Kattareeya Kumthip

HIV and HCV Cooperatively Promote Hepatic Fibrogenesis via Induction of Reactive Oxygen Species and NFκB

HIV/HCV coinfection leads to accelerated hepatic fibrosis progression, with higher rates of cirrhosis, liver failure, and liver death than does HCV mono-infection. However, the profibrogenic role of HIV on hepatocytes and hepatic stellate cells (HSC) has not been fully clarified. We hypothesized that HIV, HCV induce liver fibrosis through altered regulation of the production of extracellular matrix and matrix metalloproteinases. We examined the fibrogenesis- and fibrolysis-related gene activity in LX2 HSC and Huh7.5.1 cells in the presence of inactivated CXCR4 and CCR5 HIV, as well as HCV JFH1 virus. The role of reactive oxygen species (ROS) upon fibrosis gene expression was assessed using the ROS inhibitor. Fibrosis-related transcripts including procollagen α1(I) (CoL1A), TIMP1, and MMP3 mRNA were measured by qPCR. TIMP1 and MMP3 protein expression were assessed by ELISA. We found that inactivated CXCR4 HIV and CCR5 HIV increased CoL1A, and TIMP1 expression in both HSC and Huh7.5.1 cells; the addition of JFH1 HCV further increased CoL1A and TIMP1 expression. CXCR4 HIV and CCR5 HIV induced ROS production in HSC and Huh7.5.1 cells which was further enhanced by JFH1 HCV. The ROS inhibitor DPI abrogated HIV-and HCV-induced CoL1A and TIMP1 expression. HIV and HCV-induced CoL1A and TIMP1 expression were also blocked by NFκB siRNA. Our data provide further evidence that HIV and HCV independently regulate hepatic fibrosis progression through the generation of ROS; this regulation occurs in an NFκB-dependent fashion. Strategies to limit the viral induction of oxidative stress are warranted to inhibit fibrogenesis.

Hepatitis C Virus NS5A Disrupts STAT1 Phosphorylation and Suppresses Type I Interferon Signaling

ABSTRACT Responses to alpha interferon (IFN-α)-based treatment are dependent on both host and viral factors and vary markedly among patients infected with different hepatitis C virus (HCV) genotypes (GTs). Patients infected with GT3 viruses consistently respond better to IFN treatment than do patients infected with GT1 viruses. The mechanisms underlying this difference are not well understood. In this study, we sought to determine the effects of HCV NS5A proteins from different genotypes on IFN signaling. We found that the overexpression of either GT1 or GT3 NS5A proteins significantly inhibited IFN-induced IFN-stimulated response element (ISRE) signaling, phosphorylated STAT1 (P-STAT1) levels, and IFN-stimulated gene (ISG) expression compared to controls. GT1 NS5A protein expression exhibited stronger inhibitory effects on IFN signaling than did GT3 NS5A protein expression. Furthermore, GT1 NS5A bound to STAT1 with a higher affinity than did GT3 NS5A. Domain mapping revealed that the C-terminal region of NS5A conferred these inhibitory effects on IFN signaling. The overexpression of HCV NS5A increased HCV replication levels in JFH1-infected cells through the further reduction of levels of P-STAT1, ISRE signaling, and downstream ISG responses. We demonstrated that the overexpression of GT1 NS5A proteins resulted in less IFN responsiveness than did the expression of GT3 NS5A proteins through stronger binding to STAT1. We confirmed that GT1 NS5A proteins exerted stronger IFN signaling inhibition than did GT3 NS5A proteins in an infectious recombinant JFH1 virus. The potent antiviral NS5A inhibitor BMS-790052 did not block NS5A-mediated IFN signaling suppression in an overexpression model, suggesting that NS5As contributions to replication are independent of its subversive action on IFN. We propose a model in which the binding of the C-terminal region of NS5A to STAT1 leads to decreased levels of P-STAT1, ISRE signaling, and ISG transcription and, ultimately, to preferential GT1 resistance to IFN treatment.

A functional genomic screen reveals novel host genes that mediate interferon-alpha’s effects against hepatitis C virus

