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Background: Cytidine/uridine monophosphate kinase 2 (UMP-CMP kinase 2, CMPK2) has been reported as an antiviral interferon-stimulated gene (ISG). We previously observed that the expression of CMPK2 was significantly upregulated after Zika Virus (ZIKV) infection in A549 cells. However, the role of CMPK2 in ZIKV replication remains to be determined. Methods: CMPK2 or retinoic acid inducible gene I (RIG-I) was overexpressed by plasmid transfection or knocked down by siRNA, respectively in A549 cells before the cells were infected with ZIKV. ZIKV RNA and NS1 protein were detected by RT-qPCR and Western Blot, respectively. The activation status of Jak-STAT signaling pathway was determined by the evaluation of the phosphorylation level of STAT1 (p-STAT1, by Western Blot), interferon stimulated response element (ISRE) activity (by dual luciferase assay) and interferon stimulated gene (ISG) expression (by RT-qPCR). Results: We found that ZIKV infection induced CMPK2 expression dependent on RIG-I. Overexpression of CMPK2 inhibited while CMPK2 knockdown promoted ZIKV replication. Mechanically, we found that CMPK2 inhibited ZIKV replication probably through the activation of Jak/STAT signaling pathway as shown by the increased level of p-STAT1, enhanced activity of ISRE, and the up-regulated expression of some downstream ISGs. Conclusion: ZIKV infection induced CMPK2 expression, which activated the Jak/STAT signaling pathway to inhibit ZIKV replication. Keywords: CMPK2; ZIKV; Jak/STAT signaling pathway

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-β1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide–transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-β1–induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-β1 mRNA levels in sodium taurocholate cotransporting polypeptide–Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-β1–induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-β1–induced OCT4/Nanog-dependent pathway.

Objectives Shigella flexneri is prevalent worldwide and is the most common Shigella in many countries, causing highly contagious diarrhea which seriously threatens public health. We aimed to develop a colorimetric loop-mediated isothermal amplification (LAMP) for the rapid, accurate, and visualization detection of S. flexneri. Methods According to the screened specific genes of S. flexneri, we designed and evaluated 3 groups loop-mediated isothermal amplification (LAMP) primers, and the colorimetric LAMP reaction volume was optimized. The specificity of the colorimetric LAMP was validated by 20 S. flexneri and 96 non-S. flexneri clinical isolates. In addition, the sensitivity of the developed assay was evaluated by the serial ten-fold dilutions of plasmid DNA. Results We developed a colorimetric LAMP assay based on the specific gene hypothetical protein(Accssion: AE014073 Region: 4170556..4171068) of S. flexneri. The colorimetric LAMP method has good specificity for the detection of S. flexneri, which enables the detection of S. flexneri within 30 min, with the plasmids detection limit of 7*10° copies/μL. The results of amplification could be easily identified by color. Conclusions This colorimetric LAMP assay could be used for rapid and accurate diagnosis of S. flexneri infection, especially in remote hospitals and laboratories with under-equipped medical facilities, and in situations where an urgent diagnosis is needed.

Background: Dysregulated immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are thought to underlie the progression of coronavirus disease 2019 (COVID-19). We sought to further characterize host antiviral and cytokine gene expression in COVID-19 patients based on illness severity. Methods: In this case-control study, we retrospectively analyzed 46 recovered COVID-19 patients and 24 healthy subjects (no history of COVID-19) recruited from the Second People's Hospital of Fuyang City. Blood samples were collected from each study participant for RNA extraction and PCR. We assessed changes in antiviral gene expression between healthy controls and patients with mild/moderate (MM) and severe/critical (SC) disease. Results: We found that type I interferon signaling (IFNA2, TLR8, IFNA1, IFNAR1, TLR9, IRF7, ISG15, APOBEC3G, and MX1) and genes encoding proinflammatory cytokines (IL12B, IL15, IL6, IL12A and IL1B) and chemokines (CXCL9, CXCL11 and CXCL10) were upregulated in patients with MM and SC disease. Moreover, we found that IFNA1, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), and Fas-associated protein with death domain (FADD) were significantly downregulated (P < 0.05) in the SC group compared to the MM group. We also observed that microRNA (miR)-155 and miR-130a levels were markedly higher in the MM group compared to the SC group. Conclusion: COVID-19 is associated with the activation of host antiviral genes. Induction of the IFN system appears to be particularly important in controlling SARS-CoV-2 infection, as decreased expression of IFNA1, APOBEC3G and FADD genes in SC patients, relative to MM patients, may be associated with disease progression.

Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it has spread rapidly around the world and caused a serious global social crisis. During the epidemic, the blood collection and supply industry have been greatly impacted, due to the sharply dropped blood donors and transfusion transmission risk of SARS-CoV-2. Many infected individuals are asymptomatic and they may donate blood without awareness of the infection or before symptoms appear. In addition, viral RNAs have been detected in the blood of some patients infected with SARS-CoV-2. Although no infectious SARS-CoV-2 virus was found in the blood nor the blood components, there is a risk of transmission through blood transfusion which may endanger blood safety, especially during the pandemic period. This review briefly introduces the biological characteristics, epidemiology of SARS-CoV-2, with a particular focus on SARS-CoV-2 infection and blood safety.

BACKGROUND: Ran-specific binding protein 1 (RANBP1) is involved in the regulation of the cell cycle, while its role in hepatocellular carcinoma (HCC) is unknown. Therefore, we aimed to demonstrate the association of RANBP1 with clinicopathologic features and potential biological functions in HCC based on The Cancer Genome Atlas (TCGA) data. METHODS: We assessed RANBP1 expression and its correlation with clinicopathologic features and evaluated the prognostic value of RANBP1 with Kaplan-Meier survival analysis and the MethSurv database. Univariate and multivariate Cox regression analyses were conducted to elucidate the factors responsible for prognosis. The identification of a co-expression network and the analysis of related biological events with RANBP1 in HCC were assessed using LinkedOmics. Moreover, gene set enrichment analysis (GSEA) was employed to annotate the biological function of RANBP1. We also explored the correlation between RANBP1 and tumor immune infiltrates using a single sample GSEA (ssGSEA). RESULTS: The expression of RANBP1 was found significantly elevated in HCC and linked to advanced T stage and histopathological grade. Up-regulated RANBP1 expression was linked to poor prognosis. High DNA methylation levels of RANBP1 were significantly linked to very poor overall survival (OS). Co-expression network analysis revealed that RANBP1 was involved in ribosome, spliceosome, deoxyribonucleic acid (DNA) replication, ribonucleic acid (RNA) transport, and cell cycle. GSEA showed enrichment of G2M-checkpoint, Wingless and Int-1 (Wnt) cell signaling, and DNA repair in the RANBP1 high-expression phenotype. By using ssGSEA analysis, the increased RANBP1 expression was positively linked to the immune infiltration level of T helper cell type-1 (Th1) and negatively linked to the immune infiltration levels of T helper cell type-17 (Th17). CONCLUSIONS: Findings suggest that RANBP1 may play a pivotal role in HCC prognosis and can potentially serve as a candidate biosignature and as a therapeutic target for HCC.

