Nationwide epidemiologic and genetic surveillance of hepatitis E in Japan, 2014-2021.

Hepatitis E virus (HEV) is an emerging causative agent of acute hepatitis. To clarify the epidemiology of HEV and characterize the genetic diversity of the virus in Japan, nationwide enhanced surveillance and molecular characterization studies of HEV in Japan were undertaken from 2014 to 2021. In total, 2770 hepatitis E cases were reported, of which 88% were domestic cases, while only 4.1% represented cases following infection abroad. In addition, 57% of domestic infections occurred in males aged in their 40s-70s. For domestic cases, infection via pork meat consumption continued to be the most reported route. Analysis of the 324 sequences detected between 2016 and 2021 showed that the majority of domestic HEV strains belong to Genotype 3a (G3a) and G3b. In contrast, six of eight cases of G1 HEV reflected infection abroad. Our results suggest that HEV is circulating widely in Japan, with genotypes G3a and G3b being most prevalent. Continued surveillance is necessary to monitor future trends and changes in the epidemiology of HEV in Japan.

Seroepidemiology of hepatitis A virus infection in Japan: An area of very low endemicity.

The incidence of hepatitis A virus (HAV) infection has declined significantly worldwide, including in Japan. A nationwide seroepidemiological study on hepatitis A in Japan has taken place almost every 10 years since 1973, and the last study was performed in 2003. In the present study, we describe the latest seroepidemiological pattern of hepatitis A in Japan using 7867 serum specimens obtained from healthy individuals collected between 2013 and 2017, approximately 10 years after the last study. Among them, 223 were anti-HAV positive. About 68% of individuals aged 60 years and older had anti-HAV antibodies, whereas only 1.1% of those aged below 60 years old had immunity; thus, almost all individuals younger than 60 years of age were HAV susceptible. In comparison with previous investigations, the susceptible population has increased and aged. According to data from the National Epidemiological Surveillance of Infectious Diseases (NESID) program, between 1989 and 2016, the proportion of patients with hepatitis A aged 60 years and older continuously increased with each year. The NESID data also suggested that recently, typical large foodborne outbreaks of hepatitis A have become rare, and cases tend to be reported among at-risk groups; overseas travelers contributed to 25% of hepatitis A cases, and in 2018, the first nationwide hepatitis A outbreak that affected mostly men who have sex with men was reported. The purpose of this study was to determine the current status of HAV infection in Japan, based on both seroepidemiology and the national surveillance data from the NESID.

Calibrating Hepatitis E Virus Serological Assays Using Asymptomatic Specimens Obtained in Japan.

This study aimed to calibrate hepatitis E virus (HEV) serological assays. We optimized the previously developed in-house HEV antibody enzyme-linked immunosorbent assay (ELISA) by setting the cutoff with an in-house serological performance panel consisting of broad HEV antibody titers and subtracting nonspecific background values for anti-HEV IgM, IgA, and IgG. We also compared the assay's performance with that of commercial serological assay kits (four kits for IgM, one for IgA, and two for IgG). Although all serological assays readily detected HEV antibodies at high titers in the symptomatic hepatitis E population, considerable variations between assays were observed in the asymptomatic population. The in-house ELISA showed a higher sensitivity for HEV IgM, IgA, and IgG than the commercial kits and detected the seroconversion of HEV IgM and IgG earlier when testing a commercially available HEV seroconversion panel. The low sensitivity of the commercial kits was due to the high setting of the original cutoff, which was demonstrated by receiver operating characteristic analysis. However, the corrected cutoff value reduced assay specificity. Background subtraction is essential to achieve high specificity because the in-house ELISA without background subtraction reduced its specificity. These results indicate that asymptomatic specimens and background subtraction contribute to the optimization of HEV serological assays. IMPORTANCE Accurate diagnosis of hepatitis E virus (HEV) infection is essential for public health surveillance and for preventing HEV-contaminated blood transfusion. Anti-HEV IgM or IgA is used as a reliable marker of recent HEV infection. However, considerable variability in the sensitivity and specificity of HEV antibody detection is observed among several commercially available assay kits. In addition, none of the HEV antibody detection methods have been approved by the U.S. Food and Drug Administration (FDA). Here, we show that the in-house enzyme-linked immunosorbent assay (ELISA) could detect HEV IgM and IgA more sensitively than commercial kits in the asymptomatic population. We also suggest that the assay performance of commercial kits might be improved by optimizing the cutoff and reducing nonspecific background noise. A sensitive serological (IgM or IgA) assay in addition to HEV RNA testing will contribute to accurate diagnosis of acute HEV infection because HEV RNA-positive duration is relatively short.

