Role In Hepatitis C Virus Infection Research Articles

KIR2DL4/HLA-G polymorphisms were associated with HCV infection susceptibility among Chinese high-risk population.

Killer-cell immunoglobulin-like receptors 2DL4 (KIR2DL4) and the human leukocyte antigen class I-G (HLA-G) display vital parts in immune responses against hepatitis C virus(HCV) infection. We select four potentially functional single nucleotide polymorphisms(SNPs) of KIR/HLA to explore the associations between KIR2DL4/HLA-G genetic variants and HCV infection results.In the present case-control study, a total of 2225 HCV-infected high-risk subjects, including 1778 paid blood donors (PBD) and 447 drug users were consecutively recruited before treatment from 2011 to 2018. KIR2DL4-rs660773, KIR2DL4-rs660437, HLA-G-rs9380142, and HLA-G-rs1707 SNPs were sorted as genotypes in the subdivided groups, involving 1095 uninfected controls subjects, 432 spontaneous HCV clearance subjects and 698 HCV persistent infection subjects. After genotyping experiments using the TaqMan-MGBassay, modified logistic regression was used to calculate the correlation among the SNPs and HCV infection. The SNPs were functionally annotated using bioinformatics analysis.Following adjusting by age, sex, alanine aminotransferase, aspartate aminotransferase, IFNL3-rs12979860, IFNL3-rs8099917, and the infection route, the logistic regression analysisdiscovered that KIR2DL4-rs660773 and HLA-G-rs9380142 were correlated with vulnerability to HCV infection (all p < 0.05). In a locus-dosage way, compared with subjects carrying the rs9380142-AA or rs660773-AA genotypes, subjects with rs9380142-AG or rs660773-AG/GG (all p < 0.05) were more vulnerable to HCV infection; the overall impact of their risk genotypes (rs9380142-AGrs660773-AG/GG) was correlated with an elevated incidence of HCV infection (ptrend < 0.001). In the Haplotype analysis, patients with haplotype AG were more likely to contract HCV compared to those with the highest common AA haplotype (p = 0.002) were higher in susceptibility to infect HCV. The SNPinfo web server estimated that rs660773 is a transcription factor binding site, whereas rs9380142 is a potential microRNA-binding site.In two Chinese high-risk population (PBD and drug uesrs), KIR2DL4 rs660773-G and HLA-G rs9380142-G alleles polymorphisms are related to HCV susceptibility. KIR2DL4/HLA-G pathway genes might affect the innate immune responses by regulating KIR2DL4/HLA-G transcription and translation play a potential role in HCV infection.

Association between hepatitis C virus infections and Toll-Like Receptor 3 Gene polymorphism, Erbil city

Introduction: Globally, hepatitis C virus (HCV) has infected about 180 million people by 2019. The World Health Organization (WHO) reports that 71 million people worldwide are infected with chronic HCV. HCV is considered as a main public health problem globally, and recently it has been observed that the prevalence of this virus is declining but still populations with asymptomatic chronic HCV exists. The main objective of the present study was to determine the effects of TLR3 gene single nucleotide polymorphism (SNP) on the susceptibility of HCV infections in Erbil city of Iraq. Materials and Methods: For this purpose, samples were collected from many private hospitals, which included 50 samples from patients with HCV and 10 samples as controls from healthy people. The sample followed molecular techniques for determining toll-like receptor 3 (TLR3) SNP. Finally, the results were analyzed using Chromas-Pro software. Results: The results of the present study showed that both age and gender have a crucial role in HCV infection. About 66% of the HCV patients were males and 64% of patients were above 40 years. Conclusion: The results concluded that there was a significant relation between TLR 3 SNP (rs78726532 polymorphism) and the risk of HCV infection in population of Erbil city.

The Relevance of MicroRNAs in the Pathogenesis and Prognosis of HCV-Disease: The Emergent Role of miR-17-92 in Cryoglobulinemic Vasculitis.