Long noncoding RNAs (lncRNAs) play a critical role in the regulation of many important cellular processes. However, the mechanisms by which lncRNAs regulate viral infection and host immune responses are not well understood. We sought to explore lncRNA regulation of hepatitis C virus (HCV) infection and interferon response. We performed RNA sequencing (RNAseq) in Huh7.5.1 cells with or without interferon alpha (IFNα) treatment. Clustered regularly interspaced short palindromic repeats/Cas9 guide RNA (gRNA) was used to knock out selected genes. The promoter clones were constructed, and the activity of related interferon-stimulated genes (ISGs) were detected by the secrete-pair dual luminescence assay. We constructed the full-length and four deletion mutants of an interferon-induced lncRNA RP11-288L9.4 (lncRNA-IFI6) based on predicted secondary structure. Selected gene mRNAs and their proteins, together with HCV infection, in Huh7.5.1 cells and primary human hepatocytes (PHHs) were monitored by quantitative real-time PCR (qRT-PCR) and western blot. We obtained 7,901 lncRNAs from RNAseq. A total of 1,062 host-encoded lncRNAs were significantly differentially regulated by IFNα treatment. We found that lncRNA-IFI6 gRNA significantly inhibited HCV infection compared with negative gRNA control. The expression of the antiviral ISG IFI6 was significantly increased following lncRNA-IFI6 gRNA editing compared with negative gRNA control in Japanese fulminant hepatitis 1 (JFH1)-infected Huh7.5.1 cells and PHHs. We observed that lncRNA-IFI6 regulation of HCV was independent of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. lncRNA-IFI6 negatively regulated IFI6 promoter function through histone modification. Overexpression of the truncated spatial domain or full-length lncRNA-IFI6 inhibited IFI6 expression and increased HCV replication. Conclusion: A lncRNA, lncRNA-IFI6, regulates antiviral innate immunity in the JFH1 HCV infection model. lncRNA-IFI6 regulates HCV infection independently of the JAK-STAT pathway. lncRNA-IFI6 exerts its regulatory function via promoter activation and histone modification of IFI6 through its spatial domain.

Aim: TMEM2 has been demonstrated to suppress HBV infection by activating the Jak/STAT pathway. In this study, we sought to explore the mechanism by which TMEM2 effects on Zika virus (ZIKV) replication. Materials & methods: TMEM2 was overexpressed. Selected gene mRNA, p-STAT1 levels and interferon stimulated response element activity were examined by qRT-PCR, Western blot and luciferase assay respectively. Results: Overexpression of TMEM2 significantly inhibited ZIKV replication, upregulated MDA5 and RIG-I expression, increased IFN-β promoter activity and IFN-β expression. Overexpression of TMEM2 enhanced the Jak/STAT signaling including increased p-STAT1 level, ISRE activity as well as the expression of several antiviral interferon-stimulated genes. Conclusion: TMEM2 inhibited ZIKV replication through increased IFN-β production and enhanced activation of the Jak/STAT signaling pathway.

Previous studies demonstrated that dengue virus (DENV) infection developed resistance to type-I interferons (IFNα/β). The underlying mechanism remains unclear. USP18 is a negative regulator of IFNα/β signaling, and its expression level is significantly increased following DENV infection in cell lines and patients’ blood. Our previous study revealed that increased USP18 expression contributed to the IFN-α resistance of Hepatitis C Virus (HCV). However, the role of USP18 in DENV replication and resistance to IFN-α is elusive. In this current study, we aimed to explore the role of USP18 in DENV-2 replication and resistance to IFN-α. The level of USP18 was up-regulated by plasmid transfection and down-regulated by siRNA transfection in Hela cells. USP18, IFN-α, IFN-β expression, and DENV-2 replication were monitored by qRT-PCR and Western blot. The activation of the Jak/STAT signaling pathway was assessed at three levels: p-STAT1/p-STAT2 (Western blot), interferon-stimulated response element (ISRE) activity (Dual-luciferase assay), and interferon-stimulated genes (ISGs) expression (qRT-PCR). Our data showed that DENV-2 infection increased USP18 expression in Hela cells. USP18 overexpression promoted DENV-2 replication, while USP18 silence inhibited DENV-2 replication. Silence of USP18 potentiated the anti-DENV-2 activity of IFN-α through activation of the IFN-α-mediated Jak/STAT signaling pathway as shown by increased expression of p-STAT1/p-STAT2, enhanced ISRE activity, and elevated expression of some ISGs. Our data indicated that USP18 induced by DENV-2 infection is a critical host factor utilized by DENV-2 to confer antagonism on IFN-α.

Highlights • CircRNAs are formed by pre-mRNA through “back-splicing”. • CircRNAs regulate host immune response and virus replication. • CircRNAs have potential as diagnostic markers or treatment targets for viral infection., Circular RNAs (circRNAs) are a class of non-coding RNAs with a special covalently closed circular structure, which is formed by precursor mRNA (pre-mRNA) through “back-splicing”. CircRNAs are more stable than linear RNAs because they are resistant to exoribonucleases. Viral infections often cause abnormal expression of circRNAs, which could serve as novel biomarkers for the diagnosis of viral infections by detecting specific circRNAs in cells, body fluids, or tissues. CircRNAs also play a critical role in regulating host immune response and virus replication. Here, we reviewed the production and function of circRNAs, mainly focusing on their regulation on virus infection, to provide novel insights into the potential role of circRNAs as diagnostic marker or treatment targets for viral infection.