Isolation and Genome Sequencing of Hepatitis E Virus Genotype 1 Imported from India to Japan.

Hepatitis E virus (HEV) is the causative agent of viral hepatitis E. In Japan, HEV genotype 3 (G3) and G4 are predominantly detected, while G1, mainly imported from countries in continental Asia, is rare. In the present study, we detected a G1 HEV strain in a patient who visited Japan from India. When PLC/PRF/5 cells (subclone 4-21) were inoculated with a stool suspension from this patient, accumulation of HEV RNA was observed in the spent culture medium, indicating that HEV had been successfully isolated from this specimen. A nearly complete HEV genome was obtained by RT-PCR amplification. Phylogenetic analyses revealed that the newly isolated HEV strain, designated 9HE36c, belongs to subtype 1g of HEV G1.

Evaluation of Heat Inactivation of Human Norovirus in Freshwater Clams Using Human Intestinal Enteroids.

Foodborne disease attributed to the consumption of shellfish contaminated with human norovirus (HuNoV) is one of many global health concerns. Our study aimed to determine the conditions of the heat-inactivation of HuNoV in freshwater clams (Corbicula japonica) using a recently developed HuNoV cultivation system employing stem-cell derived human intestinal enteroids (HIEs). We first measured the internal temperature of the clam tissue in a water bath during boiling at 90 °C and found that approximately 2 min are required for the tissue to reach 90 °C. Next, GII.4 HuNoV was spiked into the center of the clam tissue, followed by boiling at 90 °C for 1, 2, 3, or 4 min. The infectivity of HuNoV in the clam tissue homogenates was evaluated using HIEs. We demonstrated that HuNoV in unboiled clam tissue homogenates replicated in HIEs, whereas infectivity was lost in all boiled samples, indicating that heat treatment at 90 °C for 1 min inactivates HuNoV in freshwater clams in our current HIE culture system. To our knowledge, this is the first study to determine the thermal tolerability of HuNoV in shellfish using HIEs, and our results could be informative for developing strategies to inactivate HuNoV in shellfish.

Comparison of the Clinical Features of Hepatitis A in People Living with HIV between Pandemics in 1999-2000 and 2017-2018 in the Metropolitan Area of Japan.

Since 2017, hepatitis A virus (HAV) infection has been an epidemic among men who have sex with men (MSM) in Japan. We have come across 11 MSM patients with hepatitis A who were also infected with HIV. In 1999-2000, we came across 5 HIV-infected patients with hepatitis A. Since the conditions of current HIV-infected patients have changed owing to the recent progress in anti-HIV therapies, we compared clinical features of hepatitis A between patients in 2017-2018 and those in 1999-2000. By comparing the background characteristics of the patients, we found that the CD4/CD8 ratio was significantly higher in the 2017-2018 group. After the onset of hepatitis, peak levels of hepatic transaminases were found to be higher in the 2017-2018 group, suggesting severe hepatocellular damage. In contrast, neither the peak level of total bilirubin nor the nadir of prothrombin time was significantly different among the 2 groups. We also analyzed the HAV genome derived from some of the recently infected patients, and found that the HAV strains were almost the same among these patients; slight differences were observed from the previously identified strain. Thus, we concluded that the recovery of immunity by recent anti-HIV therapies may result in more severe hepatocellular damages and differences in clinical features.

