Permissive Cell Lines Research Articles

Sodium taurocholate cotransporting polypeptide is the limiting host factor of hepatitis B virus infection in macaque and pig hepatocytes

Ntcp is the key host factor limiting HBV infection in cynomolgus and rhesus macaques and in pigs. In rodents (mouse, rat) and dogs, transfer of hNTCP supports viral entry but additional host factors are required for the establishment of HBV infection. This finding paves the way for the development of macaques and pigs as immunocompetent animal models to study HBV infection in vivo, immunological responses against the virus and viral pathogenesis. (Hepatology 2017;66:703-716).

Norovirus vaccines under development

Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis, including both outbreaks and endemic infections. The development of preventive strategies, including vaccines, for the most susceptible groups (children <5years of age, the elderly and individuals suffering crowding, such as military personnel and travelers) is desirable. However, NoV vaccine development has faced many difficulties, including genetic/antigenic diversity, limited knowledge on NoV immunology and viral cycle, lack of a permissive cell line for cultivation and lack of a widely available and successful animal model. Vaccine candidates rely on inoculation of virus-like particles (VLPs) formed by the main capsid protein VP1, subviral particles made from the protruding domain of VP1 (P-particles) or viral vectors with a NoV capsid gene insert produced by bioengineering technologies. Polivalent vaccines including multiple NoV genotypes and/or other viruses acquired by the enteric route have been developed. A VLP vaccine candidate has reached phase II clinical trials and several others are in pre-clinical stages of development. In this article we discuss the main challenges facing the development of a NoV vaccine and the current status of prevailing candidates.

Experimental infection of Tilapia Lake Virus (TiLV) in Nile tilapia (Oreochromis niloticus) and red tilapia (Oreochromis spp.)

Since 2015, a novel orthomyxo-like virus, tilapia lake virus (TiLV) has been associated with outbreaks of disease and massive mortality of cultured Nile and red tilapia (Oreochromis niloticus and Oreochromis spp., respectively) in Thailand. In this study, TiLV was isolated from field samples and propagated in the permissive E-11 cell line, with cytopathic effect (CPE) development within 3–5days post-inoculation. Electron micrographs of infected E-11 cells and fish tissues confirmed the rounded, enveloped virions of 60 to 80nm with characteristics very similar to those of Orthomyxoviridae. In vivo challenge studies showed that high mortality in Nile (86%) and red tilapia (66%) occurred within 4–12days post-infection. The virus was re-isolated from challenged fish tissues in the permissive cell line, and PCR analysis confirmed TiLV as a causative pathogen. The distinct histopathology of challenged fish included massive degeneration and inflammatory cell infiltration in the liver and brain as well as the presence of eosinophilic intracytoplasmic inclusions in hepatocytes and splenic cells. Our results fulfilled Koch’s postulates and confirmed that TiLV is an etiologic agent of mass mortality of tilapia in Thailand. The emergence of this virus in many countries has helped increase awareness that it is a potential threat to tilapia aquacultured in Thailand, Asia, and worldwide.

Real-Time Expression Analysis of Selected Anticarsia gemmatalis multiple nucleopolyhedrovirus Gene Promoters during Infection of Permissive, Semipermissive and Nonpermissive Cell Lines.

Baculovirus infection follows a transcriptionally controlled sequence of gene expression that occurs by activation of different viral gene promoter sequences during infection. This sequence of promoter activation may be disrupted by cellular defenses against viral infection, which might interfere with viral progeny formation. In this work, the activity of the ie1, gp64, lef-1, vp39, p6.9 and polh promoters of the Anticarsia gemmatalis multiple nucleopolyhedrovirus was assessed during infection of permissive, semipermissive and nonpermissive cell lines by a novel methodology that detects reporter protein luminescence in real-time. This technique allowed us to characterize in rich detail the AgMNPV promoters in permissive cell lines and revealed differential profiles of expression in cells with limited permissivity that correlate well with limitations in viral DNA replication. Semipermissive and nonpermissive cell lines presented delays and restrictions in late and very late promoter expression. Cells undergoing apoptosis did not inhibit late gene expression; however, viral progeny formation is severely affected. This work demonstrates the application of the real-time luminescence detection methodology and how the promoter expression profile may be used to diagnose cellular permissivity to baculovirus infection.