Globally, hepatitis B virus (HBV) infection and its related liver diseases account for 780,000 deaths every year. Outcomes of HBV infection depend on the interaction between the virus and host immune system. It is becoming increasingly apparent that Kupffer cells (KCs), the largest population of resident and monocyte-derived macrophages in the liver, contribute to HBV infection in various aspects. These cells play an important role not only in the anti-HBV immunity including virus recognition, cytokine production to directly inhibit viral replication and recruitment and activation of other immune cells involved in virus clearance but also in HBV outcome and progression, such as persistent infection and development of end-stage liver diseases. Since liver macrophages play multiple roles in HBV infection, they are directly targeted by HBV to benefit its life cycle. In the present review, we briefly outline the current advances of research of macrophages, especially the studies of their phenotypes, in chronic HBV infection.

Background: 2&rsquo, 5&rsquo, oligoadenylate synthetase 2 (OAS2) has been known as an antiviral interferon-stimulated gene (ISG). However, the role of OAS2 on Zika virus (ZIKV) replication is still unknown. In this study, we sought to explore the effect of OAS2 on ZIKV replication and its underlying mechanism. Methods: We performed RNA-Seq in A549 cells with or without ZIKV infection. OAS2 or RIG-I was overexpressed by plasmid transfection or knocked down by siRNA in A549 cells. Expression levels of mRNA and protein of selected genes were detected by RT-qPCR and Western Blot, respectively. Interferon stimulated response element (ISRE) activity was examined by dual luciferase assay. Results: We found that ZIKV infection induced OAS2 expression through a RIG-I-dependent pathway. OAS2 overexpression inhibited ZIKV replication, while OAS2 knockdown increased ZIKV replication. We observed that OAS2 inhibited ZIKV replication through enhanced IFN&beta, expression, leading to the activation of the Jak/STAT signaling pathway. Conclusion: ZIKV infection induced OAS2 expression, which in turn exerted its anti-ZIKV activities through the IFN-activated Jak/STAT signaling pathway.

Additional file 1: Supplemental Figure 1. Transfection efficiency in HepAD38 cells. HepAD38 cells were seeded at 3 × 105/ml, 2 ml per well in 6-well plates in antibiotic-free medium for 24 h before 1μg (A), 2μg (B), 4μg (C) GFP plasmid DNA or 4μg empty vector (D) was transfected into each well. Fluorescent microscopy images were taken 48 h post transfection. Supplemental Figure 2. USP18 knockdown in HepAD38 cells did not affect expression of HBV proteins. HepAD38 cells were transfected with the 20 nM siUSP18, 20 nM negative siRNA or left untreated, respectively. Forty-eight hours later, culture medium was collected to quantify HBsAg (A) and HBeAg (B) expression level by ELISA assay. Intracellular HBcAg was detected by western blot (C). Results are presented as means ± SD(n ≥ 3). Supplemental Figure 3. IFN and ISG expression in HepAD38. HepAD38 cells were grown in the medium with or without tetracycline (1 mg/ml) until confluent for 6 days. Intracellular RNA was then extracted. Expression of IFNα (A), IFNβ (B) and ISGs mRNA including MxA (C) and OAS2 (D) were detected by real-time PCR. Results are presented as means ± SD (n ≥ 3). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Supplemental Figure 4. Detection of mycoplasma in HepAD38 cells. HepAD38 cells were grown in the medium with or without tetracycline (1 mg/ml) until confluent. The supernatant was collected for detecting mycoplasma. NC, negative control; PC, positive control; Tet+, the complete HepAD38 medium with 1 mg/ml tetracycline; Tet-, the complete HepAD38 medium without tetracycline.

