Entry Inhibitors of Hepatitis C Virus.

Hepatitis C virus (HCV) infection affects approximately 1% of the world's population and is a major cause of chronic liver diseases. Although antiviral therapy consisting of direct-acting antivirals (DAAs) can cure the majority of HCV patients, it is still limited by viral resistances, drug-drug interactions, and high costs. Moreover, the role of DAAs in the prevention of occurrences of graft reinfection in HCV patients who receive liver transplantations is still under comprehensive clinical investigation, bringing the risk of recipient reinfection. HCV entry is composed of initial non-specific attachment and binding, post-binding interactions with essential host factors, internalization, and virion-cell membrane fusion to release viral RNA to cytosol. Thus, a number of novel and promising targets from either virion or cellular factors of these processes become optimal interfering elements for antiviral therapy, eliminating viral infection at the very beginning. Therefore, entry inhibitors can be supplemented into the future treatment regimens to optimize and widen the prevention and therapeutics of HCV infection. This chapter introduces the basic HCV entry processes and summarizes molecular mechanisms and research status of the current antiviral agents targeting HCV entry in preclinical and clinical study.

Lipid Droplets and Their Participation in Zika Virus Infection.

Lipid droplets (LDs) are highly conserved and dynamic intracellular organelles. Their functions are not limited to serving as neutral lipid reservoirs; they also participate in non-energy storage functions, such as cell lipid metabolism, protection from cell stresses, maintaining protein homeostasis, and regulating nuclear function. During a Zika virus (ZIKV) infection, the viruses hijack the LDs to provide energy and lipid sources for viral replication. The co-localization of ZIKV capsid (C) protein with LDs supports its role as a virus replication platform and a key compartment for promoting the generation of progeny virus particles. However, in view of the multiple functions of LDs, their role in ZIKV infection needs further elucidation. Here, we review the basic mechanism of LD biogenesis and biological functions and discuss how ZIKV infection utilizes these effects of LDs to facilitate virus replication, along with the future application strategy of developing new antiviral drugs based on the interaction of ZIKV with LDs.

Retinoic acid induced 16 deficiency exacerbates high-fat diet-induced steatohepatitis in mice.

Non-alcoholic fatty liver disease (NAFLD) associated with obesity may progress to non-alcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma (HCC). Retinoic acid induced 16 (RAI16) plays an important role in cell apoptosis and is also a potential marker for HCC. Here we aimed to test the effect of RAI16 deficiency on liver pathology in high-fat diet (HFD) fed mice. Wild type (WT) and RAI16 knockout (RAI16-/-) C57BL/6 mice were fed with HFD or chow for up to 12 months. With consumption of HFD diet, RAI16-/- mice on HFD developed much more excess fatty liver within 4 months than WT mice on HFD. The expressions of fatty acid synthesis associated molecules Ppar-γ, Srebp-1c and Fas were further increased in RAI16-/- mice compared with WT mice on HFD. Macrophage infiltration related molecules Mcp-1 and F4/80 and pro-inflammatory factor Lcn2 were significantly increased in RAI16-/- mice compared with WT mice on HFD. Conclusively, RAI16 deficiency exacerbated HFD-induced liver injury, associated with increased inflammation. These findings indicate that RAI16 plays an important role in HFD-induced liver pathology and might be considered as a target for treatment of NAFLD. SIGNIFICANCE: 1. RAI16-/- mice on HFD developed much more excess fatty liver. 2. RAI16-/- mice showed more macrophage infiltration and proinflammation.

RIP3 deficiency exacerbates inflammation in dextran sodium sulfate-induced ulcerative colitis mice model.