Development of a highly permissive cell line from spotted knifejaw (Oplegnathus punctatus) for red sea bream iridovirus

We developed a cell line (SKF-9) from spotted knifejaw (Oplegnathus punctatus), which is highly susceptible to red sea bream iridovirus (RSIV). Cytopathic effect (CPE) characterized by rounding and enlargement was clearly observed in RSIV-infected SKF-9 cells. The amount of viral production of SKF-9 cells was approximately 100 times greater than that of GF cells, which is conventionally used for RSIV research. The viral titer was not decreased through passages using SKF-9 cells, whereas a considerable reduction in the titer was observed when GF cells were used. On the other hand, control of appropriate passage numbers (<50) of the cell line was important to maintain the high permissiveness of SKF-9. Mortality rate was 100% when culture supernatant of RSIV propagated with SKF-9 cells was injected into spotted knifejaw with dilutions of 100, 10−5, and 10−6. Thus, the SKF-9 cell line can be a useful tool for research of RSIV (and ISKNV) and for vaccine production.

Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells

ABSTRACTThe Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1), was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO) cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin) were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.

Carbon Monoxide Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by the Cyclic GMP/Protein Kinase G and NF-κB Signaling Pathway.

PRRSV causes great economic losses each year to the swine industry worldwide. Carbon monoxide (CO), a metabolite of HO-1, has been shown to have antimicrobial and antiviral activities in infected cells. Our previous research demonstrated that HO-1 can suppress PRRSV replication. Here we show that endogenous CO produced through HO-1 catalysis mediates the antiviral effect of HO-1. CO inhibits PRRSV replication by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-κB signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks.

IDENTIFICATION OF A HIGHLY TRANSFECTABLE CELL LINE PERMISSIVE TO PORCINE EPIDEMIC DIARRHEA VIRUS INFECTION AND REPLICATION

Vero cell line has been routinely used for isolation and propagation of porcine epidemic diarrhea virus (PEDV), but isolation rate of PEDV in Vero cells has been demonstrated low, and the virus may gradually lose host infectivity upon in vitro passages. Besides, the entry mechanism of PEDV into Vero cells is also known to be cellular receptor (porcine aminopeptidase N; pAPN; CD13)-independent, which suggests that Vero cells might not be a suitable cell line for studying the interaction of PEDV with its receptor. To explore alternatives, a HEK 293 cell line stably expressing pAPN (HEK 293-pAPN) was established, and its susceptibility to PEDV infection was compared with Vero, HEK 293, and PK-15 cells. Interestingly, cytopathic effects characterized by cell fusion and multinuclear syncytial cells were observed in HEK 293, HEK 293-pAPN, and Vero cells after 48 h of inoculation, but not in PK-15 cells. Moreover, evidence of PEDV replication in these cell lines was also confirmed by immunocytochemistry staining and real-time quantitative PCR analysis. In conclusion, we demonstrated that HEK 293 cells, regardless of overexpression of pAPN molecules, are permissive to PEDV. HEK 293 cell line with its highly transfectable characteristic might not only serve as an alternative tool for studying the entry mechanisms of PEDV, but also a replication permissive cell line for virus rescue such as from the infectious clone of PEDV. Furthermore, the identification of a cell line of human origin, HEK 293, permissive to PEDV raises the concern of possible interspecies transmission.

Is gibbon ape leukaemia virus still a threat?

Abstract In the late 1960s and early 1970s, an outbreak of lymphoma and leukaemia in gibbons (Hylobatidae), attributed to the retrovirus gibbon ape leukaemia virus (GALV), was widely reported in the literature. The virus was identified in captive gibbon colonies in Thailand, the USA and Bermuda. The virus is a known cell culture contaminant and, in particular, research into HIV can be impeded by expression of GALV particles in HIV permissive cell lines. In this review, we bring together published work, laboratory records from early GALV research, correspondence about the transportation of gibbons during the 1960s and 1970s, phylogenetic analyses, laboratory screening and zoological records for the first time, to discover more about the origin and transmission of GALV. Based on this evidence, we suggest that GALV may have been transmitted to gibbons as an iatrogenic event and was never widespread. Instead, all infected gibbons were probably transported from the site of the original outbreak, housed with gibbons from this site or infected with material derived from gibbons from this site. We also propose that GALV is not an ongoing pathogen of captive gibbons.

