inhibitor.

We describe a study leading to the discovery of compound 11, a pan-genotypic hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor with excellent potency, metabolic stability, and pharmacokinetics (PK). Compound 11 incorporating a 4-silapiperidine group was discovered by further optimizing our previous lead with a triethylsilyl moiety. It displayed great potency against genotype 1 subtype a (GT1a), -1b, -2a, -3a, -4a, -5a, and -6a with an EC50 range of 0.33-17 pM and improved potency against the resistance-associated variant GT1a_M28T. Pharmacokinetics (PK) study indicated that compound 11 has reasonable PK exposures with a high liver distribution in preclinical animal species (mouse, rat, and dog). The results of a 14 day repeat-dose toxicity study identified the safety of compound 11.

Hepatitis C Virus NAT-Positive Solid Organ Allografts Transplanted Into Hepatitis C Virus-Negative Recipients: A Real-World Experience.

Hepatitis C virus (HCV)-viremic organs are underutilized, and there is limited real-world experience on the transplantation of HCV-viremic solid organs into recipients who are HCV negative. Patients listed or being evaluated for solid organ transplant after January 26, 2018, were educated and consented by protocol on the transplantation of HCV-viremic organs. All recipients were HCV nucleic acid test and anti-HCV antibody negative at the time of transplant and received an HCV-viremic organ. The primary outcome was sustained virological response (SVR) at 12 weeks after completion of direct-acting antiviral (DAA) therapy (SVR12 ). Seventy-seven patients who were HCV negative underwent solid organ transplantation from a donor who was HCV viremic. No patients had evidence of advanced hepatic fibrosis. Treatment regimen and duration were at the discretion of the hepatologist. Sixty-four patients underwent kidney transplant (KT), and 58 KT recipients had either started or completed DAA therapy. Forty-one achieved SVR12 , 10 had undetectable viral loads but are not eligible for SVR12 , and 7 remain on treatment. One KT recipient was a nonresponder because of nonstructural protein 5A resistance. Four patients underwent liver transplant and 2 underwent liver-kidney transplant. Three patients achieved SVR12 , 1 has completed DAA therapy, and 2 remain on treatment. Six patients underwent heart transplant and 1 underwent heart-kidney transplant. Six patients achieved SVR12 and 1 patient remains on treatment. Limited data exist on the transplantation of HCV-viremic organs into recipients who are HCV negative. Our study is the largest to describe a real-world experience of the transplantation of HCV-viremic organs into recipients who are aviremic. In carefully selected patients, the use of HCV-viremic grafts in the DAA era appears to be efficacious and well tolerated.

Elbasvir/Grazoprevir for HCV Infection in Russia: A Randomized Trial.

PurposeHepatitis C virus (HCV) infection is a major healthcare concern in Russia, where almost 5 million individuals are viremic. Elbasvir/grazoprevir is a fixed-dose combination therapy for the treatment of HCV genotype 1 and genotype 4 infection. The present analysis aimed to assess the safety and efficacy of elbasvir/grazoprevir in individuals with HCV infection enrolled at Russian study sites in the C-CORAL study.Patients and MethodsC-CORAL (Protocol PN-5172-067; NCT02251990) was a Phase 3, placebo-controlled, double-blind study conducted throughout Asia and Russia. Treatment-naive participants with chronic HCV infection were randomly assigned to receive immediate or deferred treatment with elbasvir 50 mg/grazoprevir 100 mg once daily for 12 weeks. Participants in the immediate-treatment group received elbasvir/grazoprevir for 12 weeks, and those in the deferred-treatment group received placebo for 12 weeks, followed by open-label elbasvir/grazoprevir for 12 weeks. The primary endpoint was sustained virologic response at 12 weeks after completion of therapy (SVR12).ResultsOne hundred and nineteen Russian participants were randomized (immediate-treatment group, n=88; deferred-treatment group, n=31). Most participants were white (99%) with HCV genotype 1b infection (97%) and mild-to-moderate (F0–F2) fibrosis (70%). SVR12 was achieved by 98.9% participants in the immediate-treatment group and by 100% of those receiving deferred elbasvir/grazoprevir in the deferred-treatment group. One participant relapsed with nonstructural protein 5A (NS5A) L28M and Y93H resistance-associated substitutions at baseline and at time of failure. Drug-related adverse events were reported by 19% of participants receiving elbasvir/grazoprevir in the immediate-treatment group and by 16% of those receiving placebo in the deferred-treatment group. No serious adverse event or deaths occurred, and no participant discontinued treatment owing to an adverse event.ConclusionElbasvir/grazoprevir for 12 weeks was highly effective in treatment-naive Russian individuals with HCV genotype 1b infection.

