Cytoplasmic Translocation of Polypyrimidine Tract-Binding Protein and Its Binding to Viral RNA during Japanese Encephalitis Virus Infection Inhibits Virus Replication

Japanese encephalitis virus (JEV) has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5′- and 3′-non-coding regions (NCRs). The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB) interacts in vitro with both the 5′-NCR of the positive-sense genomic RNA - 5NCR(+), and its complementary sequence in the negative-sense replication intermediate RNA - 3NCR(-). The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(-) RNA with viral RNA-dependent RNA polymerase (NS5 protein), an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA)-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.     

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

The machinery for hepatitis C virus (HCV) RNA replication is still poorly characterized. The relationship between HCV RNA replication and translation is also not clear. We have previously shown that a cellular protein polypyrimidine-tract-binding protein (PTB) binds to HCV RNA at several different sites and modulates HCV translation in several ways. Here we show that PTB also participates in RNA replication. By bromouridine triphosphate (BrUTP) labeling and confocal microscopy of cells harboring an HCV replicon, we showed that the newly synthesized HCV RNA was localized to distinct structures in the cytoplasm, which also contain PTB. Membrane flotation analysis demonstrated that a fraction of cytoplasmic PTB was associated with a detergent-resistant membrane (DRM) structure consisting of lipid rafts, which also contained HCV nonstructural proteins and the human vesicle-associated membrane protein-associated protein (hVAP-33). PTB in the DRM was resistant to protease digestion, but became sensitive after treatment with the raft-disrupting agents. PTB in the DRM consisted of multiple isoforms and the brain-specific paralog. By using small interfering RNA (siRNA) of PTB, we showed that silencing of the endogenous PTB reduced the replication of HCV RNA replicon. In a cell-free, de novo HCV RNA synthesis system, HCV RNA synthesis was inhibited by anti-PTB antibody. These studies together indicated that PTB is a part of the HCV RNA replication complex and participates in viral RNA synthesis. Thus, PTB has dual functions in HCV life cycle, including translation and RNA replication.     

hnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'NTR of the HCV RNA genome

Like other members of the Flaviviridae family, the 3' non-translated region (NTR) of the hepatitis C virus (HCV) is believed to function in the initiation and regulation of viral RNA replication by interacting with components of the viral replicase complex. To inves-tigate the possibility that host components may also participate in this process, we used UV cross-linking assays to determine if any cellular proteins could bind specifically to the 3'NTR RNA. We demonstrate the specific interaction of two host proteins with the extensive pyrimidine-rich region within the HCV 3'NTR. One host protein migrates as a doublet with a molecular weight of 57 kDa and is immunoreactive with antisera specific for polypyrimidine tract-binding protein (PTB), and the other protein (35 kDa) is recognized by a monoclonal antibody specific for heterogeneous nuclear ribonucleoprotein C (hnRNP C). These results suggest that recognition of the large pyrimidine-rich region by PTB and hnRNP C may play a role in the initiation and/or regulation of HCV RNA replication.     

Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication

Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly.     

Hepatitis C Virus Infection Causes Iron Deficiency in Huh7.5.1 Cells

Patients with chronic hepatitis C virus (HCV) infection frequently develop systemic iron overload, which exacerbates morbidity. Nevertheless, iron inhibits HCV replication in cell culture models and thereby exerts antiviral activity. We hypothesized that the cellular iron status is crucial for the establishment of HCV infection. We show that HCV infection of permissive Huh7.5.1 hepatoma cells promotes an iron deficient phenotype. Thus, HCV leads to increased iron regulatory protein (IRP) activity, accumulation of IRP2 and suppression of transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) in the host. These data suggest that HCV regulates cellular iron levels to bypass iron-mediated inhibition in viral replication.     

17β-estradiol inhibits the production of infectious particles of hepatitis C virus

Persistent infection with hepatitis C virus causes serious liver diseases, such as chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma. The male gender is one of the critical factors in progression of hepatic fibrosis due to chronic HCV infection; thus female hormones may play a role in delaying the progression of hepatic fibrosis. It has also been reported that women are more likely than men to clear HCV in the acute phase of infection. These observations lead the present authors to the question: do female hormones inhibit HCV infection? In this study using HCV J6/JFH1 and Huh‐7.5 cells, the possible inhibitory effect(s) of female hormones such as 17β‐estradiol (the most potent physiological estrogen) and progesterone on HCV RNA replication, HCV protein synthesis and production of HCV infectious particles (virions) were analyzed. It was found that E₂, but not P₄, significantly inhibited production of the HCV virion without inhibiting HCV RNA replication or HCV protein synthesis. E₂–mediated inhibition of HCV virion production was abolished by a nuclear estrogen receptor (ER) antagonist ICI182780. Moreover, treatment with the ERα‐selective agonist 4, 4′, 4″‐ (4‐propyl‐[1H]‐pyrazole‐1, 3, 5‐triyl)trisphenol (PPT), but not with the ERβ‐selective agonist 2, 3‐bis (4‐hydroxyphenyl)‐propionitrile (DPN) or the G protein‐coupled receptor 30 (GPR30)‐selective agonist 1‐(4‐[6‐bromobenzo 1, 3 dioxol‐5‐yl]‐3a, 4, 5, 9b‐tetrahydro‐3H‐cyclopenta [c] quinolin‐8‐yl)‐ethanone (G‐1), significantly inhibited HCV virion production. Taken together, the present results suggest that the most potent physiological estrogen, E₂, inhibits the production of HCV infectious particles in an ERα–dependent manner.     

Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.     

Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication

Hepatitis C virus (HCV) infection is treated with interferon (IFN)-based therapy. The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2alpha) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2alpha phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively, target eIF2- and eIF3-dependent steps in the viral RNA translation initiation process.     

Replication Vesicles are Load- and Choke-Points in the Hepatitis C Virus Lifecycle

Hepatitis C virus (HCV) infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. To understand how the virus establishes its tightly controlled intracellular RNA replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular HCV RNA replication. We therefore quantitatively measured viral RNA and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly validated the model using additional, independent experiments. Model analysis was used to predict the efficacy of different classes of inhibitors and identified sensitive substeps of replication that could be targeted by current and future therapeutics. A protective replication compartment proved to be essential for sustained RNA replication, balancing translation versus replication and thus effectively limiting RNA amplification. The model predicts that host factors involved in the formation of this compartment determine cellular permissiveness to HCV replication. In gene expression profiling, we identified several key processes potentially determining cellular HCV replication efficiency.     

Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators

Background

Hepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide. There is no protective vaccine currently available for HCV, therefore the development of novel strategy to prevent chronic infection is important. We reported earlier that a recombinant human antibody clone blocks viral NS3 helicase activity and inhibits replication of HCV 1b virus. This study was performed further to explore the mechanism of action of this recombinant antibody and to determine whether or not this antibody inhibits replication and infectivity of a highly efficient JFH1 HCV 2a virus clone.

Results

The antiviral effect of intracellular expressed antibody against the HCV 2a virus strain was examined using a full-length green fluorescence protein (GFP) labeled infectious cell culture system. For this purpose, a Huh-7.5 cell line stably expressing the NS3 helicase gene specific IgG1 antibody was prepared. Replication of full-length HCV-GFP chimera RNA and negative-strand RNA was strongly inhibited in Huh-7.5 cells stably expressing NS3 antibody but not in the cells expressing an unrelated control antibody. Huh-7.5 cells stably expressing NS3 helicase antibody effectively suppressed infectious virus production after natural infection and the level of HCV in the cell free supernatant remained undetectable after first passage. In contrast, Huh-7.5 cells stably expressing an control antibody against influenza virus had no effect on virus production and high-levels of infectious HCV were detected in culture supernatants over four rounds of infectivity assay. A recombinant adenovirus based expression system was used to demonstrate that Huh-7.5 replicon cell line expressing the intracellular antibody strongly inhibited the replication of HCV-GFP RNA.

Conclusion

Recombinant human anti-HCV NS3 antibody clone inhibits replication of HCV 2a virus and infectious virus production. Intracellular expression of this recombinant antibody offers a potential antiviral strategy to inhibit intracellular HCV replication and production.     

Polypyrimidine Tract Binding Protein Functions as a Negative Regulator of Feline Calicivirus Translation

Background

Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5′ or 3′ extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5′end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle.

Principal Findings

We have demonstrated that PTB interacts with at least two binding sites within the 5′end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication.

Conclusions

Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may contribute to the stimulation of viral RNA replication via clearance of ribosomes from viral RNA.     

Influenza A Virus Host Shutoff Disables Antiviral Stress-Induced Translation Arrest

Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication.     

Inhibition of hepatitis C virus replication through adenosine monophosphate-activated protein kinase-dependent and -independent pathways

Persistent infection with hepatitis C virus (HCV) is closely correlated with type 2 diabetes. In this study, replication of HCV at different glucose concentrations was investigated by using J6/JFH1-derived cell-adapted HCV in Huh-7.5 cells and the mechanism of regulation of HCV replication by AMP-activated protein kinase (AMPK) as an energy sensor of the cell analyzed. Reducing the glucose concentration in the cell culture medium from 4.5 to 1.0 g/L resulted in suppression of HCV replication, along with activation of AMPK. Whereas treatment of cells with AMPK activator 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) suppressed HCV replication, compound C, a specific AMPK inhibitor, prevented AICAR's effect, suggesting that AICAR suppresses the replication of HCV by activating AMPK in Huh-7.5 cells. In contrast, compound C induced further suppression of HCV replication when the cells were cultured in low glucose concentrations or with metformin. These results suggest that low glucose concentrations and metformin have anti-HCV effects independently of AMPK activation.     

Host sphingolipid biosynthesis as a target for hepatitis C virus therapy

An estimated 170 million individuals worldwide are infected with hepatitis C virus (HCV), a serious cause of chronic liver disease. Current interferon-based therapy for treating HCV infection has an unsatisfactory cure rate, and the development of more efficient drugs is needed. During the early stages of HCV infections, various host genes are differentially regulated, and it is possible that inhibition of host proteins affords a therapeutic strategy for treatment of HCV infection. Using an HCV subgenomic replicon cell culture system, here we have identified, from a secondary fungal metabolite, a lipophilic long-chain base compound, NA255 (1), a previously unknown small-molecule HCV replication inhibitor. NA255 prevents the de novo synthesis of sphingolipids, major lipid raft components, thereby inhibiting serine palmitoyltransferase, and it disrupts the association among HCV nonstructural (NS) viral proteins on the lipid rafts. Furthermore, we found that NS5B protein has a sphingolipid-binding motif in its molecular structure and that the domain was able to directly interact with sphingomyelin. Thus, NA255 is a new anti-HCV replication inhibitor that targets host lipid rafts, suggesting that inhibition of sphingolipid metabolism may provide a new therapeutic strategy for treatment of HCV infection.     

Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.