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease. The receptor-interacting protein kinase 3 (RIP3) was reported to be involved in many inflammatory disease. However, the mechanism of RIP3 in the pathogenesis of UC is still unclear. To investigate the effects and possible mechanism of RIP3 in UC pathogenesis, RIP3-/- mice was used in dextran sulfate sodium (DSS)-induced colitis model. It was found that by DSS-induced colitis, RIP3-/- mice showed significantly enhanced colitis symptoms, including increased weight loss, colon shortening, and colonic mucosa damage and severity, but decreased production of interleukin 6 and interleukin 1ß. The results showed that RIP3 deficiency could not ameliorate but exacerbate the severity of colitis. On the mechanism, it was found that messenger RNA expressions of several repair-associated cytokines including interleukin 6, interleukin 22, cyclooxygenase 2, epithelial growth factor receptor ligand Epiregulin and matrix metalloproteinase 10 were siginificant decreased in RIP3-/- mice. Thus, RIP3-/- mice exhibited an impaired tissue repair in response to DSS. In a conclusion, RIP3 deficiency exerted detrimental effects in DSS induced colitis partially because of the impaired repair-associated cytokines expression.

Trachelogenin, a novel inhibitor of hepatitis C virus entry through CD81.

Although much progress has been made in antiviral agents against hepatitis C virus (HCV) in recent years, novel HCV inhibitors with improved efficacy, optimized treatment duration and more affordable prices are still urgently needed. Here, we report the identification of a natural plant-derived lignan, trachelogenin (TGN), as a potent entry inhibitor of HCV without genotype specificity, and with low cytotoxicity. TGN was extracted and purified from Caulis trachelospermi, a traditional Chinese herb with anti-inflammatory and analgesic effects. A crucial function of TGN was the inhibition of HCV entry during a post-binding step without affecting virus replication, translation, assembly and release. TGN blocked virus infection by interfering with the normal interactions between HCV glycoprotein E2 and the host entry factor CD81, which are key processes for valid virus entry. In addition, TGN diminished HCV cell-to-cell spread and exhibited additional synergistic effects when combined with IFN or telaprevir. In conclusion, this study highlights the effect of a novel HCV entry inhibitor, TGN, which has a target that differs from those of the current antiviral agents. Therefore, TGN is a potential candidate for future cocktail therapies to treat HCV-infected patients.

Inhibition of STAT Pathway Impairs Anti-Hepatitis C Virus Effect of Interferon Alpha.

BACKGROUND/AIMS: Signal transducer and activator of transcription (STAT) pathway plays an important role in antiviral efficacy of interferon alpha (IFN-α). IFN-α is the main therapeutic against hepatitis C virus (HCV) infection. We explored effects of IFN-α on HCV replication and antiviral gene expression by targeting STAT. METHODS: In response to IFN-α, STAT status, HCV replication, and antiviral gene expression were analyzed in human hepatoma Huh7.5.1 cells before and after cell culture-derived HCV infection. RESULTS: IFN-α treatment induced expression and phosphorylation of STAT1 and STAT2 in Huh7.5.1 cells. Pretreatment of Huh7.5.1 cells with a mAb to IFN alpha receptor (IFNAR) 2 decreased IFN-α-dependent phosphorylation of STAT1 and STAT2, whereas pretreatment with an IFNAR1 mAb increased such phosphorylation, suggesting that IFNAR mediates IFN-α-triggered STAT signaling. During HCV infection, STAT1 and STAT2 phosphorylation could be rescued by IFN-α and IFN-α-induced phosphorylation of STAT1 and STAT2 was impaired. Inhibition of STAT pathway by Jak inhibitor I significantly enhanced HCV RNA replication and viral protein expression. Antiviral genes coding for IFN regulatory factor 9 and IFN-stimulated gene 15 were up-regulated by IFN-α during HCV infection but such up-regulation was abrogated by Jak inhibitor I. CONCLUSION: These results establish that activation of STAT pathway is essential for anti-HCV efficacy of IFN-α. Impairment of IFN-α-triggered STAT signaling by HCV may account for evading IFN-α response.

Interferon alpha antagonizes STAT3 and SOCS3 signaling triggered by hepatitis C virus.