BACKGROUND & AIMS The precise mechanisms by which IFN exerts its antiviral effect against HCV have not yet been elucidated. We sought to identify host genes that mediate the antiviral effect of IFN-α by conducting a whole-genome siRNA library screen. METHODS High throughput screening was performed using an HCV genotype 1b replicon, pRep-Feo. Those pools with replicate robust Z scores ≥2.0 entered secondary validation in full-length OR6 replicon cells. Huh7.5.1 cells infected with JFH1 were then used to validate the rescue efficacy of selected genes for HCV replication under IFN-α treatment. RESULTS We identified and confirmed 93 human genes involved in the IFN-α anti-HCV effect using a whole-genome siRNA library. Gene ontology analysis revealed that mRNA processing (23 genes, p=2.756e-22), translation initiation (nine genes, p=2.42e-6), and IFN signaling (five genes, p=1.00e-3) were the most enriched functional groups. Nine genes were components of U4/U6.U5 tri-snRNP. We confirmed that silencing squamous cell carcinoma antigen recognized by T cells (SART1), a specific factor of tri-snRNP, abrogates IFN-αs suppressive effects against HCV in both replicon cells and JFH1 infectious cells. We further found that SART1 was not IFN-α inducible, and its anti-HCV effector in the JFH1 infectious model was through regulation of interferon stimulated genes (ISGs) with or without IFN-α. CONCLUSIONS We identified 93 genes that mediate the anti-HCV effect of IFN-α through genome-wide siRNA screening; 23 and nine genes were involved in mRNA processing and translation initiation, respectively. These findings reveal an unexpected role for mRNA processing in generation of the antiviral state, and suggest a new avenue for therapeutic development in HCV.

Kinetic differences in the induction of interferon stimulated genes by interferon-α and interleukin 28B are altered by infection with hepatitis C virus

Several genome‐wide association studies (GWAS) have identified a genetic polymorphism associated with the gene locus for interleukin 28B (IL28B), a type III interferon (IFN), as a major predictor of clinical outcome in hepatitis C. Antiviral effects of the type III IFN family have previously been shown against several viruses, including hepatitis C virus (HCV), and resemble the function of type I IFN including utilization of the intracellular Janus kinase signal transducer and activator of transcription (JAK‐STAT) pathway. Effects unique to IL28B that would distinguish it from IFN‐α are not well defined. By analyzing the transcriptomes of primary human hepatocytes (PHH) treated with IFN‐α or IL28B, we sought to identify functional differences between IFN‐α and IL28B to better understand the roles of these cytokines in the innate immune response. Although our data did not reveal distinct gene signatures, we detected striking kinetic differences between IFN‐α and IL28B stimulation for interferon stimulated genes (ISGs). While gene induction was rapid and peaked at 8 hours of stimulation with IFN‐α in PHH, IL28B produced a slower, but more sustained increase in gene expression. We confirmed these findings in the human hepatoma cell line Huh7.5.1. Interestingly, in HCV‐infected cells the rapid response after stimulation with IFN‐α was blunted, and the induction pattern resembled that caused by IL28B. Conclusion: The kinetics of gene induction are fundamentally different for stimulations with either IFN‐α or IL28B in hepatocytes, suggesting distinct roles of these cytokines within the immune response. Furthermore, the observed differences are substantially altered by infection with HCV. (Hepatology 2014;59:1250‐1261)

Correlation between mutations in the core and NS5A genes of hepatitis C virus genotypes 1a, 1b, 3a, 3b, 6f and the response to pegylated interferon and ribavirin combination therapy.

Summary.  Several studies have reported correlation between mutations in core and NS5A proteins of hepatitis C virus (HCV) and response to interferon (IFN) therapy. In particular, mutations in NS5A protein have been shown to correlate with responsiveness to IFN treatment of HCV‐1b in Japanese patients. This study investigated whether amino acid (aa) mutations in the core and NS5A proteins of HCV‐1a, 1b, 3a, 3b and 6f correlated with the response to pegylated interferon (Peg‐IFN) plus ribavirin (RBV) therapy in Thai patients. The entire sequences of core and NS5A of HCV from 76 HCV‐infected patients were analysed in comparison with corresponding reference sequences. The data revealed that the number of aa mutations in full‐length NS5A, its C‐terminus, IFN sensitivity‐determining region, variable region 3 (V3) and V3 plus flanking region of HCV‐1b NS5A protein were significantly higher in responders than in the treatment failure group (P =0.010, 0.031, 0.046, 0.020 and 0.006, respectively). Similar results were found in a putative protein kinase R binding domain region in HCV‐6f NS5A protein (P =0.022). Moreover, specific aa substitutions in NS5A that appeared to be associated with responders or the treatment failure group were observed at positions 78 and 305 for HCV‐1b (P =0.028), 64 and 52 for HCV‐1a (P =0.033) and 6f (P =0.045). Nevertheless, analysis of aa sequences of core protein revealed highly conserved sequences among HCV genotypes and no significant differences between the viruses from responders and the treatment failure group. Our findings indicate that mutations in aa residues of NS5A of HCV‐1a, 1b and 6f correlated well with responsiveness to Peg‐IFN and RBV combination therapy.

HCV induces transforming growth factor β1 through activation of endoplasmic reticulum stress and the unfolded protein response.

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.

Molecular characterization of norovirus GII.17 detected in healthy adult, intussusception patient, and acute gastroenteritis children in Thailand.