Hepatitis C virus (HCV) is a major public health problem. HCV is a hepatotropic and lymphotropic virus that leads to hepatocellular carcinoma (HCC) and lymphoproliferative disorders such as cryoglobulinemic vasculitis (CV) and non-Hodgkin’s lymphoma (NHL). The molecular mechanisms by which HCV induces these diseases are not fully understood. MicroRNAs (miRNAs) are small non-coding molecules that negatively regulate post-transcriptional gene expression by decreasing their target gene expression. We will attempt to summarize the current knowledge on the role of miRNAs in the HCV life cycle, HCV-related HCC, and lymphoproliferative disorders, focusing on both the functional effects of their deregulation as well as on their putative role as biomarkers, based on association analyses. We will also provide original new data regarding the miR 17-92 cluster in chronically infected HCV patients with and without lymphoproliferative disorders who underwent antiviral therapy. All of the cluster members were significantly upregulated in CV patients compared to patients without CV and significantly decreased in those who achieved vasculitis clinical remission after viral eradication. To conclude, miRNAs play an important role in HCV infection and related oncogenic processes, but their molecular pathways are not completely clear. In some cases, they may be potential therapeutic targets or non-invasive biomarkers of tumor progression.

Hepatitis C virus alternative reading frame protein (ARFP): Production, features, and pathogenesis.

Earlier observation suggests that hepatitis C virus (HCV) is a single-stranded RNA virus which encodes at least 10 viral proteins. F protein is a novel protein which has been discovered recently. These studies suggest three mechanisms for the production of this protein concerning ribosomal frameshift at codon 10, initial translation at codons 26 and 85 or 87. In this study, the association between protein F and chronicity of hepatocellular carcinoma (HCC) has been reviewed. Evidence suggests that humoral immune system can recognize this protein and produce antibodies against it. By detecting antibodies in infected people, investigators found that F protein might have a role in HCV infection causing chronic cirrhosis and HCC as higher prevalence was found in patients with mentioned complications. The increment of CD4+, CD25+, and FoxP3+ T cells, along with CD8+ T cells with low expression of granzyme B, also leads to weaker responses of the immune system which helps the infection to become chronic. Moreover, it contributes to the survival of the virus in the body through affecting the production of interferon. F protein also might play roles in the disease development, resulting in HCC. The existence of F protein affects cellular pathways through upregulating p53, c-myc, cyclin D1, and phosphorylating Rb. This review will summarize these effects on immune system and related mechanisms in cellular pathways.

STAT3 signaling pathway plays importantly genetic and functional roles in HCV infection.

BackgroundHepatitis C virus (HCV) infection is an extensive health problem, which leads to serious liver diseases. Host genetic polymorphisms were associated with HCV infection, progression, and treatment effect of patients. Signal transducers and activators of transcription 3 (STAT3) signaling pathway was important to HCV infection, but no genetic association was studied between STAT3 signaling pathway and HCV infection.MethodsTo investigate the genetic and functional roles of the STAT3 signaling pathway, we collected 394 HCV patients and 395 normal controls to genotype 25 signal nucleotide polymorphisms (SNPs) of six genes (Interleukin 6 [IL6, OMIM 147620], Interleukin 6 receptor [IL6R, OMIM 147880], Hepatocyte nuclear factor 1 alpha [HNF1A, OMIM 142410], Hepatocyte nuclear factor 4 alpha [HNF4A, OMIM 600281], STAT3 [OMIM 102582], and ATP binding cassette subfamily C member 2 [ABCC2, OMIM 601107]). Then expression level of these genes were analyzed in HCV infected cells with or without IL6 transfection.ResultsOur results identified that the SNPs in STAT3 signaling pathway were associated with HCV infection or biochemical features of Yunnan HCV patients. Genotype AA of rs4075015 (IL6R) and GG of rs3212172 (HNF4A) increased the risk of HCV infection (p = 0.024 and 0.029), but the genotype AA of rs7553796 (IL6R) played a protective role in HCV infection (p = 0.0008). High‐density lipoprotein cholesterol (HDL‐C) and Glutamyl transpeptidase (GGT) level were associated with genotypes of rs4845617 (IL6R, p = 0.045) and rs1053023 (STAT3, p = 0.034), respectively. Cell assays suggested that IL6 transfection could suppress HCV proliferation. RNA and protein levels of the IL6R, HNF1A, STAT3, and ABCC2 genes significantly increased after HCV infection.ConclusionWe identified STAT3 signaling pathway influenced HCV infection and biochemical characteristics of HCV patients through genetic and functional aspects.