We aimed to investigate regulation of signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) by interferon alpha (IFN-α) and to analyze the relationship between STAT3 and SOCS3 during hepatitis C virus (HCV) infection. Changes in STAT3 and SOCS3 were analyzed at both mRNA and protein levels in human hepatoma cells infected with HCV (J6/JFH1). At 72h of HCV infection, STAT3 expression was decreased with sustained phosphorylation, and IFN-α increased such decrease and phosphorylation. HCV increased SOCS3 expression, while IFN-α impaired such increase, indicating different regulation of STAT3 and SOCS3 by IFN-α. IFN-α-induced expression and phosphorylation of upstream kinases of the JAK/STAT pathway, Tyk2 and Jak1, were suppressed by HCV. Moreover, knockdown of STAT3 by RNA interference led to decreases in HCV RNA replication and viral protein expression, without affecting either the expression of Tyk2 and Jak1 or the SOCS3 induction in response to IFN-α. These results show that IFN-α antagonizes STAT3 and SOCS3 signaling triggered by HCV and that STAT3 regulation correlates inversely with SOCS3 induction by IFN-α, which may be important in better understanding the complex interplay between IFN-α and signal molecules during HCV infection.

Two conserved histidines (His490 and His621) on the E2 glycoprotein of hepatitis C virus are critical for CD81-mediated cell entry.

Hepatitis C virus (HCV) entry is a sequential and multi-step process that includes receptor interactions followed by pH-dependent membrane fusion. Specific and conserved histidine residues on the viral envelope proteins are involved in most pH-induced virus entries. In the case of HCV, some conserved histidines on the E1 and E2 proteins have been investigated in HCV pseudotype particle (HCVpp) systems. However, the roles of these histidines in cell-culture-derived HCV particle (HCVcc) systems remain unclear due to the different aspects of the viral life cycle emphasized by the two systems. In this study, the role of two conserved histidines (His490 and His621, located in domains II and III of E2, respectively) in HCV infection was evaluated in the context of JFH-1-based HCVcc using alanine substitutions. The infectivity of the H490A mutant decreased in spite of comparable initial RNA replication, protein expression and assembly efficiency as WT virus. The H621A mutant did not affect viral protein expression, but exhibited no obvious infectivity; there were fewer core proteins in the culture supernatant compared with WT virus, indicating the partially deficient virus assembly. The HCV receptor CD81-binding ability of the two mutant E2s was assessed further using enzyme immunoassays. The CD81-binding activity of H490A-E2 was reduced, and H621A-E2 was unable to bind to CD81. These data revealed the crucial role played by His490 and His621 in HCV infection, particularly during CD81 binding in cell entry. These results also contributed to the mechanical identification of the histidines involved in pH-dependent HCV entry.

The role of lipid rafts in the early stage of Enterovirus 71 infection.

BACKGROUND/AIMS: Although it has been widely accepted that Enterovirus 71 (EV71) enters permissive cells via receptor-mediated endocytosis, the details of entry mechanism for EV71 still need more exploration. This study aimed to investigate the role of lipid rafts in the early stage of EV71 Infection. METHODS: The effect of cholesterol depletion or addition of exogenous cholesterol was detected by immunofluorescence assays and quantitative real-time PCR. Effects of cholesterol depletion on the association of EV71 with lipid rafts were determined by flow cytometry and co-immunoprecipitation assays. Localization and internalization of EV71 and its receptor were assayed by confocal microscpoy and sucrose gradient analysis. The impact of cholesterol on the activation of phosphoinositide 3'-kinase/Akt signaling pathway during initial virus infection was analyzed by Western-blotting. RESULTS: Disruption of membrane cholesterol by a pharmacological agent resulted in a significant reduction in the infectivity of EV71. The inhibitory effect could be reversed by the addition of exogenous cholesterol. Cholesterol depletion post-infection did not affect EV71 infection. While virus bound equally to cholesterol-depleted cells, EV71 particles failed to be internalized by cholesterol-depleted cells. EV71 capsid protein co-localized with cholera toxin B, a lipid-raft-dependent internalization marker. CONCLUSION: Lipid rafts play a critical role in virus endocytosis and in the activation of PI3K/Akt signaling pathway in the early stage of EV71 infection.

Involvement of ERK pathway in interferon alpha-mediated antiviral activity against hepatitis C virus.