The machinery for endocytosis of epidermal growth factor receptor coordinates the transport of incoming hepatitis B virus to the endosomal network.

Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4ß (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.

Hepatitis B Virus Genotype-Dependent Vulnerability of Infected Cells to Immune Reaction in the Early Phase of Infection.

Infection with hepatitis B virus (HBV) genotype (GT)-A has been reported to predispose patients to chronic infection. To explore the immune responses in infection with different HBV genotypes and clarify the genotype-dependent pathogenicity, a system mimicking the immune reaction during the early phase of HBV infection is indispensable. To this end, we established a coculture system with the replication-competent HBV molecular clone-transfected HepG2 cells and immortalized human natural killer (NK) cells, NK-92MI. Using this system, we evaluated HBV genotype dependency in NK functions and cell death of HBV positive HepG2 cells induced by NK cells or administration of tumor necrosis factor (TNF) by use of flow cytometry. After coculture with NK cells, we found that GT-A-positive HepG2 cells exhibited lower susceptibility to NK cell-induced cell death than GT-B- or GT-C-positive HepG2 cells. The NK responses of degranulation and cytokine production were not different among transfected HBV genotypes in cocultured cells. The expression levels of death receptors in HBV-transfected HepG2 cells were not different. In GT-A-positive cells, a similar low susceptibility was detected by the external administration of TNF, although relatively higher susceptibility was observed in GT-B- and GT-C-positive cells than in GT-A-positive cells. The activation of caspase signaling was revealed to be responsible for this genotype-dependent susceptibility. In conclusion, our results indicate that the HBV genotype does not influence the NK cell function itself but rather cell vulnerability through the TNF signal pathway. This observation may explain the high chronicity rate of HBV GT-A strains even in adult infections.

Integrin α3 is involved in non-enveloped hepatitis E virus infection.

Hepatitis E virus (HEV) causes acute and fulminant hepatitis worldwide. Although enveloped (e) and non-enveloped (ne) forms of HEV have been discovered, host factors involved in infection, including receptors, remain to be elucidated. Here, we identified integrin α3 (encoded by ITGA3), a protein that binds and responds to the extracellular matrix, as an essential host factor for HEV infection. Integrin α3 expression was lower in four HEV-non-permissive cell subclones than in an HEV-permissive subclone. ITGA3 knockout cells lost HEV permissibility, suggesting that integrin α3 is critical for HEV infection. Stable expression of integrin α3 in an HEV-non-permissive subclone provided permissibility only to infection by neHEV; expression of integrin α3 lacking the ectodomain did not. Direct interaction between neHEV and the integrin α3 ectodomain was confirmed by co-precipitation using a soluble integrin α3-Fc. These results strongly suggest that integrin α3 is a key molecule for cellular attachment and entry of neHEV.

Adenosine deaminase acting on RNA-1 (ADAR1) inhibits hepatitis B virus (HBV) replication by enhancing microRNA-122 processing.

Adenosine deaminases acting on RNA-1 (ADAR1) involves adenosine to inosine RNA editing and microRNA processing. ADAR1 is known to be involved in the replication of various viruses, including hepatitis C and D. However, the role of ADAR1 in hepatitis B virus (HBV) infection has not yet been elucidated. Here, for the first time, we demonstrated ADAR1 antiviral activity against HBV. ADAR1 has two splicing isoforms in human hepatocytes: constitutive p110 protein and interferon-α (IFN-α)-responsive p150 protein. We found that overexpression of ADAR1 decreased HBV RNA in an HBV culture model. A catalytic-site mutant ADAR1 also decreased HBV RNA levels, whereas another adenosine deaminases that act on the RNA (ADAR) family protein, ADAR2, did not. Moreover, the induction of ADAR1 by stimulation with IFN-α also reduced HBV RNA levels. Decreases in endogenous ADAR1 expression by knock-down or knock-out increased HBV RNA levels. A major hepatocyte-specific microRNA, miRNA-122, was found to be positively correlated with ADAR1 expression, and exogenous miRNA-122 decreased both HBV RNA and DNA, whereas, conversely, transfection with a miRNA-122 inhibitor increased them. The reduction of HBV RNA by ADAR1 expression was abrogated by p53 knock-down, suggesting the involvement of p53 in the ADAR1-mediated reduction of HBV RNA. This study demonstrated, for the first time, that ADAR1 plays an antiviral role against HBV infection by increasing the level of miRNA-122 in hepatocytes.