HCV chronically infects over 71 million people worldwide and is one of the leading causes of advanced liver diseases. Type I interferons (IFN-α/β) play critical role in host antiviral innate immunity. IFN-α/β exerts its anti-HCV effects through the activation of the JAK/STAT signaling pathway leading to the induction of a few hundred interferon-stimulated genes (ISGs). The interplay between ISG and HCV infection remains partially understood. In this review, we summarized the role of ISGs in HCV infection and interferon anti-HCV activity.

Background and Objectives With a prevalence of approximately 71 million infected individuals globally, chronic hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitis leading to hepatocellular carcinoma (HCC). This review aimed to summarize the general history of HCV and provide a perspective on future journey of HCV. Materials and Methods We searched for articles published in periodicals, monographs and edited books with the key words including ‘non-A, non-B hepatitis’, ‘HCV’, ‘interferon’ and ‘direct-antiviral agents (DAAs)’. And the data emphasized HCV emergence, identification, treatment, epidemiology as well as the cellular and animal models leading to the eradication of HCV infections, were summarized. Results and Conclusion The battle against hepatitis C is destined to be recorded in history as one of science's remarkable success. Although the revolutionary direct-antiviral agents (DAAs) are able to cure more than 95% of HCV patients, access to diagnosis and therapy remains improved. More efforts should be made to promote HCV screening, treatment delivery and vaccine development.

Ubiquitin-specific protease 18 (USP18) as a negative regulator of the Jak/STAT signaling pathway plays an important role in the host innate immune response. USP18 has been shown to bind to the type I interferon receptor subunit 2 (IFNAR2) to down-regulate the Jak/STAT signaling. In this study, we showed that insulin receptor substrate (IRS)-4 functioned as a novel USP18-binding protein. Co-precipitation assays revealed that two regions (amino acids 335-400 and 1094-1257) of IRS4 were related to bind to the C- terminal region of USP18. IRS4 binding to USP18 diminished the inhibitory effect of USP18 on Jak/STAT signaling. IRS4 over-expression enhanced while IRS4 knock-down suppressed the Jak/STAT signaling in the presence of IFN-a stimulation. As such, IRS4 increased IFN-a-mediated anti-HCV activity. Mechanistically, IRS4 promoted the IFN-a-induced Jak/STAT signaling by interact with USP18. These results suggested that IRS4 binds to USP18 to diminish the blunting effect of USP18 on IFN-a-induced Jak/STAT signaling. Our findings indicated that IRS4 is a novel USP18-binding protein that can be used to boost the host innate immunity to control HCV, and potentially other viruses that are sensitive to IFN-a.

Background Hepatitis B virus (HBV) infection remains as one of the major public health problems in the world. Type I interferon (IFN) plays an essential role in antiviral defense by induced expression of a few hundred interferon stimulated genes (ISGs), including ubiquitin-specific protease 18 (USP18). The expression level of USP18 was elevated in the pretreatment liver tissues of chronic hepatitis B(CHB) patients who did not respond to IFN treatment. Thus, this study was designed to investigate the effects of USP18 on HBV replication/production. Methods The levels of wild type USP18(WT-USP18) and USP18 catalytically inactive form C64S were up-regulated by plasmids transfection in HepAD38 cells, respectively. Real-time PCR and ELISA were used to quantify HBV replication. Type I IFN signaling pathway was monitored at three levels: p-STAT1 (western Blot), interferon stimulated response element (ISRE) activity (dual luciferase assay) and ISGs expression (real time PCR). Results Our data demonstrated that overexpression of either WT-USP18 or USP18-C64S inactive mutant increased the intracellular viral pgRNA, total DNA, cccDNA, as well as HBV DNA levels in the culture supernatant, while silencing USP18 led to opposite effect on HBV production. In addition, upregulated WT-USP18 or USP18-C64S suppressed ISRE activity and the expression levels of p-STAT1 and ISGs. Conclusion USP18 promoted HBV replication via inhibiting type I IFN signaling pathway, which was independent of its protease activity.