Interferon alpha (IFN-α) is the key component of the therapy for hepatitis C virus (HCV) infection. IFN-α exerts anti-HCV activity by targeting certain signaling pathways. Using infectious HCV culture system in human hepatoma Huh7.5.1 cells, we analyzed functional relevance of extracellular signal-regulated kinase (ERK) pathway for IFN-α-mediated anti-HCV activity. IFN-α treatment resulted in activation of ERK pathway by increasing phosphorylation of c-Raf, MEK, and ERK1/2 in Huh7.5.1 cells, whereas HCV impaired such activation. IFN-α-dependent ERK1/2 phosphorylation was blocked by MEK inhibitor U0126. Pharmacological inhibition of ERK1/2 by U0126 or siRNA-mediated knockdown of ERK1/2 resulted in suppressive effects on HCV RNA levels and expression of HCV nonstructural protein 3 and envelope protein 2, establishing an important role for ERK pathway in HCV replication. Moreover, induction of a set of antiviral genes by IFN-α was enhanced in HCV-infected Huh7.5.1 cells due to the ERK1/2 knockdown, suggesting that impairment of ERK signaling may potentiate HCV inhibition by IFN-α. These results demonstrate that ERK pathway is involved in IFN-α-mediated antiviral activity against HCV.

Inhibition of IRS-1 by hepatitis C virus infection leads to insulin resistance in a PTEN-dependent manner.

BACKGROUND: Hepatitis C virus (HCV) infection was recently recognized as an independent risk factor for insulin resistance (IR), the onset phase of type 2 diabetes mellitus (T2DM). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulates PI3K/Akt signaling pathway, which is critical for IR development and progression of cirrhosis to hepatocellular carcinoma (HCC). Here, we investigate the role of PTEN in HCV-associated IR and explored the mechanisms by which HCV regulates PTEN. METHODS: Western blotting was used to detect the levels of insulin signaling pathway components, including insulin receptor substrate-1 (IRS-1), phosphorylated IRS-1 (pIRS-1) at serine 307 (Ser307), both phosphorylated Akt (pAkt) and total Akt. A time-course experiment measuring activation of the insulin signaling pathway was performed to assess the effect of HCV infection on insulin sensitivity by examining the phosphorylation levels of Akt and GSK3ß, a downstream target of Akt. Huh7.5.1 cells were transduced with a lentiviral vector expressing PTEN or PTEN shRNA, and IRS-1 and pIRS-1 (Ser307) levels were determined in both HCV-infected and uninfected cells. The pc-JFH1-core plasmid was constructed to explore the underlying mechanisms by which HCV regulated PTEN and therefore IRS-1 levels. RESULTS: HCV infection inhibited the insulin signaling pathway by reducing the levels of IRS-1 and pAkt/Akt while increasing phosphorylation of IRS-1 Ser307. In addition, HCV infection decreased the sensitivity to insulin-induced stimulation by inhibiting Akt and GSK3ß phosphorylation. Furthermore, PTEN mRNA and protein levels were reduced upon HCV infection as well as transfection with the pc-JFH1-core plasmid. The reduction in IRS-1 level observed in HCV-infected cells was rescued to a limited extent by overexpression of PTEN, which in turn slightly reduced pIRS-1 (Ser307) level. In contrast, IRS-1 level were significantly decreased and phosphorylation of IRS-1 at Ser-307 was strongly enhanced by PTEN knockdown, suggesting that both reduction in IRS-1 level and increase in IRS-1 phosphorylation at Ser307 upon HCV infection occurred in a PTEN-dependent manner. CONCLUSIONS: HCV infection suppresses the insulin signaling pathway and promotes IR by repressing PTEN, subsequently leading to decreased levels of IRS-1 and increased levels of pIRS-1 at Ser307. The findings provide new insight on the mechanism of HCV-associated IR.

Small nucleolar RNA 113-1 suppresses tumorigenesis in hepatocellular carcinoma.

