Activation of the Ras/Raf/MEK pathway facilitates hepatitis C virus replication via attenuation of the interferon-JAK-STAT pathway

Hepatitis C virus (HCV) is a major cause of chronic liver diseases worldwide, often leading to the development of hepatocellular carcinoma (HCC). Constitutive activation of the Ras/Raf/MEK pathway is responsible for approximately 30% of cancers. Here we attempted to address the correlation between activation of this pathway and HCV replication. We showed that knockdown of Raf1 inhibits HCV replication, while activation of the Ras/Raf/MEK pathway by V12, a constitutively active form of Ras, stimulates HCV replication. We further demonstrated that this effect is regulated through attenuation of the interferon (IFN)-JAK-STAT pathway. Activation of the Ras/Raf/MEK pathway downregulates the expression of IFN-stimulated genes (ISGs), attenuates the phosphorylation of STAT1/2, and inhibits the expression of interferon (alpha, beta, and omega) receptors 1 and 2 (IFNAR1/2). Furthermore, we observed that HCV infection activates the Ras/Raf/MEK pathway. Thus, we propose that during HCV infection, the Ras/Raf/MEK pathway is activated, which in turn attenuates the IFN-JAK-STAT pathway, resulting in stimulation of HCV replication.     

Activation of the alternative complement pathway by mannose-binding lectin via a C2-bypass pathway

MBL is a serum lectin that activates the lectin pathway of the complement system. MBL forms complexes with three types of MASPs. Upon binding to Salmonella serogroup C-specific oligosaccharide, MBL activates the alternative pathway via a C2-bypass pathway without involving MASP-2, C2 or C4. We demonstrate that mannan-bound MBL activates the alternative pathway via a C2-bypass pathway that requires MASP-2 and C4. Thus, depending on the ligands to which MBL binds, there may be two distinct MBL-mediated C2-bypass pathways.     

Activation and evasion of the antiviral 2'-5' oligoadenylate synthetase/ribonuclease L pathway by hepatitis C virus mRNA

Chronic hepatitis C virus (HCV) infections are a significant cause of morbidity and mortality worldwide. Interferon-alpha2b treatment, alone or in combination with ribavirin, eliminates HCV from some patients, but patients infected with HCV genotype 1 viruses are cured less frequently than patients infected with HCV genotype 2 or 3 viruses. We report that HCV mRNA was detected and destroyed by the interferon-regulated antiviral 2'-5' oligoadenylate synthetase/ ribonuclease L pathway present in cytoplasmic extracts of HeLa cells. Ribonuclease L cleaved HCV mRNA into fragments 200 to 500 bases in length. Ribonuclease L cleaved HCV mRNA predominately at UA and UU dinucleotides within loops of predicted stem-loop structures. HCV mRNAs from relatively interferon-resistant genotypes (HCV genotypes 1a and 1b) have fewer UA and UU dinucleotides than HCV mRNAs from more interferon-sensitive genotypes (HCV genotypes 2a, 2b, 3a, and 3b). HCV 2a mRNA, with 73 more UA and UU dinucleotides than HCV 1a mRNA, was cleaved by RNase L more readily than HCV 1a mRNA. In patients, HCV 1b mRNAs accumulated silent mutations preferentially at UA and UU dinucleotides during interferon therapy. These results suggest that the sensitivity of HCV infections to interferon therapy may correlate with the efficiency by which RNase L cleaves HCV mRNA.     

Molecular structure of the Japanese hepatitis C viral genome

The amino acid sequence of the polyprotein deduced from the nucleotide sequence of the Japanese hepatitis C virus genome (N. Kato et. al. (1990) Proc. Natl. Acad. Sci. USA 87, 9524–9528)indicated that this virus is a member of a new class of positive-stranded RNA viruses. Several domains of this polyprotein also showed weak homology with those of flaviviruses and pestiviruses including the chymotrypsin-like serine proteinase. NTPase and RNA-dependent RNA polymerase     

High-resolution functional profiling of hepatitis C virus genome

Hepatitis C virus is a leading cause of human liver disease worldwide. Recent discovery of the JFH-1 isolate, capable of infecting cell culture, opens new avenues for studying HCV replication. We describe the development of a high-throughput, quantitative, genome-scale, mutational analysis system to study the HCV cis-elements and protein domains that are essential for virus replication. An HCV library with 15-nucleotide random insertions was passaged in cell culture to examine the effect of insertions at each genome location by insertion-specific fluorescent-PCR profiling. Of 2399 insertions identified in 9517 nucleotides of the genome, 374, 111, and 1914 were tolerated, attenuating, and lethal, respectively, for virus replication. Besides identifying novel functional domains, this approach confirmed other functional domains consistent with previous studies. The results were validated by testing several individual mutant viruses. Furthermore, analysis of the 3' non-translated variable region revealed a spacer role in virus replication, demonstrating the utility of this approach for functional discovery. The high-resolution functional profiling of HCV domains lays the foundation for further mechanistic studies and presents new therapeutic targets as well as topological information for designing vaccine candidates.     