Epidermal growth factor receptor is a host-entry cofactor triggering hepatitis B virus internalization.

Sodium taurocholate cotransporting polypeptide (NTCP) is a host cell receptor required for hepatitis B virus (HBV) entry. However, the susceptibility of NTCP-expressing cells to HBV is diverse depending on the culture condition. Stimulation with epidermal growth factor (EGF) was found to potentiate cell susceptibility to HBV infection. Here, we show that EGF receptor (EGFR) plays a critical role in HBV virion internalization. In EGFR-knockdown cells, HBV or its preS1-specific fluorescence peptide attached to the cell surface, but its internalization was attenuated. PreS1 internalization and HBV infection could be rescued by complementation with functional EGFR. Interestingly, the HBV/preS1-NTCP complex at the cell surface was internalized concomitant with the endocytotic relocalization of EGFR. Molecular interaction between NTCP and EGFR was documented by immunoprecipitation assay. Upon dissociation from functional EGFR, NTCP no longer functioned to support viral infection, as demonstrated by either (i) the introduction of NTCP point mutation that disrupted its interaction with EGFR, (ii) the detrimental effect of decoy peptide interrupting the NTCP-EGFR interaction, or (iii) the pharmacological inactivation of EGFR. Together, these data support the crucial role of EGFR in mediating HBV-NTCP internalization into susceptible cells. EGFR thus provides a yet unidentified missing link from the cell-surface HBV-NTCP attachment to the viral invasion beyond the host cell membrane.

Evaluation of Heating Conditions for Inactivation of Hepatitis E Virus Genotypes 3 and 4.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis throughout the world. HEV genotypes 1 through 4 infect humans, whereas genotypes 3 and 4 (Gt3 and Gt4) also infect other animals. In developed countries, the main HEV infection route is by foodborne transmission, resulting from the consumption of undercooked meat. It is important to know the criteria for HEV control in daily cooking. In this study, we assessed the heat conditions required to inactivate HEV Gt3 and Gt4 in culture supernatants and spiked minced pork meat. HEV inactivation was determined by measuring viral RNA amplification in PLC/PRF/5 cell culture. In our cell culture assay, an inoculum containing HEV titer that is equivalent to >105 genome RNA copies can be determined as infectious. The internal temperature of pork during heating was measured to represent that achieved during cooking. Both HEV Gt3 and Gt4 were inactivated in culture supernatants heated at >65°C for 5 min and at >80°C for 1 min and in minced meat at 70°C for 5 min. Inoculated culture supernatant contained 108 HEV genome RNA copies (103 infectious units [IU]); therefore, it was indicated that HEV titer decreased >3 log IU after heating. In a comparison of Gt3 and Gt4, Gt4 showed slightly greater heat stability than Gt3. Boiling showed superior heating efficacy compared with roasting, and pork liver was slightly easier to heat than pork loin. Heating for 5 min by both boiling and roasting increased the internal temperature of pork products to more than 70°C. Although our data revealed that HEV Gt4 was slightly more heat stable than Gt3, both genotypes were inactivated by the appropriate heating conditions. Therefore, the risk of HEV foodborne infection could be mitigated by the appropriate cooking of pork meat. It is also important that both the supplier and the consumer are cognizant of the risk of HEV foodborne infection from livestock products.

Interferon sensitivity-determining region of hepatitis C virus influences virus production and interferon signaling.