GSK2818713, a Novel Biphenylene Scaffold-Based Hepatitis C NS5A Replication Complex Inhibitor with Broad Genotype Coverage.

Pan-genotype NS5A inhibitors underpin hugely successful hepatitis C virus (HCV) therapy. The discovery of GSK2818713 (13), a nonstructural protein 5A (NS5A) HCV inhibitor characterized by a significantly improved genotype coverage relative to first-generation NS5A inhibitor daclatasvir (DCV), is detailed herein. The SAR analysis revealed cooperative potency effects of the biphenylene, bicyclic pyrrolidine (Aoc), and methyl-threonine structural motifs. Relative to DCV, 13 improved activity against genotype 1a (gt1a) and gt1b NS5A variants as well as HCV chimeric replicons containing NS5A fragments from genotypes 2-6. Long-term treatment of subgenomic replicons with 13 potently and durably decreased HCV RNA levels for gt1a, gt2a, and gt3a. These properties, suitable pharmacokinetics, and the lack of cross-resistance resulted in the selection of 13 as a preclinical candidate.

Real-world effectiveness and safety of sofosbuvir and nonstructural protein 5A inhibitors for chronic hepatitis C genotype 1, 2, 3, 4, or 6: a multicentre cohort study

BackgroundWe investigated real-world effectiveness and safety of sofosbuvir and the nonstructural protein 5A inhibitors in the treatment of patients infected with hepatitis C virus (HCV) genotypes 1, 2, 3, 4, or 6.MethodsWe analyzed data from 1021 patients with HCV infection (506 with genotype 1; 16 with genotype 2; 314 with genotype 3; 13 with genotype 4; 166 with genotype 6) who received 12 to 24 weeks of daclatasvir plus sofosbuvir (n = 767), ledipasvir/sofosbuvir (n = 197), or sofosbuvir/velpatasvir (n = 57), with or without ribavirin in 12 centers across Thailand to estimate sustained virologic response at post-treatment week 12 (SVR12).ResultsOverall, SVR12 rate was 98.0% (95% confidence interval [CI], 96.7–98.8%) with daclatasvir plus sofosbuvir, 97.9% (95% CI, 94.8–99.2%) with ledipasvir/sofosbuvir, and 96.5% (95% CI, 88.1–99.0%) with sofosbuvir/velpatasvir. SVR12 was achieved by 99.2% (95% CI, 97.9–99.7%) of subjects with genotype 1 infection, 100% (95% CI, 78.5–100%) of those with genotype 2 infection, 96.7% (95% CI, 94.0–98.2%) of those with genotype 3 infection, 90.9% (95% CI, 62.3–98.4%) of those with genotype 4 infection, and 96.7% (95% CI 92.5–98.6%) of those with genotype 6 infection. Patients with advanced liver disease were at risk of treatment failure. Only four patients discontinued treatment before week 4 due to non-hepatic adverse events.ConclusionsIn this large cohort of patients with various HCV genotypes managed in the real-world practice setting, daclatasvir plus sofosbuvir, ledipasvir/sofosbuvir, and sofosbuvir/velpatasvir achieved high SVR rates with good safety profile, comparable to those observed in clinical trials.

Retreatment of chronic hepatitis C virus genotype-4 patients after non-structural protein 5A inhibitors' failure: efficacy and safety of different regimens.

Nonstructural protein 5A (NS5A) is an important regimen for the treatment of chronic hepatitis C virus (HCV) genotype-4 infected patients. Retreatments for NS5A virologic failure are limited. The aim of this study is to provide real-life data regarding the effectiveness and safety of retreatment with different regimens after NS5A regimen virologic failure in GT4 patients. A total of 524 HCV patients (mean age 48 ± 11 years, 71% males), with virologic failure to sofosbuvir+daclatasvir, n = 450 and sofosbuvir/ledipasvir, n = 74 were included in this study. Patients were retreated with sofosbuvir + ombitasvir/paritaprevir/ritonavir + ribavirin, n = 278 and sofosbuvir + simeprevir + daclatasvir + ribavirin, n = 246. Response was evaluated 12 weeks after the end of treatment (SVR12). Overall, SVR12 was 95.2% [95% confidence interval (CI) 93.3%-97.1%]. In sofosbuvir + ombitasvir/paritaprevir/ritonavir + ribavirin and sofosbuvir + simeprevir + daclatasvir + ribavirin, SVR12s were 94.9% (95% CI 92.5%-97.4%) and 95.5% (95% CI 92.8%-98%), respectively. In liver cirrhosis patients, SVR12s were 96.4% (95% CI 90.7%-100%) and 98% (95% CI 94.9%-100%), respectively. Relapse in the sofosbuvir + ombitasvir/paritaprevir/ritonavir + ribavirin was n = 14 patients, and n = 11 patients in sofosbuvir + simeprevir + daclatasvir + ribavirin. Three patients developed hepatic encephalopathy, haematemesis, lower limb oedema, and one patient died in the SOF + OBV/PTV/RTV + RIB. In the sofosbuvir + simeprevir + daclatasvir + ribavirin, three patients developed hepatocellular carcinoma and one patient died. No treatment discontinuation due to anaemia. Salvage treatment for NS5A-treatment failure is effective and well tolerated in genotype-4 patients, in both noncirrhotic and compensated cirrhotic groups.