PB1832 MATRIX CHEMOTHERAPY FOR PRIMARY CNS LYMHPOMA COMPLICATED WITH HCV INFECTION AND SUCCESSFUL ERADICATION OF BOTH DISEASES

Background:There is a well‐known connection between Hepatitis C virus (HCV) infection and B‐cell lymphomas (BCL). HCV infected patients have an increased risk of developing BCL. Hypothetically, lymphomagenesis can be induced by chronic antigen stimulation (analogous to Helicobacter pylori associated gastric marginal zone lymphoma) and/or a direct pro‐oncogenic effect of intracellular HCV proteins. Several studies have demonstrated that antiviral therapy may induce remission and improve prognosis of HCV‐associated lymphomas. On the other hand, HCV infection can cause sever complication during the immunochemotherapy and stem cell transplantation. These patients are at risk for HCV reactivation, accelerated liver disease progression, drug‐induced liver toxicity. Thus, HCV‐infected patients often suffer treatment delays, reduced dose intensity, and many times are excluded from bone marrow transplantation. Accordingly, there is some evidence of negative prognostic impact of HCV infection on survival.Aims:To draw attention to the HCV caused complications during lymphoma treatment.Methods:Here we present the case of a HCV‐infected patient with primary central nervous system (CNS) lymphoma complicated with HCV infection and review the relevant literature.Results:A 56‐year old woman with CNS lymphoma was treated with MATRIX (methotrexate, cytarabine, thiotepa, and rituximab). Initial serum serology showed reactivity to HBcAg and HCV. Polymerase chain reaction (PCR) detected no hepatits B virus DNA, however the copy number for hepatitis C virus (HCV) RNA was 104 850 000 IU/ml. She received HBV prophylaxis with lamivudine and subsequently entecavir against reactivation of HBV. After the 2nd cycle of chemotherapy her liver function tests became severely abnormal (AST: 514 U/L ALT: 264 U/L, ALP: 145 U/L, GGT: 1328 U/L, total Bi: 74.6 umol/L, conjugated Bi: 63.5 umol/L). The patient was asymptomatic regarding the lymphoma and in complete remission according to MRI scans. Further systemic chemotherapy had to be halted. The HBV DNA was still undetectable by PCR. Drug induced or mechanical cholestasis and the role of ongoing HCV infection was contemplated. The biliary tracts were not dilated on ultrasound scans. She had an ERCP with sphincterotomy and sofosbuvir plus ledipasvir plus ribavirin treatment for HCV was started. Meanwhile, intrathecal methotrexate therapy was administered 3‐weekly to bridge till next chemotherapy. The antiviral therapy was completed without any side effects and at 3 months, her HCV RNA PCR was negative with entirely normal liver function. The patient achieved sustained virological response. Two more cycles of MATRIX was delivered and an autologous stem cell transplantation with BCNU and thiothepa conditioning. Liver function remained normal ever since.Summary/Conclusion:This is a rare case where the role of HCV infection in liver damage during systemic chemotherapy is highly possible. We would like to draw attention to the liver vulnerability caused by chronic HCV infection. Nowadays HCV eradication is feasible with novel direct acting antiviral therapies, HCV‐infection should not contraindicate the adequate chemotherapy.