We previously identified that miR-130a downregulates HCV replication through two independent pathways: restoration of host immune responses and regulation of pyruvate metabolism. In this study, we further sought to explore host antiviral target genes regulated by miR-130a. We performed a RT&sup2, Profiler&trade, PCR array to identify the host antiviral genes regulated by miR-130a. The putative binding sites between miR-130a and its downregulated genes were predicted by miRanda. miR-130a and predicted target genes were over-expressed or knocked down by siRNA or CRISPR/Cas9 gRNA. Selected gene mRNAs and their proteins, together with HCV replication in JFH1 HCV-infected Huh7.5.1 cells were monitored by qRT-PCR and Western blot. We identified 32 genes that were significantly differentially expressed more than 1.5-fold following miR-130a overexpression, 28 of which were upregulated and 4 downregulated. We found that ATG5, a target gene for miR-130a, significantly upregulated HCV replication and downregulated interferon stimulated gene expression. miR-130a downregulated ATG5 expression and its conjugation complex with ATG12. ATG5 and ATG5-ATG12 complex affected interferon stimulated gene (ISG) such as MX1 and OAS3 expression and subsequently HCV replication. We concluded that miR-130a regulates host antiviral response and HCV replication through targeting ATG5 via the ATG5-dependent autophagy pathway.

Chronic hepatitis B virus infection continues to be a major health burden worldwide. It can cause various degrees of liver damage and is strongly associated with the development of liver cirrhosis and hepatocellular carcinoma. Covalently closed circular DNA in the nucleus of infected cells cannot be disabled by present therapies which may lead to HBV persistence and relapse. In this review, we summarized the current knowledge on hepatitis B virus covalently closed circular DNA and its potential role as a therapeutic target.

Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication. IMPORTANCE We identified that miR-130a regulates the target gene PKLR and its subsequent effect on pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized, miRNA-regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral life cycle and prevent establishment of persistent infection for HCV, HBV, and potentially other viral infections.

HCV infection is a major world health problem, leading to both end-stage liver disease and primary liver cancer. Great efforts have been made in developing new therapies for HCV infection; however, combination therapy with pegylated IFN-α and ribavirin (pegIFN-RBV) remains the first choice of treatment for chronic HCV infection in most countries. The treatment response to pegIFN-RBV remains relatively low. Understanding the molecular mechanisms of persistent HCV infection and pegIFN-RBV resistance will suggest ways of improving the current standard of care and offers new antiviral therapies for both HCV and other viral infections. Recent data suggest that increased expression of hepatic IFN-stimulated genes (ISGs) before treatment is associated with treatment nonresponse in patients chronically infected with HCV. Although ISGs are generally antiviral in nature, in the case of HCV, the virus may exploit some of them to its benefit. This is not unique to HCV: Blockade of type I IFN signaling has been shown to control persistent LCMV infection. Thus, in certain viral infections, preactivation or overactivation of type I IFN signaling may contribute to viral persistence. In this review, we briefly summarize the findings from high-throughput gene expression profiling from patients chronically infected with HCV, then focus on a novel ubiquitin-like signaling pathway (ISG15/USP18) and its potential role in HCV persistence. Finally, the role of activation of endogenous type I IFN signaling in persistent HCV infection will be discussed in the context of recent studies indicating that blocking IFN signaling controls persistent LCMV infection.

Hepatitis C virus (HCV) infection is a major global health problem. There is no effective vaccine and the current treatment regimen with pegylated interferon α and ribavirin is associated with significant adverse events. Therefore, there is an urgent need to identify new antiviral targets for HCV therapy. In recent years, a growing number of microRNAs (miRNAs) have been reported to be able to regulate HCV replication and infection by interacting with the HCV genome directly or by regulating host innate immunity to build a nonspecific antiviral state within cells. In this review, we discuss HCV virology and standard of care followed by miRNA in general, and then give a brief overview of miRNAs involved in HCV infection and discuss their potential application as a therapeutic option for the treatment of HCV infection.