Transduction of skin-migrating dendritic cells by human adenovirus 5 occurs via an actin-dependent phagocytic pathway.

Dendritic cells (DC) are central to the initiation of immune responses, and various approaches have been used to target vaccines to DC in order to improve immunogenicity. Cannulation of lymphatic vessels allows for the collection of DC that migrate from the skin. These migrating DC are involved in antigen uptake and presentation following vaccination. Human replication-deficient adenovirus (AdV) 5 is a promising vaccine vector for delivery of recombinant antigens. Although the mechanism of AdV attachment and penetration has been extensively studied in permissive cell lines, few studies have addressed the interaction of AdV with DC. In this study, we investigated the interaction of bovine skin-migrating DC and replication-deficient AdV-based vaccine vectors. We found that, despite lack of expression of Coxsackie B–Adenovirus Receptor and other known adenovirus receptors, AdV readily enters skin-draining DC via an actin-dependent endocytosis. Virus exit from endosomes was pH independent, and neutralizing antibodies did not prevent virus entry but did prevent virus translocation to the nucleus. We also show that combining adenovirus with adjuvant increases the absolute number of intracellular virus particles per DC but not the number of DC containing intracellular virus. This results in increased trans-gene expression and antigen presentation. We propose that, in the absence of Coxsackie B–Adenovirus Receptor and other known receptors, AdV5-based vectors enter skin-migrating DC using actin-dependent endocytosis which occurs in skin-migrating DC, and its relevance to vaccination strategies and vaccine vector targeting is discussed.

Characterization of a Chikungunya virus strain isolated from banked patients' sera.

BackgroundChikungunya virus (CHIKV) is a prevalent mosquito-borne pathogen that is emerging in many parts of the globe causing significant human morbidity. Here, we report the isolation and characterization of an infectious CHIKV from banked serum specimens of suspected patients from the 2009 epidemic in Thailand.MethodsStandard plaque assay was used for CHIKV isolation from the banked serum specimens. Isolated CHIKV was identified base on E1 structural gene sequence. Growth kinetic, infectivity, cell viability and cytokine gene expression throughout CHIKV infection in a permissive cell line, 293T cells, was performed using several approaches, including standard plaque assay, immunofluorescence assay, classical MTT assay, and quantitative real-time PCR. Two tailed Student’s t test was used for evaluation statistically significance between the mean values of the groups.ResultsBased on the E1 structural gene sequence and phylogenetic analysis, we identified the virus as the CHIK/SBY8/10 isolate from Indonesia. Assessment of the growth kinetics, cytopathic effects as well as its ability to induce cellular immune responses suggested that the currently isolated CHIK/SBY8/10 virus is relatively more virulent than a known CHIKV vaccine strain, which also induces more dramatic proinflammatory responses.ConclusionsOur studies further add to the infectivity of a less-studied yet infectious CHIKV isolate as well as underscored the importance and utility of 293T cells as an excellent cell culture model for studying viral growth, CHIKV-induced inflammatory cellular responses and cell death. Together, these studies provide novel information on the CHIKV biology, infectivity and virus-cell interaction, which would help develop novel interventions against the infection.

Abstract 4356: Identification of the anthrax toxin receptor (ANTXR1) as the high affinity cellular receptor for Seneca Valley Virus (SVV)