Interactions between Viperin, Vesicle-Associated Membrane Protein A, and Hepatitis C Virus Protein NS5A Modulate Viperin Activity and NS5A Degradation.

The radical SAM enzyme, viperin, exerts a wide range of antiviral effects through both the synthesis of the antiviral nucleotide 3'-deoxy-3',4'-didehydro-CTP (ddhCTP) and through its interactions with various cellular and viral proteins. Here we investigate the interaction of viperin with hepatitis C virus nonstructural protein 5A (NS5A) and the host sterol regulatory protein, vesicle-associated membrane protein A (VAP-33). NS5A and VAP-33 form part of the viral replication complex that is essential for replicating the RNA genome of the hepatitis C virus. Using transfected enzymes in HEK293T cells, we show that viperin binds independently to both NS5A and the C-terminal domain of VAP-33 (VAP-33C) and that this interaction is dependent on the proteins being colocalized to the ER membrane. Coexpression of VAP-33C and NS5A resulted in changes to the catalytic activity of viperin that depended upon viperin being colocalized to the ER membrane. The viperin-NS5A-VAP-33C complex exhibited the lowest specific activity, indicating that NS5A may inhibit viperin's ability to synthesize ddhCTP. Coexpression of viperin with NS5A was also found to significantly reduce cellular NS5A levels, most likely by increasing the rate of proteasomal degradation. An inactive mutant of viperin, unable to bind the iron-sulfur cluster, was similarly effective at reducing cellular NS5A levels.

Antibody Responses against Enterovirus Proteases are Potential Markers for an Acute Infection.

Background: Enteroviruses are a group of common non-enveloped RNA viruses that cause symptoms ranging from mild respiratory infections to paralysis. Due to the abundance of enterovirus infections it is hard to distinguish between on-going and previous infections using immunological assays unless the IgM fraction is studied. Methods: In this study we show using Indirect ELISA and capture IgM ELISA that an IgG antibody response against the nonstructural enteroviral proteins 2A and 3C can be used to distinguish between IgM positive (n = 22) and IgM negative (n = 20) human patients with 83% accuracy and a diagnostic odds ratio of 30. Using a mouse model, we establish that the antibody response to the proteases is short-lived compared to the antibody response to the structural proteins in. As such, the protease antibody response serves as a potential marker for an acute infection. Conclusions: Antibody responses against enterovirus proteases are shorter-lived than against structural proteins and can differentiate between IgM positive and negative patients, and therefore they are a potential marker for acute infections.

The prokaryotic expression, polyclonal antibody preparation, and subcellular localization of the transmembrane protein NS2A of the duck Tembusu virus

Duck Tembusu virus (DTMUV) is a single-stranded, positive-sense RNA arbovirus, belonging to the genus Flavivirus, the family Flaviviridae. As a transmembrane protein, non-structural protein 2A (NS2A) plays an important role in virion assembly, replication complex and antagonizing host immune response. Since NS2A protein contains many hydrophobicaminoacids, it is hard to gain the full-length protein of NS2A for prokaryotic expression. Therefore, to make a deep study, prokaryotic expression and polyclonal antibody preparation of truncated DTMUV NS2A was performed. The truncated NS2A gene (178-450 bp) was obtained, and sub-cloned into the prokaryotic vector pGEX-4T-1 (pGEX-4T-1-NS2A178-450bp). Subsequently, the recombinant GST-NS2A60-150aa protein was successfully expressed in E. coli BL21 (DE3) with the induction by 0.3 mmol/l isopropyl β-D-thiogalactoside (IPTG) for 6 h at 37°C. The GST-NS2A60-150aa protein was extracted from the gel. The BALB/c mice were immunized with the purified recombinant NS2A protein to prepare polyclonal antibodies against the truncated NS2A protein. The titer of the polyclonal antibodies, determined by ELISA analysis, was 1:128,000. The specificity of the polyclonal antibodies (mPAb-DTMUV-NS2A60-150aa) were verified by Western blot analysis. Furthermore, the indirect immunofluorescence (IFA) was performed to explore the subcellular localization of NS2A. NS2A protein was, in the transfected cells, located mainly around nucleus in the endoplasmatic reticulum. Taken together, our study provided a useful tool for the further exploration of the biological functions and molecular mechanism of DTMUV NS2A. Keywords: duck Tembusu virus; non-structural protein 2A; prokaryotic expression; polyclonal antibodies; subcellular location.