Activation of the ERK pathway in osteoblastic cells, role of gremlin and BMP-2

Gremlin is a glycoprotein that binds and antagonizes the actions of bone morphogenetic proteins (BMPs) -2, -4, and -7. Gremlin appears to activate the extracellular regulated kinase (ERK) pathway in endothelial and tumor cells, and as a consequence to have direct cellular effects. To determine whether gremlin has direct effects in osteoblasts, independent of its BMP binding activity, we examined its effects in ST-2 murine stromal cell lines and in primary cultures of murine calvarial osteoblasts. Gremlin did not activate Signaling mothers against decapentaplegic (Smad), and suppressed the BMP-2 induced Smad 1/5/8 phosphorylation and the transactivation of the BMP/Smad reporter construct 12xSBE-Oc-pGL3, confirming its BMPs antagonizing activity. Neither gremlin nor BMP-2 induced ERK 1/2 activation in ST-2 cells or calvarial osteoblasts. Moreover, slight changes in culture conditions induced the phosphorylation of ERK independent from BMP or gremlin exposure. In conclusion, gremlin inhibits BMP-2 signaling and activity, and does not have independent actions on ERK signaling in osteoblasts. Consequently, gremlin activity in osteoblasts can be attributed only to its BMP antagonizing effects.     

Activation of the N-Ras-PI3K-Akt-mTOR pathway by hepatitis C virus: control of cell survival and viral replication

The hepatitis C virus (HCV) replication complex is localized within detergent-resistant membranes or lipid rafts. We analyzed the protein contents of detergent-resistant fractions isolated from Huh7 cells expressing a self-replicating full-length HCV-1b genome. Using two-dimensional gel electrophoresis followed by mass spectrometry, we identified N-Ras as one of the proteins in which expression was increased in the detergent-resistant fractions from HCV genomic replicon clones compared to control cells. N-Ras is an activator of the phosphatidylinositol-3-kinase (PI3K)-Akt pathway. We found that the activities of PI3K and Akt, as well as the activity of their downstream target, mTOR, in the HCV-replicating cells were increased. Both PI3K-Akt- and mTOR-dependent pathways have been shown to promote cell survival. In agreement with this, HCV replicon cells were resistant to serum starvation-induced apoptosis. We also characterized the role of this pathway in HCV replication. Reduction of N-Ras expression by transfection of N-Ras small interfering RNA (siRNA) resulted in increased replication of HCV. We observed a similar increase in HCV replication in cells treated with the PI3K inhibitor LY294002 and in cells transfected with mTOR siRNA. Taken together, these data suggest that increased N-Ras levels in subcellular sites of HCV replication and stimulation of the prosurvival PI3K-Akt pathway and mTOR by HCV not only protect cells against apoptosis but also contribute to the maintenance of steady-state levels of HCV replication. These effects may contribute to the establishment of persistent infection by HCV.     

Activation and function of the mTORC1 pathway in mast cells

Little is known about the signals downstream of PI3K which regulate mast cell homeostasis and function following FcepsilonRI aggregation and Kit ligation. In this study, we investigated the role of the mammalian target of rapamycin complex 1 (mTORC1) pathway in these responses. In human and mouse mast cells, stimulation via FcepsilonRI or Kit resulted in a marked PI3K-dependent activation of the mTORC1 pathway, as revealed by the wortmannin-sensitive sequential phosphorylation of tuberin, mTOR, p70S6 kinase (p70S6K), and 4E-BP1. In contrast, in human tumor mast cells, the mTORC1 pathway was constitutively activated and this was associated with markedly elevated levels of mTORC1 pathway components. Rapamycin, a specific inhibitor of mTORC1, selectively and completely blocked the FcepsilonRI- and Kit-induced mTORC1-dependent p70S6K phosphorylation and partially blocked the 4E-BP1 phosphorylation. In parallel, although rapamycin had no effect on FcepsilonRI-mediated degranulation or Kit-mediated cell adhesion, it inhibited cytokine production, and kit-mediated chemotaxis and cell survival. Furthermore, Rapamycin also blocked the constitutive activation of the mTORC1 pathway and inhibited cell survival of tumor mast cells. These data provide evidence that mTORC1 is a point of divergency for the PI3K-regulated downstream events of FcepsilonRI and Kit for the selective regulation of mast cell functions. Specifically, the mTORC1 pathway may play a critical role in normal and dysregulated control of mast cell homeostasis.     

Perturbation of autophagic pathway by hepatitis C virus

Autophagy removes long-lived proteins and damaged organelles and is important for maintaining cellular homeostasis. It can also serve in innate immunity to remove intracellular pathogens. As such, viruses have evolved different mechanisms to subvert this innate immune response. We have recently demonstrated that hepatitis C virus (HCV) can also suppress autophagic protein degradation by suppressing the fusion between autophagosomes and lysosomes. This suppression causes the accumulation of autophagosomes and enhances HCV RNA replication.(1) Our further analysis indicated that the induction of autophagosomes by HCV is dependent on the unfolded protein response (UPR). Our studies thus delineate a molecular pathway by which HCV induces autophagosomes. The prolonged perturbation of the autophagic pathway by HCV likely plays an important role in HCV pathogenesis.     