BACKGROUND: Emerging evidence suggests that small nucleolar RNAs (snoRNAs) are involved in tumorigenesis. The roles of small nucleolar RNA 113-1 (SNORD113-1) on the development of hepatocellular carcinoma (HCC) remain unknown. METHODS: The expression of SNORD113-1 was measured in 112 HCC tumor tissues using quantitative RT-PCR and compared with expression levels from with paired non-tumor tissues. The effects of SNORD113-1 on HCC tumorigenesis were investigated in HepG2 and Huh7 cells as well as a xenograft nude mouse model. CpG methylation within the promoter region of the SNORD113-1 gene was identified using Sodium bisulfite sequencing. Cancer pathway reporter investigate the mechanism by which SNORD113-1 suppressed tumorigenesis. RESULTS: SNORD113-1 expression was significantly downregulated in HCC tumors compared with adjacent non-tumor tissues, and downregulation of SNORD113-1 in HCC tumors was significantly associated with worse survival of patients. In addition, CpG methylation at the promoter region of the SNORD113-1 gene was higher in HCC tumors than adjacent non-tumor tissues. Functionally, SNORD113-1 suppressed cancer cell growth in HepG2 and Huh7 cells and in a xenograft nude mouse model. Furthermore, SNORD113-1 inactivated the phosphorylation of ERK1/2 and SMAD2/3 in MAPK/ERK and TGF-ß pathways. CONCLUSIONS: SNORD113-1 functions as a tumor suppressor role in HCC and may be important as a potential diagnostic and therapeutic target for HCC.

Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent, dynamin and caveola-mediated endocytosis pathway.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of the most common agents of viral encephalitis. The infectious entry process of JEV into host cells remains largely unknown. Here, we present a systemic study concerning the cellular entry mechanism of JEV to B104 rat neuroblastoma cells. It was observed that JEV internalization was inhibited by chloroquine and ammonium chloride, both of which can elevate the pH of acidic organelles. However, JEV entry was not affected by chlorpromazine, overexpression of a dominant-negative form of EPS 15 protein, or silencing of the clathrin heavy chain by small interfering RNA (siRNA). These results suggested that JEV entry depended on the acidic intracellular pH but was independent of clathrin. We found that endocytosis of JEV was dependent on membrane cholesterol and was inhibited by inactivation of caveolin-1 with siRNA or dominant-negative mutants. It was also shown, by using the inhibitor dynasore, the K44A mutant, and specific siRNA, that dynamin was required for JEV entry. Phagocytosis or macropinocytosis did not play a role in JEV internalization. In addition, we showed that JEV entry into the neuroblastoma cells is not virus strain specific by assessing the effect of the pharmacological inhibitors on the internalization of JEV belonging to different genotypes. Taken together, our results demonstrate that JEV enters B104 cells through a dynamin-dependent caveola-mediated uptake with a pH-dependent step, which is distinct from the clathrin-mediated endocytosis used by most flaviviruses.

Interferon alpha and ribavirin collaboratively regulate p38 mitogen-activated protein kinase signaling in hepatoma cells.

Signaling events triggered by interferon alpha (IFN-α) and ribavirin are involved in anti-hepatitis C virus (HCV) action. The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in HCV pathogenesis. Effects of IFN-α and ribavirin on p38 MAPK signaling were investigated in human hepatoma cells. Type I IFN receptor 2 (IFNAR2) mediated IFN-α-induced p38 MAPK phosphorylation. Also, p38 MAPK phosphorylation was enhanced by ribavirin. Treatment for 48 h with a combination of IFN-α and ribavirin increased p38 MAPK phosphorylation, whereas the treatment for 72 h reduced p38 MAPK phosphorylation. Cell culture-derived HCV (HCVcc) infection dramatically increased p38 MAPK phosphorylation and such phosphorylation was inhibited by IFN-α or ribavirin. Moreover, siRNA-mediated knockdown of p38 MAPK resulted in enhancement of ribavirin-dependent HCV RNA replication. These results suggest that regulation of p38 MAPK signaling by IFN-α and ribavirin might contribute to anti-HCV action.

MiR-138 induces cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma.