The number of amino acid substitutions in the interferon (IFN) sensitivity-determining region (ISDR) of hepatitis C virus (HCV) NS5A is a strong predictor for the outcome of IFN-based treatment. To assess the involvement of ISDR in the HCV life cycle and to clarify the molecular mechanisms influencing IFN susceptibility, we used recombinant JFH-1 viruses with NS5A of the genotype 1b Con1 strain (JFH1/5ACon1) and with NS5A ISDR containing 7 amino acid substitutions (JFH1/5ACon1/i-7mut), and compared the virus propagation and the induction of interferon-stimulated genes (ISGs). By transfecting RNAs of these strains into HuH-7-derived cells, we found that the efficiency of infectious virus production of JFH1/5ACon1/i-7mut was attenuated compared with JFH1/5ACon1. After transfecting full-length HCV RNA into HepaRG cells, the mRNA expression of ISGs was sufficiently induced by IFN treatment in JFH1/5ACon1/i-7mut-transfected but not in JFH1/5ACon1-transfected cells. These data suggested that the NS5A-mediated inhibition of ISG induction was deteriorated by amino acid substitutions in the ISDR. In conclusion, using recombinant JFH-1 viruses, we demonstrated that HCV NS5A is associated with infectious virus production and the inhibition of IFN signaling, and amino acid substitutions in the NS5A ISDR deteriorate these functions. These observations explain the strain-specific evasion of IFN signaling by HCV.

Resistance mutations of hepatitis B virus in entecavir-refractory patients.

The emergence of resistance mutations in the reverse transcriptase gene of hepatitis B virus (HBV) is associated with treatment failure. Entecavir (ETV) is one of the most potent anti-HBV reagents; it has a very low resistance rate and is used as the first-line treatment for chronic hepatitis B. In this study, we isolated HBVs in 4 ETV-refractory patients (2 with viral breakthrough, 1 with partial virological response, and 1 with flare-up) and assessed ETV resistance using replication-competent 1.38-fold HBV genome-length molecular clones. The full genome sequences of infected HBVs in ETV-refractory patients were determined. The HBV molecular clones were generated with the patient-derived sequences. After transfection of these molecular clones into HepG2 cells, viral replications and ETV susceptibilities were evaluated by measuring the amount of intracellular core-particle-associated HBV DNA using Southern blotting and real-time polymerase chain reaction. Among these cases, ETV-resistant variants were detected in 2 patients with viral breakthrough and responsible amino acid mutations in reverse transcriptase were successfully identified in these variants. No ETV-resistant mutation was detected in the other cases. The identified ETV-resistant mutations did not confer resistance to tenofovir disoproxil fumarate. Conclusion: The HBV replication model with patient-derived sequences is useful for assessing replication efficiency, susceptibility to anti-HBV reagents, and responsible resistance mutations and can aid in choosing the appropriate treatment strategy for treatment-failure cases of chronic hepatitis B. (Hepatology Communications 2017;1:110-121).

Effects of Resistance-Associated NS5A Mutations in Hepatitis C Virus on Viral Production and Susceptibility to Antiviral Reagents.

Direct-acting antivirals (DAAs) for hepatitis C virus (HCV) have potent anti-HCV effects but may provoke resistance-associated variants (RAVs). In this study, we assessed the characteristics of these RAVs and explored efficacious anti-HCV reagents using recombinant HCV with NS5A from a genotype 1b strain. We replaced the NS5A of JFH1 with that of Con1 (JFH1/5ACon1) and introduced known NS5A inhibitor resistance mutations (L31M, L31V, L31I and Y93H) individually or in combination. Susceptibilities against anti-HCV reagents were also investigated. RAVs with Y93H exhibited high extracellular core antigen levels and infectivity titers. Variants with any single mutation showed mild to moderate resistance against NS5A inhibitors, whereas variants with double mutations at both L31 and Y93 showed severe resistance. The variants with mutations exhibited similar levels of susceptibility to interferon (IFN)-α, IFN-λ1, IFN-λ3 and Ribavirin. Variants with the Y93H mutation were more sensitive to protease inhibitors compared with JFH1/5ACon1. In conclusion, the in vitro analysis indicated that the Y93H mutation enhanced infectious virus production, suggesting advantages in the propagation of RAVs with this mutation. However, these RAVs were susceptible to protease inhibitors. Thus, a therapeutic regimen that includes these reagents is a promising means to eradicate these RAVs.

ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2.

Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation.

Amino Acid Polymorphisms in Hepatitis C Virus Core Affect Infectious Virus Production and Major Histocompatibility Complex Class I Molecule Expression.

Amino acid (aa) polymorphisms in the hepatitis C virus (HCV) genotype 1b core protein have been reported to be a potent predictor for poor response to interferon (IFN)-based therapy and a risk factor for hepatocarcinogenesis. We investigated the effects of these polymorphisms with genotype 1b/2a chimeric viruses that contained polymorphisms of Arg/Gln at aa 70 and Leu/Met at aa 91. We found that infectious virus production was reduced in cells transfected with chimeric virus RNA that had Gln at aa 70 (aa70Q) compared with RNA with Arg at aa 70 (aa70R). Using flow cytometry analysis, we confirmed that HCV core protein accumulated in aa70Q clone transfected cells, and it caused a reduction in cell-surface expression of major histocompatibility complex (MHC) class I molecules induced by IFN treatment through enhanced protein kinase R phosphorylation. We could not detect any effects due to the polymorphism at aa 91. In conclusion, the polymorphism at aa 70 was associated with efficiency of infectious virus production, and this deteriorated virus production in strains with aa70Q resulted in the intracellular accumulation of HCV proteins and attenuation of MHC class I molecule expression. These observations may explain the strain-associated resistance to IFN-based therapy and hepatocarcinogenesis of HCV.

ZNF10 inhibits HIV-1 LTR activity through interaction with NF-κB and Sp1 binding motifs.

Kruppel-associated box-containing zinc finger (KRAB-ZNF) genes constitute the single largest gene family of transcriptional repressors in the genomes of higher organisms. In this study, we isolated 52 cDNA clones of KRAB-ZFPs from U1 cell lines and screened them to identify which were capable of regulating HIV-1 gene expression. We identified 5 KRAB-ZFPs that suppressed ⩾50% of HIV-1 LTR. Of the 5 identified KRAB-ZFPs, the expression of ZNF10 significantly enhanced the transcriptional repression activity of the LTR compared with other ZNFs. In addition, the depletion of endogenous ZNF10 led to the activation of HIV-1 LTR. The repressor activity of ZNF10 was required for TRIM28, SETDB1 and HP1-gamma binding. These results indicate that ZNF10 could be involved in a potent intrinsic antiretroviral defense.

A new role for PGA1 in inhibiting hepatitis C virus-IRES-mediated translation by targeting viral translation factors.

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit the replication of a wide variety of DNA and RNA viruses in different mammalian cell types. We investigated a new role for prostaglandin A1 (PGA1) in the inhibition of hepatitis C virus (HCV)-IRES-mediated translation. PGA1 exhibited dose-dependent inhibitory effects on HCV translation in HCV replicon cells. Furthermore, repetitive PGA1 treatment demonstrated the potential to safely induce the suppression of HCV translation. We also validated a new role for PGA1 in the inhibition of HCV-IRES-mediated translation by targeting cellular translation factors, including the small ribosomal subunit (40S) and eukaryotic initiation factors (eIFs). In pull-down assays, biotinylated PGA1 co-precipitated with the entire HCV IRES RNA/eIF3-40S subunit complex. Moreover, the interactions between PGA1 and the elongation factors and ribosomal subunit were dependent upon HCV IRES RNA binding, and the PGA1/HCV IRES RNA/eIF3-40S subunit complex inhibited HCV-IRES-mediated translation. The novel mechanism revealed in this study may aid in the search for more effective anti-HCV drugs.