Ubiquitin-independent degradation of hepatitis C virus F protein

Hepatitis C virus (HCV) F protein is encoded by the +1 reading frame of the viral genome. It overlaps with the core protein coding sequence, and multiple mechanisms for its expression have been proposed. The full-length F protein that is synthesized by translational ribosomal frameshift at codons 9 to 11 of the core protein sequence is a labile protein. By using a combination of genetic, biochemical, and cell biological approaches, we demonstrate that this HCV F protein can bind to the proteasome subunit protein α3, which reduces the F-protein level in cells in a dose-dependent manner. Deletion-mapping analysis identified amino acids 40 to 60 of the F protein as the α3-binding domain. This α3-binding domain of the F protein together with its upstream sequence could significantly destabilize the green fluorescent protein, an otherwise stable protein. Further analyses using an F-protein mutant lacking lysine and a cell line that contained a temperature-sensitive E1 ubiquitin-activating enzyme indicated that the degradation of the F protein was ubiquitin independent. Based on these observations as well as the observation that the F protein could be degraded directly by the 20S proteasome in vitro, we propose that the full-length HCV F protein as well as the F protein initiating from codon 26 is degraded by an ubiquitin-independent pathway that is mediated by the proteasome subunit α3. The ability of the F protein to bind to α3 raises the possibility that the HCV F protein may regulate protein degradation in cells.     

Activation of the 5-lipoxygenase pathway in E-mast cells by peanut agglutinin

Mouse E-mast cells were differentiated and grown by culturing bone marrow cells in medium containing WEHI-3-conditioned medium. These cells possess surface receptors to the following agglutinins: peanut (PNA), concanavalin A (Con A), and soybean (Sb). One to 200 micrograms of PNA/10(6) E-mast cells selectively stimulated the generation of leukotriene C4 (LTC4) in the absence of beta-hexosaminidase release. Exposure of 10(6) E-mast cells to 1 to 200 micrograms Con A or Sb had no effect either on preformed mediator release or on the generation of leukotrienes. LTC4 was quantitated by integrated UV absorbance after resolution by reverse phase high pressure liquid chromatography. The optimum release of LTC4 (13.2 ng/10(6) cells) was achieved by 50 micrograms of PNA/10(6) cells. The response is characterized by the inhibition by excess amounts of PNA. The amount of LTC4 generated during optimal PNA stimulation is lower than the amount produced after stimulation by IgE-antigen or by calcium ionophore A23187 (19.8 ng and 148 ng, respectively). The release of LTC4 began within 5 min after PNA stimulation, and reached a plateau within 45 to 60 min at 37 degrees C. This kinetic pattern is similar to that observed after calcium ionophore A23187 stimulation of these cells. The results suggest that PNA is capable of selectively activating the 5-lipoxygenation of arachidonic acid without affecting beta-hexosaminidase secretion. Apparently, separate biochemical events may serve to mobilize each class of mediators.     

Structure of the 3' terminus of the hepatitis C virus genome.

Hepatitis C virus (HCV), a positive-strand RNA virus, has been considered to have a poly(U) stretch at the 3' terminus of the genome. We previously found a novel 98-nucleotide sequence downstream from the poly(U) stretch on the HCV genome by primer extension analysis of the 5' end of the antigenomic-strand RNA in infected liver (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215: 744-749, 1995). Here, we show that the novel sequence is a highly conserved 3' tail of the HCV genome. We repeated primer extension analyses with four HCV-infected liver samples and found the 98-nucleotide sequence in all the samples. Furthermore, experiments in which RNA oligonucleotide was ligated to the 3' end of the HCV genome existing in infectious serum revealed nearly identical 3' termini with no extra sequence downstream from the 98-nucleotide sequence, suggesting that this sequence is the tail of the HCV genome. This tail sequence was highly conserved among individuals and even between the two most genetically distant HCV types, II/1b and III/2a. Computer modeling predicted that the tail sequence can form a conserved stem-and-loop structure. These results suggest that the novel 3' tail is a common structure of the HCV genome that plays an important role in initiation of genomic replication.     

Interaction of hepatitis C virus F protein with prefoldin 2 perturbs tubulin cytoskeleton organization

By use of the yeast two-hybrid system, hepatitis C virus (HCV) F protein was found to interact with a cellular protein named prefoldin 2. The interaction was confirmed by confocal immunofluorescence microscopy as well as coimmunoprecipitation experiments. Prefoldin 2 is a subunit of a hexameric molecular chaperone complex, named prefoldin, which delivers nascent actin and tubulin proteins to the eukaryotic cytosolic chaperonin for facilitated folding. Functional prefoldin spontaneously assembles from its six subunits (prefoldin 1-6). In the yeast three-hybrid system, it was found that expression of HCV F protein impeded the interaction between prefoldin 1 and 2. By performing immunofluorescence experiment and non-denaturing gel electrophoresis, it was shown that expression of HCV F protein resulted in aberrant organization of tubulin cytoskeleton. Since HCV replication requires intact microtubule and actin polymerization, HCV F protein may serve as a modulator to prevent high level of HCV replication and thus contributes to viral persistence in chronic HCV infection.