All viral infections subvert the host immune response. Targeting the host mechanisms that are modulated by viral infection offers new avenues for antiviral drug development. Host ubiquitin and multiple ubiquitin-like modifiers (Ubls) are commonly altered by, or important for, viral infection. Protein modification by ubiquitin or Ubls contributes to numerous cellular processes, such as protein degradation, signal transduction, protein relocalization and pathogen-host interactions. This post-translational modification plays an essential role for viral life cycles and host antiviral mechanisms. Some Ubls, such as ISG15 and SUMO, have been shown to modulate virus infections and are potential targets for therapeutic manipulation. Hepatitis C virus (HCV) is a positive-stranded RNA virus that predominantly infects hepatocytes. Recent data suggest that ISG15 might be a potential drug target for anti-HCV therapy. Inhibition of ISG15 expression and/or ISG15 conjugation (ISGylation) provides a rationale for the design of new anti-HCV drugs.

HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1 and PERK. HCV increases TGF-β1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-β1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2 and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-β1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-β1 expression. ROS, ER stress, NFκB and TGF-β1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-β1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-β1 activation. We conclude that HCV activates NFκB and TGF-β1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-β1.

Hepatitis B virus (HBV) is an important account of infectious hepatitis and interferon (IFN) remains one of the best treatment options. Activation of type I IFN signaling pathway leads to expressions of IFN-stimulated genes (ISGs) which play important roles in antiviral and immunomodulatory responses to HBV or hepatitis C virus (HCV) infection. Our previous studies indicated that ISG15 and its conjugation (ISGylation) were exploited by HCV to benefit its replication and persistent infection. This study was designed to assess the role of ISG15 and ISGylation in HBV infectionin vitro. The levels of ISG15 and ISGylation were upregulated by ISG15 plasmid transfection into HepG2.2.15 cells. Decreased ISGylation was achieved by siRNA targeting UBE1L, the only E1 activating enzyme for ISGylation. Overexpression of ISG15 and subsequent ISGylation significantly increased the levels of HBV DNA in the culture supernatants although the intracellular viral replication remained unaffected. Silencing UBE1L, with decreased ISGylation achieved, abrogated this ISGylation-mediated promoting effect. Our data indicated that overexpression of ISG15 stimulated HBV production in an ISGylation-dependent manner. Identification of ISG15-conjugated proteins (either HBV viral or host proteins) may reveal promising candidates for further antiviral drug development.

Calcitriol, the bioactive metabolite of vitamin D, was reported to inhibit HCV production in a synergistic fashion with interferon, a treatmentin vitro. Our previous study established that miR-130a inhibits HCV replication by restoring the host innate immune response. We aimed to determine whether there is additive inhibitory effect of calcitriol and miR-130a on HCV replication. Here we showed that calcitriol potentiates the anti-HCV effect of miR-130a in both Con1b replicon and J6/JFH1 culture systems. Intriguingly, this potentiating effect of calcitriol on miR-130a was not through upregulating the expression of cellular miR-130a or through increasing the miR-130a-mediated IFNα/βproduction. All these findings may contribute to the development of novel anti-HCV therapeutic strategies although the antiviral mechanism needs to be further investigated.

HCV infection is a major world health problem, leading to both end-stage liver disease and primary liver cancer. Great efforts have been made in developing new therapies for HCV infection; however, combination therapy with pegylated IFN-α and ribavirin (pegIFN-RBV) remains the first choice of treatment for chronic HCV infection in most countries. The treatment response to pegIFN-RBV remains relatively low. Understanding the molecular mechanisms of persistent HCV infection and pegIFN-RBV resistance will suggest ways of improving the current standard of care and offers new antiviral therapies for both HCV and other viral infections. Recent data suggest that increased expression of hepatic IFN-stimulated genes (ISGs) before treatment is associated with treatment nonresponse in patients chronically infected with HCV. Although ISGs are generally antiviral in nature, in the case of HCV, the virus may exploit some of them to its benefit. This is not unique to HCV: Blockade of type I IFN signaling has been shown to control persistent LCMV infection. Thus, in certain viral infections, preactivation or overactivation of type I IFN signaling may contribute to viral persistence. In this review, we briefly summarize the findings from high-throughput gene expression profiling from patients chronically infected with HCV, then focus on a novel ubiquitin-like signaling pathway (ISG15/USP18) and its potential role in HCV persistence. Finally, the role of activation of endogenous type I IFN signaling in persistent HCV infection will be discussed in the context of recent studies indicating that blocking IFN signaling controls persistent LCMV infection.