Assessment of the role of interleukin 17A and interleukin 17F in chronic hepatitis C virus infection in Egyptian patients

Background/purposeof the study Chronic hepatitis C virus (HCV) infection is considered one of the major health problems. About 170 million patients were infected with HCV worldwide. Till few years, Egypt was considered the highest HCV prevalent country worldwide, with predominant genotype number 4. The host immunity plays a major role in HCV infection with evolving data confirming the role of T-helper 17 cells in the formation of chronic HCV infection. The aim of our work was to determine the role of interleukins 17A (IL17A), 17F (IL17F) in the formation of chronic hepatitis C infection.Patients and methodsWe classify the patients into two groups: the first group included 51 chronic HCV patients who did not take antiviral therapy (the study group) and the second group included 51 healthy blood donors (as a control group). The levels of IL17A and IL17F in the serum of both groups were measured using the sandwich enzyme-linked immunosorbent assay method.ResultsThe serum values of IL17A were higher in patients with chronic HCV than the other group with the mean values being 52.9±32.6 pg/ml in the patient group and 17.1 ±10.4 pg/ml in the control group. IL17F was slightly higher in the HCV patient group than the control group, but it was statistically insignificant. Moreover, there were significant positive correlations between IL17A and alanine aminotransferase, viral load, and degree of liver fibrosis.ConclusionPatients with chronic HCV infections had a higher serum level of IL17A than the normal persons and it is positively correlated with alanine aminotransferase, viral load, and degree of liver fibrosis. This suggests its pivotal role in the formation of chronic HCV infection; so, it can be used as a new marker for disease progression due to its positive correlation with the severity of liver injury.

Rab13 Is Involved in the Entry Step of Hepatitis C Virus Infection.

Membrane transport probably participates in the lifecycle of hepatitis C virus (HCV). Rab proteins are essential host factors for HCV RNA replication, but these proteins' roles in other steps of the HCV lifecycle are not clear. The tight junction (TJ) plays a key role in HCV infection. Rab13 regulates the endocytic recycling of the TJ-associated proteins. Here we investigated whether Rab13 is involved in the HCV entry step. We used HuH-7-derived RSc cells and Li23-derived D7 cells. To evaluate the effect of Rab13 in HCV infection, we transfected the cells with siRNA targeting Rab13 before HCV infection. The down-regulation of Rab13 inhibited HCV infection. The D7 cells had showed a greater inhibitory effect against HCV infection compared to that in the RSc cells by Rab13 knockdown. Next, to evaluate the effect of Rab13 after infection, we inoculated the cells with HCV before transfection of the siRNA. The down-regulation of Rab13 did not show any effects after HCV infection. We further examined whether Rab13 would influence HCV RNA replication by using HCV replicon-harboring cells. The results revealed that Rab13 did not affect the step of HCV RNA replication. These results suggest that Rab13 plays an important role in the step of HCV entry.

HCV Infection and Interferon-Based Treatment Induce p53 Gene Transcription in Chronic Hepatitis C Patients.

It is suggested that the tumor suppressor p53 gene, classified as an interferon-stimulated gene, is implicated in the interferon (IFN)-mediated innate immunity against viruses. This study aimed to examine the transcriptional response of the p53 gene to hepatitis C virus (HCV) infection and IFN-based therapy in chronic hepatitis C (CHC) patients. The study included 65 CHC patients (HCV genotype 1), treated with pegylated IFN-α and ribavirin, and 51 healthy individuals. p53 gene expression was quantified by real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMCs). Analyses were performed before and at weeks 4 and 12 of treatment. p53 gene expression was significantly upregulated in CHC patients compared with healthy controls and at week 4 of therapy. No significant differences in p53 mRNA expression between rapid virologic responders, complete early virologic responders, and nonresponders were observed. No significant correlation was found between p53 gene expression and viral load. The results obtained indicate that HCV infection and IFN-based treatment induces p53 gene transcription in PBMCs. The p53 gene may therefore play a role in HCV infection but is not directly involved in treatment-induced HCV elimination. Moreover, variations in p53 gene expression do not determine on-treatment response in patients with chronic HCV genotype 1 infection.