Noroviruses (NoVs) have been recognized as a leading cause of sporadic cases and outbreaks of acute gastroenteritis in all age groups. During the surveillance of NoVs in Chiang Mai, Thailand, four cases of the novel GII.17 NoVs were sporadically detected by RT-PCR in 2014-2015. The first case of GII.17 was detected in a healthy adult who worked for a restaurant. The second case was found in a pediatric patient who admitted to the hospital with intussusception. The third and fourth cases were found in acute gastroenteritis children. Phylogenetic analysis clearly demonstrated that GII.17 NoVs detected in this study were genetically closely related with the novel GII.17 Kawasaki reference strains. These four GII.17 NoV positive specimens were also tested by two immunochromatographic test kits in order to evaluate the sensitivity for GII.17 NoV detection. The viral loads in those specimens were determined by real-time RT-PCR. The sensitivity of GII.17 NoV detection varies by individual test kits and also depending on the amount of the viruses contained in the fecal specimens. In summary, our study reported the detection of novel GII.17 NoVs in a wide range of subjects with and without diarrhea. Therefore, continued comprehensive screening and genetic molecular characterization of NoV strains circulating in this area need to be further investigated.

SOCS1 abrogates IFN’s antiviral effect on hepatitis C virus replication

Suppressor of cytokine signaling 1 (SOCS1) and suppressor of cytokine signaling 3 (SOCS3) have been thought to block type I interferon (IFN) signaling. We have previously reported that SOCS3 suppresses HCV replication in an mTOR-dependent manner. However, the relationship between SOCS1 and HCV replication remains unclear. Here, we found that overexpression of SOCS1 alone did not have an effect on HCV RNA replication. However, suppression of HCV replication by IFN-α was rescued by SOCS1 overexpression. The upregulation of HCV replication by SOCS1 overexpression in the presence of IFN is likely a result of the impairment of IFN signaling by SOCS1 and subsequent induction of ISGs. Knockdown of SOCS1 alone with specific shRNA enhanced the antiviral effect of IFN compared with negative control. Thus, SOCS1 acts as a suppressor of type I IFN function against HCV.

Wide variety of recombinant strains of norovirus GII in pediatric patients hospitalized with acute gastroenteritis in Thailand during 2005 to 2015

Norovirus (NoV) has been reported as being a common cause of acute gastroenteritis both in children and adults worldwide. Of the many variants, NoV GII.4 is the most predominant genotype. One of the mechanisms that drives the evolution and emergence of new variants of NoV is homologous recombination. This study describes the genetic recombination involved in cases of NoV GII detected in pediatric patients with acute gastroenteritis in Chiang Mai, Thailand during 2005 to 2015. From a total of 1938 stool samples, 3 (0.15%) were positive for NoV GI and 298 (15.38%) were identified as NoV GII. The genotypes detected in this study were GI.6, GI.14, GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.12, GII.13, GII.14, GII.15, GII.16, GII.17, GII.20, and GII.21. The NoV recombinant strains were verified by analysis of the partial sequence of ORF1 (RdRp)/ORF2 (capsid) junction. Phylogenetic analyses of partial ORF1 and ORF2 regions resulted in the identification of 21 (6.98%) NoV recombinant strains. Among these, 9 recombination patterns were detected in this study; GII.Pe/GII.4, GII.Pg/GII.1, GII.Pg/GII.12, GII.P7/GII.6, GII.P7/GII.14, GII.P12/GII.4, GII.P16/GII.2, GII.P16/GII.13, and GII.P21/GII.3. The findings demonstrated the wide variety of recombinant strains of NoV GII strains detected in pediatric patients admitted to the hospitals with acute gastroenteritis in Chiang Mai, Thailand during the past decade.

Detection and characterization of Aichi virus 1 in pediatric patients with diarrhea in Thailand

Kobuvirus is a newly discovered virus that belongs to the Kobuvirus genus in Picornaviridae family, which comprised of three species including Aichivirus A, Aichivirus B, and Aichivirus C. The kobuvirus isolated from human has been classified as Aichi virus 1 and belongs to Aichivirus A species. The present study aimed to assess the epidemiology and to perform molecular characterization of Aichi virus 1 in children admitted to hospitals with acute gastroenteritis in Chiang Mai, Thailand. A total of 923 fecal specimens collected from January, 2011 to December, 2013 were screened for the presence of Aichi virus 1 by RT semi‐nested PCR. Out of 923 fecal specimens tested, Aichi virus 1 was detected with the prevalence of 2.6% (24/923). Of these, 0.3% (3/923) was genotype A and 2.3% (21/923) were genotype B. It is interesting to note that the genotype A showed the nucleotide sequence closely related to the Aichi virus reference strain isolated from sewage in Tunisia, while genotype B was most closely related to other human Aichi virus B reference strains. The results suggest that Aichi virus 1 of both genotypes A and B are circulating in pediatric patients in Thailand. J. Med. Virol. 89:234–238, 2017.