The deregulation of microRNA (miRNA) is frequently associated with a variety of cancers, including hepatocellular carcinoma (HCC). In this study, we identified 10 upregulated miRNAs (miR-217, miR-518b, miR-517c, miR-520g, miR-519a, miR-522, miR-518e, miR-525-3p, miR-512-3p and miR-518a-3p) and 10 downregulated miRNAs (miR-138, miR-214, miR-214#, miR-27a#, miR-199a-5p, miR-433, miR-511, miR-592, miR-483-5p and miR-483-3p) by Taqman miRNAs array and quantitative real-time PCR (qRT-PCR) confirmation. Additionally, we investigated the expression and possible role of miR-138 in HCC. qRT-PCR results showed that miR-138 was downregulated in 77.8%(14/18) of HCC tissues compared with adjacent non-tumor tissues. Overexpression of miR-138 reduced cell viability and colony formation by induction of cell arrest in HCC cell lines and inhibited tumor cell growth in xenograft nude mice. The use of miR-138 inhibitor increased cell viability and colony formation in HCC cell lines and tumor cell growth in xenograft nude mice. Using TargetScan predictions, CCND3 was defined as a potential direct target of miR-138. Furthermore, CCND3 protein expression was observed to be negatively correlated with miR-138 expression in HCC tissues. The dual-luciferase reporter gene assay results showed that CCND3 was a direct target of miR-138. The use of miR-138 mimic or inhibitor could decrease or increase CCND3 protein levels in HCC cell lines. We conclude that the frequently downregulated miR-138 can regulate CCND3 and function as a tumor suppressor in HCC. Therefore, miR-138 may serve as a useful therapeutic agent for miRNA-based HCC therapy.

Retinoic acid induced 16 enhances tumorigenesis and serves as a novel tumor marker for hepatocellular carcinoma.

Our previous work identified downregulated miR-483-5p in hepatocellular carcinoma (HCC). This study aims to identify the target of miR-483-5p, evaluate the potential value of this target as a tumor marker for HCC and explore the role of this target in HCC tumorigenesis. Upregulated retinoic acid induced 16 (RAI16) (17/18 cases) was negatively correlated with downregulated miR-483-5p (14/18 cases) in HCC tissues. The dual-luciferase reporter assay showed that RAI16 is a target of miR-483-5p. Immunohistochemistry analysis showed RAI16 was moderate or strong staining in tumor tissues but negative or weak staining in adjacent non-tumor tissues. The sensitivity and specificity of RAI16 for HCC diagnosis were 70.6 and 93.6%, respectively, and increased to 80.9 and 92.0% when combined with glypican-3. Finally, overexpression or knockdown of RAI16 increased or decreased cell viability and colony formation in HCC cell lines and enhanced or inhibited tumor cell growth in xenograft nude mice. Mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and transforming growth factor-ß pathways were mostly affected by RAI16. RAI16 could activate the phosphorylation of ERK1/2 and SMAD2/3. In conclusion, RAI16 may serve as a useful therapeutic agent for HCC gene therapy and tumor marker for HCC diagnosis.

Association of heat-shock protein 70 with lipid rafts is required for Japanese encephalitis virus infection in Huh7 cells.

Japanese encephalitis virus (JEV) is an enveloped flavivirus and the most common agent of viral encephalitis. It enters cells through receptor-mediated endocytosis and low pH-triggered membrane fusion. Although lipid rafts, cholesterol-enriched lipid-ordered membrane domains, have been shown to participate in JEV entry, the mechanisms of the early events of JEV infection, including the cellular receptors of JEV, remain largely unknown. In the current study, it was demonstrated that heat-shock protein 70 (HSP70), rather than other members of the HSP70 family, was required for JEV entry into a human cell line. Cell-surface expression of HSP70 and a direct interaction between JEV envelope (E) protein and HSP70 were observed. Biochemical fractionation showed that HSP70 clearly migrated into the raft fraction after virus infection and co-fractioned with E protein. Depletion of cholesterol shifted the E protein and HSP70 to a non-raft membrane and decreased JEV entry without affecting virus binding to host cells. Notably, recruitment of HSP70 into lipid rafts was required for activation of the phosphoinositide 3-kinase/Akt signalling pathway in the early stage of JEV infection. These results indicate that lipid rafts facilitate JEV entry, possibly by providing a convenient platform to concentrate JEV and its receptors on the host-cell membrane.

Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection.

Hepatitis C virus (HCV)-related research has been hampered by the lack of appropriate small-animal models. It has been reported that tree shrews, or tupaias (Tupaia belangeri), can be infected with serum-derived HCV. However, these reports do not firmly establish the tupaia as a reliable model of HCV infection. Human CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), and occludin (OCLN) are considered essential receptors or coreceptors for HCV cell entry. In the present study, the roles of these tupaia orthologs in HCV infection were assessed. Both CD81 and SR-BI of tupaia were found to be able to bind with HCV envelope protein 2 (E2). In comparison with human CD81, tupaia CD81 exhibited stronger binding activity with E2 and increased HCV pseudoparticle (HCVpp) cell entry 2-fold. The 293T cells transfected with tupaia CLDN1 became susceptible to HCVpp infection. Moreover, simultaneous transfection of the four tupaia factors into mouse NIH 3T3 cells made the cells susceptible to HCVpp infection. HCVpp of diverse genotypes were able to infect primary tupaia hepatocytes (PTHs), and this infection could be blocked by either anti-CD81 or anti-SR-BI. PTHs could be infected by cell culture-produced HCV (HCVcc) and did produce infectious progeny virus in culture supernatant. These findings indicate that PTHs possess all of the essential factors required for HCV entry and support the complete HCV infection cycle. This highlights both the mechanisms of susceptibility of tupaia to HCV infection and the possibility of using tupaia as a promising small-animal model in HCV study.

Zika virus infection triggers lipophagy by stimulating the AMPK-ULK1 signaling in human hepatoma cells.

Zika virus (ZIKV) is a globally transmitted mosquito-borne pathogen, and no effective treatment or vaccine is available yet. Lipophagy, a selective autophagy targeting lipid droplets (LDs), is an emerging subject in cellular lipid metabolism and energy homeostasis. However, the regulatory mechanism of lipid metabolism and the role of lipophagy in Zika virus infection remain largely unknown. Here, we demonstrated that ZIKV induced lipophagy by activating unc-51-like kinase 1 (ULK1) through activation of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) in Huh7 cells. Upon ZIKV infection, the average size and triglyceride content of LDs significantly decreased. Moreover, ZIKV infection significantly increased lysosomal biosynthesis and LD-lysosome fusion. The activities of AMPK at Thr-172 and ULK1 at Ser-556 were increased in ZIKV-infected cells and closely correlated with lipophagy induction. Silencing of AMPK expression inhibited ZIKV infection, autophagy induction, and LD-lysosome fusion and decreased the triglyceride content of the cells. The activities of mammalian target of rapamycin (mTOR) at Ser-2448 and ULK1 at Ser-757 were suppressed independently of AMPK during ZIKV infection. Therefore, ZIKV infection triggers AMPK-mediated lipophagy, and the LD-related lipid metabolism during ZIKV infection is mainly regulated via the AMPK-ULK1 signaling pathway.

Interferon alpha regulates MAPK and STAT1 pathways in human hepatoma cells.

BACKGROUND: Signaling events triggered by interferon (IFN) account for the molecular mechanisms of antiviral effect. JAK-STAT pathway plays a critical role in IFN signaling, and other pathways are also implicated in IFN-mediated antiviral effect. Changes in mitogen-activated protein kinase (MAPK) and STAT1 pathways were evaluated in human hepatoma cells Huh7 and HepG2 upon IFN alpha treatment. RESULTS: Phosphorylation of ERK was significantly and specifically up-regulated, whereas enhanced phosphorylation of upstream kinase MEK was unobservable upon IFN alpha treatment. A mild increase in p38 MAPK, SAPK/JNK and downstream target ATF-2 phosphorylation was detectable after exposure to IFN alpha, indicating differential up-regulation of the MAPK signaling cascades. Moreover, STAT1 phosphorylation was strongly enhanced by IFN alpha. CONCLUSION: IFN alpha up-regulates MAPK and STAT1 pathways in human hepatoma cells, and may provide useful information for understanding the IFN signaling.