Single-nucleotide polymorphism of Toll-like receptor 4 and interleukin-10 in response to interferon-based therapy in Egyptian chronic hepatitis C patients.

Egypt has the highest prevalence of hepatitis C virus (HCV) in the world. It has been suggested that not only the virus but also the interaction between the virus and the host immune system is important in determining the course of the infection and the response to interferon (IFN)-based therapy. While the adaptive immune system plays a critical role in HCV infection, the innate immune system has only been recognized recently. Toll-like receptors (TLRs) form the cornerstone of the innate immune response. Interleukin-10 (IL-10) is one of the upstream regulators of TLR4. A possible interplay between TLR4 and IL-10 has been suggested. The present study aimed to investigate the role of single-nucleotide polymorphisms (SNPs) in TLR4 and IL-10-1082 and the expression levels of these proteins in predicting the response to treatment in chronic HCV patients. A total of 83 chronic HCV-infected Egyptian patients treated with peg-IFN-α2b-ribavirin combination therapy and 40 healthy subjects were included in this study. SNPs in the TLR4 rs2149356 and IL-10-1082 genes and their serum levels were assessed. Within the responders group, T/T and A/A genotypes were the significantly most frequent genotypes of TLR4 and IL-10-1082, respectively. Moreover, a higher frequency of T/T and A/A was found to be associated with lower serum TLR4 and IL-10 levels in our responder patients. In addition, subjects with the T/T genotype in the healthy control group had a lower serum TLR4 level than those with other genotypes. We conclude that the SNPs TLR4 rs2149356-T/T and IL-10-1082-A/A may be important predictors for HCV therapy.

Dynamic Interaction of Stress Granules, DDX3X, and IKK-α Mediates Multiple Functions in Hepatitis C Virus Infection.

The ubiquitous ATP-dependent RNA helicase DDX3X is involved in many cellular functions, including innate immunity, and is a pivotal host factor for hepatitis C virus (HCV) infection. Recently, we showed that DDX3X specifically recognizes the HCV 3' untranslated region (UTR), leading to the activation of IKK-α and a cascade of lipogenic signaling to facilitate lipid droplet biogenesis and viral assembly (Q. Li, V. Pene, S. Krishnamurthy, H. Cha, and T. J. Liang, Nat Med 19:722-729, 2013, http://dx.doi.org/10.1038/nm.3190). The interaction of DDX3X with HCV core protein seems to be dispensable for its proviral role. In this study, through systematic imaging and biochemical and virologic approaches, we identified a dynamic association between DDX3X and various cellular compartments and viral elements mediating multiple functions of DDX3X in productive HCV infection. Upon HCV infection, the HCV 3'UTR interacts with DDX3X and IKK-α, which redistribute to speckle-like cytoplasmic structures shown to be stress granules (SGs). As viral proteins accumulate in infected cells, DDX3X granules together with SG-associated proteins redistribute and colocalize with HCV core protein around lipid droplets (LDs). IKK-α, however, does not relocate to the LD but translocates to the nucleus. In HCV-infected cells, various HCV nonstructural proteins also interact or colocalize with DDX3X in close proximity to SGs and LDs, consistent with the tight juxtaposition of the replication complex and the assembly site at the surface of LDs. Short interfering RNA (siRNA)-mediated silencing of DDX3X and multiple SG components markedly inhibits HCV infection. Our data suggest that DDX3X initiates a multifaceted cellular program involving dynamic associations with HCV RNA and proteins, IKK-α, SG, and LD surfaces for its crucial role in the HCV life cycle. IMPORTANCE DDX3X is a proviral host factor for HCV infection. Recently, we showed that DDX3X binds to the HCV 3'UTR, activating IKK-α and cellular lipogenesis to facilitate viral assembly (Q. Li et al., Nat Med 19:722-729, 2013, http://dx.doi.org/10.1038/nm.3190). Here, we report associations of DDX3X with various cellular compartments and viral elements that mediate its multiple functions in the HCV life cycle. Upon infection, the HCV 3'UTR redistributes DDX3X and IKK-α to speckle-like cytoplasmic structures shown to be SGs. Subsequently, interactions between DDX3X, SG, and HCV proteins facilitate the translocation of DDX3X-SG complexes to the LD surface. HCV nonstructural proteins are shown to colocalize with DDX3X in close proximity to SGs and LDs, consistent with the tight juxtaposition of the HCV replication complex and assembly site at the LD surface. Our data demonstrate that DDX3X initiates a multifaceted cellular program involving dynamic associations with HCV elements, IKK-α, SGs, and LDs for its critical role in HCV infection.

Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web

The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.

Immunological mechanisms controlling hepatitis C virus infection

Hepatitis C virus (HCV) infection is a significant global health problem, affecting over 150 million people worldwide. There is increasing evidence that a small percentage of individuals exposed to the HCV have the capacity to generate a strong cellular as well as humeral immune response against the virus and avoid persistent infection, and perhaps do so repeatedly after re-exposure. While the critical role of the adaptive immune system in HCV infection is well-established, the importance of the innate immune system has been recognized in more recent years. The immune system has many weapons to combat the HCV infection. These include type I interferons, HCV specific CD4+ cells and CD8+ T cells, cytokine production, natural killer cells, dentritic cells, and the production of anti-HCV neutralizing antibodies. Toll-like receptors form an important element of the innate immune response, and there is considerable evidence for their crucial role in HCV infection. In order to limit the availability of the cellular components for viral amplification, apoptosis occurs. It involves caspases, the key effectors of apoptotic cell death. This article reviews what the immune system does, when HCV attacks the body.

Single Nucleotide Polymorphisms of Toll-Like Receptor 7 and Toll-Like Receptor 9 in Hepatitis C Virus Infection Patients from Central China

PurposeTo analyze the correlation of polymorphisms of toll-like receptor 7 (TLR7) (rs179009) and toll-like receptor 9 (TLR9) (rs187084) in hepatitis C virus (HCV) infections in the Han population.Materials and MethodsThe genotypes of TLR7IVS2-151 in HCV infection were detected by Sanger sequencing using polymerase chain reaction-restriction fragment length polymorphism to determine the TLR9 T-1486C single nucleotide polymorphisms (SNP) for all enrolled patients.ResultsWe found no significant difference between males with spontaneous clearance of HCV versus those chronically infected [χ2=2.71, p=0.10, odd ratios (OR)=0.58, 95% confidence interval (CI) 0.31-1.11]. However, significant differences were found for the distribution of TLR7 (rs179009) in females (χ2=9.46, p=0.01). In females, a significant difference was also found between chronic hepatitis C and those with spontaneous clearance of HCV in terms of TLR7 IVS2-151G/A allele frequencies (χ2=9.50, p=0.00, OR=0.46, 95% CI 0.28-0.75). In HCV-infected patients, no significant association was found between the frequency of TLR9 genotypes and alleles.ConclusionThe site of TLR7 IVS2-151 (rs179009) G/A may be a factor for susceptibility of chronic HCV in the female Han population. TLR9T-1486C (rs18084) SNP may not play a major role in HCV infection. However, individual risk profiles for HCV infection did vary by sex and this relationship should be further investigated.