Abstract Small cell lung cancer (SCLC) is an extremely aggressive and almost universally lethal form of lung cancer, which accounts for approximately 15% of all lung cancer diagnoses annually. Novel and more effective therapies are desperately needed for SCLC, as the standard treatment has not improved in almost three decades. Recently, oncolytic viruses have shown strong anti-cancer efficacies in tumors with high levels of selectivity for cancer cells. The oncolytic picornavirus, Seneca Valley Virus (SVV), has previously been shown to infect and lyse cancer cells with neuroendocrine features, notably SCLC and pediatric brain tumors. Pre-clinical mouse models and early phase clinical trials have confirmed the ability of SVV to home directly to the tumor through the vasculature, specifically infect tumor cells without affecting normal cells, and replicate to high viral titers intratumorally even after the production of neutralizing antibodies. However, the clinical development of SVV has been hindered by the inability to identify patients who would benefit from SVV virotherapy because essential host proteins for viral entry, including the cellular receptor, have not been characterized. Using genome wide loss-of-function CRISPR screens in the SCLC cell line H446 and haploid cell line HAP1, we identified the anthrax toxin receptor 1 (ANTXR1) as an essential host protein for SVV. Interestingly, ANTXR1 is also one of the receptors for the toxin of Bacillus anthracis and has been previously described as being upregulated in tumor endothelial cells. Secondary screens performed in both cell lines confirmed gene knockout of ANTXR1 conferred resistance to SVV. We established that in multiple permissive SCLC and pediatric cancer cell lines that loss of ANTXR1 protein expression using CRISPR gene knockout leads to a loss of SVV permissivity. Re-expression of ANTXR1 in ANTXR1 knockout cell lines was sufficient to rescue SVV permissivity. Furthermore, the expression of the ANTXR1 protein in two non-permissive SCLC cell lines led to a conversion in SVV permissivity. A direct and high affinity interaction was confirmed between SVV and ANTXR1 using co-immunoprecipitation assays. Additionally, we discovered that ANTXR1 is the major binding determinant for SVV on cells using a binding assay with fluorescently labeled SVV. Lastly we determined using previously published gene expression data from the Cancer Cell Line Encyclopedia (CCLE) that non-permissive lines which do express ANTXR1 can be explained by the presence of an intact and active innate immune response pathway. These findings identify ANTXR1 as the high-affinity cellular receptor for SVV. Furthermore, these data identify a predictive biomarker for SVV permissivity that could lead to defined selection criteria for subsequent clinical trials testing SVV in both SCLC and pediatric brain cancer patients. Citation Format: Linde A. Miles, J.T. Poirier, Charles M. Rudin. Identification of the anthrax toxin receptor (ANTXR1) as the high affinity cellular receptor for Seneca Valley Virus (SVV). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4356.

Innate immune defects in HIV permissive cell lines

BackgroundPrimary CD4+ T cells and cell lines differ in their permissiveness to HIV infection. Impaired innate immunity may contribute to this different phenotype.FindingsWe used transcriptome profiling of 1503 innate immunity genes in primary CD4+ T cells and permissive cell lines. Two clusters of differentially expressed genes were identified: a set of 249 genes that were highly expressed in primary cells and minimally expressed in cell lines and a set of 110 genes with the opposite pattern. Specific to HIV, HEK293T, Jurkat, SupT1 and CEM cell lines displayed unique patterns of downregulation of genes involved in viral sensing and restriction. Activation of primary CD4+ T cells resulted in reversal of the pattern of expression of those sets of innate immunity genes. Functional analysis of prototypical innate immunity pathways of permissive cell lines confirmed impaired responses identified in transcriptome analyses.ConclusionIntegrity of innate immunity genes and pathways needs to be considered in designing gain/loss functional genomic screens of viral infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0275-8) contains supplementary material, which is available to authorized users.

Select membrane proteins modulate MNV-1 infection of macrophages and dendritic cells in a cell type-specific manner

Noroviruses cause gastroenteritis in humans and other animals, are shed in the feces, and spread through the fecal-oral route. Host cellular expression of attachment and entry receptors for noroviruses is thought to be a key determinant of cell tropism and the strict species-specificity. However, to date, only carbohydrates have been identified as attachment receptors for noroviruses. Thus, we investigated whether host cellular proteins play a role during the early steps of norovirus infection. We used murine norovirus (MNV) as a representative norovirus, since MNV grows well in tissue culture and is a frequently used model to study basic aspects of norovirus biology. Virus overlay protein binding assay followed by tandem mass spectrometry analysis was performed in two permissive cell lines, RAW264.7 (murine macrophages) and SRDC (murine dendritic cells) to identify four cellular membrane proteins as candidates. Loss-of-function studies revealed that CD36 and CD44 promoted MNV-1 binding to primary dendritic cells, while CD98 heavy chain (CD98) and transferrin receptor 1 (TfRc) facilitated MNV-1 binding to RAW 264.7 cells. Furthermore, the VP1 protruding domain of MNV-1 interacted directly with the extracellular domains of recombinant murine CD36, CD98 and TfRc by ELISA. Additionally, MNV-1 infection of RAW 264.7 cells was enhanced by soluble rCD98 extracellular domain. These studies demonstrate that multiple membrane proteins can promote efficient MNV-1 infection in a cell type-specific manner. Future studies are needed to determine the molecular mechanisms by which each of these proteins affect the MNV-1 infectious cycle.