The hepatitis C viral nonstructural protein 5A stabilizes growth-regulatory human transcripts.

The hepatitis C viral nonstructural protein 5A stabilizes growth-regulatory human transcripts.

Real world study of daclatavir combined with sofosbuvir treatment in chronic hepatitis C

Objective To investigate the efficacy and safety of daclatavir combined with sofosbuvir treatment in chronic hepatitis C (CHC) in the real world. Methods A total of 56 CHC patients administrated with daclatavir (60 mg/d) combined with sofosbuvir (400 mg/d) in Huzhou Central Hospital from February to June in 2018 were enrolled. All patients were administrated with daclatavir combined with sofosbuvir for 12 weeks and followed up for 24 weeks. The virological response and the effect of antiviral therapy on hepatic fibrosis were analyzed. Non-structural protein 5A (NS5A) region mutation sequence was detected by Sanger method. Safety and the adverse events were observed. The t test, chi-square test and Mann-Whitney U test were used to analyze the data. Results Hepatitis C virus (HCV) RNA of all patients treated with daclatavir and sofosbuvir was undectable after eight-week treatment. Sustained virological response at 12 weeks post-treatment (SVR12) was 98.1% (52/53). Gender, globulin, insulin, triglyceride and hemoglobin were correlated with virus clearance (χ2= 4.47, t=2.51, U=1.98, U=2.32 and t=2.03, respectively, all P 0.05). During the treatment, patients developed dizziness, fatigue, nausea and vomiting, panic, insomnia, sleepiness and sexual function enhancement. Conclusion Daclatavir in combination with sophobuvir shows high virological response and good safety in the treatment of chronic hepatitis C, and liver fibrosis is improved after clearance of HCV. Key words: Hepatitis C, chronic; Daclatasvir; Sofosbuvir; Real world study

TAF and TDF attenuate liver fibrosis through NS5ATP9, TGFβ1/Smad3, and NF-κB/NLRP3 inflammasome signaling pathways.

This study aimed to investigate the roles and mechanisms of tenofovir alafenamide fumarate (TAF)/tenofovir disoproxil fumarate (TDF) in treating liver fibrosis. The effects of TAF/TDF on carbon tetrachloride (CCl4)-induced liver fibrosis in C57BL/6 wild-type or nonstructural protein 5A transactivated protein 9 (NS5ATP9) knockout mice were studied. The differentiation, activation, and proliferation of LX-2 cells after TAF/TDF treatment were tested in vitro. The expression of NS5ATP9 and activities of transforming growth factor-β1 (TGFβ1)/Sekelsky mothers against decapentaplegic homolog 3 (Smad3) and NF-κB/NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome signaling pathways were detected in TAF/TDF-treated mice and LX-2 cells. The genes related to extracellular matrix accumulation were detected in vivo and in vitro after NS5ATP9 silencing or knockout. TAF/TDF significantly inhibited CCl4-induced liver fibrosis in mice, and regulated the differentiation, activation, and proliferation of hepatic stellate cells (HSCs). Furthermore, TAF/TDF suppressed the activities of TGFβ1/Smad3 and NF-κB/NLRP3 inflammasome signaling pathways in vivo and in vitro. NS5ATP9 inhibited liver fibrosis through TGFβ1/Smad3 and NF-κB signaling pathways. TAF/TDF upregulated the expression of NS5ATP9 in vivo and in vitro. Finally, TAF/TDF could only show marginal therapeutic effects when NS5ATP9 was silenced and knocked out in vivo and in vitro. TAF/TDF prevented progression and promoted reversion of liver fibrosis through assembling TGFβ1/Smad3 and NF-κB/NLRP3 inflammasome signaling pathways via upregulating the expression of NS5ATP9. TAF/TDF also regulated the differentiation, activation, and proliferation of HSCs. The findings provided strong evidence for the role of TAF/TDF as a new promising therapeutic strategy in liver fibrosis.