A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Identification of transferrin receptor 1 as a hepatitis C virus entry factor

Hepatitis C virus (HCV) is a liver tropic pathogen that affects ∼170 million people worldwide and causes liver pathologies including fibrosis, cirrhosis, steatosis, iron overload, and hepatocellular carcinoma. As part of a project initially directed at understanding how HCV may disrupt cellular iron homeostasis, we found that HCV alters expression of the iron uptake receptor transferrin receptor 1 (TfR1). After further investigation, we found that TfR1 mediates HCV entry. Specifically, functional studies showed that TfR1 knockdown and antibody blocking inhibit HCV cell culture (HCVcc) infection. Blocking cell surface TfR1 also inhibited HCV pseudoparticle (HCVpp) infection, demonstrating that TfR1 acts at the level of HCV glycoprotein-dependent entry. Likewise, a TfR1 small-molecule inhibitor that causes internalization of surface TfR1 resulted in a decrease in HCVcc and HCVpp infection. In kinetic studies, TfR1 antibody blocking lost its inhibitory activity after anti-CD81 blocking, suggesting that TfR1 acts during HCV entry at a postbinding step after CD81. In contrast, viral spread assays indicated that HCV cell-to-cell spread is less dependent on TfR1. Interestingly, silencing of the TfR1 trafficking protein, a TfR-1 specific adaptor protein required for TfR1 internalization, also inhibited HCVcc infection. On the basis of these results, we conclude that TfR1 plays a role in HCV infection at the level of glycoprotein-mediated entry, acts after CD81, and possibly is involved in HCV particle internalization.

Prevalence and factors associated with HCV infection among elderly individuals in a southern Brazilian city

Few Latin American studies have assessed the prevalence of hepatitis C virus (HCV) infection in elderly individuals, in whom the highest rates are expected. We aimed to investigate the prevalence of and factors associated with HCV infection in elderly residents in the municipality of Tubarão, Santa Catarina. This cross-sectional study included 820 individuals (aged ≥ 60 years) who were selected by simple random sampling. The presence of anti-HCV antibodies was tested by chemiluminescence, and HCV RNA detection was performed for the anti-HCV-reactive subjects. Those individuals who were anti-HCV reactive but had undetectable HCV RNA levels were tested using a third-generation recombinant immunoblot assay. The variables were compared using the chi-squared test or Fisher's exact test, and those variables with p < 0.05 were included in the logistic regression model. The mean patient age was 68.6 years (SD 7.0 years); 39% were men, and 92% were Caucasian. Eighteen subjects were anti-HCV positive. Among these individuals, 4 were characterized as false-positives, leaving 14 (1.7%) individuals with confirmed infections for analysis. HCV infection was associated with an age older than 65 years, households with 3 or more residents and the previous transfusion of blood products. In the logistic regression analysis, the following variables were independently associated with HCV infection: households with 3 or more residents (OR 7.9, 95% CI 1.7-35.9, p = 0.008) and previous blood transfusion (OR 6.2, 95% CI 2.1-18.6, p = 0.001). The HCV prevalence in the elderly population in the municipality of Tubarão was higher than that found in previous studies of blood donors in the same region. Although exposure to contaminated blood products remained important, other transmission routes, such as household transmission, could play a role in HCV infection.

NS5ATP9 Contributes to Inhibition of Cell Proliferation by Hepatitis C Virus (HCV) Nonstructural Protein 5A (NS5A) via MEK/Extracellular Signal Regulated Kinase (ERK) Pathway

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions and viral pathogenesis. However, on the impact of cell growth, there have been different study results. NS5ATP9, also known as KIAA0101, p15PAF, L5, and OEACT-1, was first identified as a proliferating cell nuclear antigen-binding protein. Earlier studies have shown that NS5ATP9 might play an important role in HCV infection. The aim of this study is to investigate the function of NS5ATP9 on hepatocellular carcinoma (HCC) cell lines proliferation under HCV NS5A expression. The results showed that overexpression of NS5ATP9 inhibited the proliferation of Bel7402 cells, whereas knockdown of NS5ATP9 by interfering RNA promoted the growth of HepG2 cells. Under HCV NS5A expression, RNA interference (RNAi) targeting of NS5ATP9 could reverse the inhibition of HepG2 cell proliferation, suggesting that NS5ATP9 might be an anti-proliferation gene that plays an important role in the suppression of cell growth mediated by HCV NS5A via MEK/ERK signaling pathway. These findings might provide new insights into HCV NS5A and NS5ATP9.