MicroRNA profile analysis of host cells before and after wild human rotavirus infection.

Rotavirus infection is an important cause of acute gastroenteritis in children, but the interaction between rotavirus and host cells is not completely understood. We isolated a wildtype (wt) rotavirus strain, ZTR-68(P [8] G1), which is derived from an infant with diarrhea in southwest China in 2010. In this study, we investigated host cellular miRNA expression profiles changes in response to ZTR-68 in early stage of infection to investigate the role of miRNAs upon rotavirus infection. Differentially expressed miRNAs were identified by deep sequencing and qRT-PCR and the function of their targets predicted by Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. A total of 36 candidate miRNAs were identified. Comparative analysis indicated that 29 miRNAs were significantly down-regulated and 7 were up-regulated after infection. The data were provided contrasting the types of microRNAs in two different permissive cell lines (HT29 and MA104). The target assays results showed that mml-miR-7 and mml-miR-125a are involved in anti-rotavirus and virus-host interaction in host cells. These results offer clues for identifying potential candidates in vector-based antiviral strategies. J. Med. Virol. 88:1497-1510, 2016. © 2016 Wiley Periodicals, Inc.

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

ABSTRACTTo assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrPSc accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrPres isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrPSc accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.

Influenza a Viruses use Multivalent Sialic Acid Clusters for Cell Binding and Receptor Activation

Binding of influenza A virus to the host cell is mediated by the major viral surface protein hemagglutinin (HA). HA recognises sialic acid (SA), a plasma membrane glycan that functions as the specific primary virus attachment factor. Since, sialic acid alone can not fulfil a signalling function, the virus needs to activate downstream factors in order to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase (RTK) family, was shown to be involved and transmit virus entry signals. However, how influenza viruses engage with and activate EGFR is largely unknown. Using quantitative super-resolution microscopy (STORM and PALM), we show that SA and EGFR are organized in submicrometer clusters in the apical plasma membrane of different permissive cell lines. We found that the local SA concentration, a parameter that directly influences virus-cell binding, strongly increases towards the cluster center, thereby representing a multivalent virus-binding platform. By using 2-color super-resolution microscopy, we show that SA and EGFR clusters co-localize, eventually allowing SA-mediated virus-EGFR interaction. Finally, we show that virus binding activates EGFR and that this process occurs without a major EGFR redistribution, suggesting the activation of preformed clusters. Further, our quantitative characterization of SA distribution on the host cell membrane, enabled us to simulate 2D virus trajectories, which could be confirmed by live cell single-virus tracking. Taken together, our results provide a quantitative functional model of the initial events of influenza virus infection and provide insights into how the virus finds and activates EGFR.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development.

Type I and type II interferon responses in two human liver cell lines (Huh-7 and HuH6).

Most studies investigating the biology of Hepatitis C virus (HCV) have used the human hepatoma cell line Huh-7 or subclones thereof, as these are the most permissive cell lines for HCV infection and replication. Other cell lines also support replication of HCV, most notably the human hepatoblastoma cell line HuH6. HCV replication in cell culture is generally highly sensitive to interferons (IFNs) and differences in the IFN-mediated inhibition of virus replication may reflect alterations in the IFN-induced antiviral response inherent to different host cells. For example, HCV replication is highly sensitive to IFN-γ treatment in Huh-7, but not in HuH6 cells. In this study, we used microarray-based gene expression profiling to compare the response of Huh-7 and HuH6 cells to stimulation with IFN-α and IFN-γ. Furthermore, we determined whether the resistance of HCV replication in HuH6 cells can be linked to differences in the expression profile of IFN-regulated genes. Although both cells lines responded to IFNs with rapid changes in gene expression, thereby demonstrating functional type I and type II signaling pathways, differences were observed for a number of genes. Raw and normalized expression data have been deposited in GEO under accession number GSE68927.

Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines

Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.