Understanding the Biological Context of NS5A–Host Interactions in HCV Infection: A Network-Based Approach

Hepatitis C virus (HCV) is a major cause of chronic liver disease. HCV NS5A protein plays an important role in HCV infection through its interactions with other HCV proteins and host factors. In an attempt to further our understanding of the biological context of protein interactions between NS5A and host factors in HCV pathogenesis, we generated an extensive physical interaction map between NS5A and cellular factors. By combining a yeast two-hybrid assay with comprehensive literature mining, we built the NS5A interactome composed of 132 human proteins that interact with NS5A. These interactions were integrated into a high-confidence human protein interactome (HPI) with the help of the TargetMine data warehouse system to infer an overall protein interaction map linking NS5A with the components of the host cellular networks. The NS5A-host interactions that were integrated with the HPI were shown to participate in compact and well-connected cellular networks. Functional analysis of the NS5A "infection" network using TargetMine highlighted cellular pathways associated with immune system, cellular signaling, cell adhesion, cellular growth and death among others, which were significantly targeted by NS5A-host interactions. In addition, cellular assays with in vitro HCV cell culture systems identified two ER-localized host proteins RTN1 and RTN3 as novel regulators of HCV propagation. Our analysis builds upon the present understanding of the role of NS5A protein in HCV pathogenesis and provides potential targets for more effective anti-HCV therapeutic intervention.

Cytokeratin 8 is increased in hepatitis C virus cells and its ectopic expression induces apoptosis of SMMC7721 cells

To investigate cytokeratin 8 (CK8) overexpression during hepatitis C virus (HCV) infection and its pathogenesis, and the effect of ectopic CK8 expression on hepatoma cell lines. We successfully established an in vitro HCV cell culture system (HCVcc) to investigate the different expression profiles of CK8 in Huh-7-HCV and Huh-7.5-HCV cells. The expression of CK8 at the mRNA level was determined by real-time polymerase chain reaction (RT-PCR). The expression of CK8 at the protein level was evaluated by Western blotting. We then constructed a eukaryotic expression combination vector containing the coding sequence of human full length CK8 gene. CK8 cDNA was amplified by reverse transcription-PCR and inserted into pEGFP-C1 and the positive clone pEGFP-CK8 was obtained. After confirming the sequence, the recombinant plasmid was transfected into SMMC7721 cells with lipofectamine2000 and CK8 expression was detected using inverted fluorescence microscopy, RT-PCR and Western blotting. Besides, we identified biological function of CK8 on SMMC7721 cells, including cell proliferation, cell cycle and apoptosis detection. RT-PCR showed that the expression level of CK8 in Huh-7-HCV and Huh-7.5-HCV cells was 2.88 and 2.95 times higher than in control cells. Western blot showed that CK8 expression in Huh-7-HCV and Huh-7.5-HCV cells was 2.53 and 3.26 times higher than that in control cells, respectively. We found that CK8 at mRNA and protein levels were both significantly increased in HCVcc. CK8 was up-regulated in SMMC7721 cells. CK8 expression at the mRNA level was significantly upregulated in SMMC7721/pEGFP-CK8 cells. CK8 expression in SMMC7721/ pEGFP-CK8 cells was 2.69 times higher than in SMMC7721 cells, and was 2.64 times higher than in SMMC7721/pEGFP-C1 cells. CK8 expression at the protein level in SMMC7721/pEGFP-CK8 cells was 2.46 times higher than in SMMC7721 cells, and was 2.29 times higher than in SMMC7721/pEGFP-C1 cells. Further analysis demonstrated that forced expression of CK8 slowed cell growth and induced apoptosis of SMMC7721 cells. CK8 up-regulation might have a functional role in HCV infection and pathogenesis, and could be a promising target for the treatment of HCV infection.