Autophagy has been shown to facilitate replication or production of hepatitis C virus (HCV); nevertheless, how HCV induces autophagy remains unclear. Here, we demonstrate that HCV nonstructural protein 4B (NS4B) alone can induce autophagy signaling; amino acid residues 1 to 190 of NS4B are sufficient for this induction. Further studies showed that the phosphorylation levels of S6K and 4E-BP1 were not altered, suggesting that the mTOR/S6 kinase pathway and mTOR/4E-BP1 pathway did not contribute to NS4B-or HCV-induced autophagy. Inhibition of Rab5 function by silencing Rab5 or overexpressing dominant-negative Rab5 mutant (S34N) resulted in significant reduction of NS4B-or HCV-induced autophagic vesicle formation. Moreover, the autophagy induction was impaired by inhibition of class III phosphoinositide 3-kinase (PI 3-kinase) Vps34 function. Finally, the coimmunoprecipitation assay indicated that NS4B formed a complex with Rab5 and Vps34, supporting the notion that Rab5 and Vps34 are involved in NS4B-induced autophagy. Taken together, these results not only reveal a novel role of NS4B in autophagy but also offer a clue to the mechanism of HCV-induced autophagy.Hepatitis C virus (HCV) infections are a growing public health burden, with more than 180 million people infected worldwide. A striking feature of HCV infection is its tendency toward chronicity, often of significant liver disease, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (48). HCV is a positive-stranded RNA virus and classified into six genotypes (20). Its 9.6-kb genome encodes a single polyprotein, which is proteolytically processed into structural proteins (core, E1, E2, and p7), primarily forming the viral nucleocapsid and envelope, as well as nonstructural proteins (nonstructural protein 2 [NS2], NS3, NS4A, NS4B, NS5A, and NS5B) (35). Nonstructural proteins NS3 to NS5B are components of the membrane-associated HCV replication complex (16). NS3 is a bifunctional protein containing protease and helicase/nucleoside triphosphatase (NTPase) activities, and NS4A serves as a cofactor for NS3 protease. NS4B protein is known to induce formation of the membranous web that serves as the site for viral RNA replication. NS5A is required for RNA replication; phosphorylation of NS5A plays an important role in the HCV life cycle. NS5B is the RNA-dependent RNA polymerase (39). Although the roles of HCV proteins have been investigated, there is a great need for more understanding of the virus-host interaction and critical cellular players in the HCV life cycle that could be harnessed for anti-HCV therapy.

Open reading frame 1 (ORF1) of potexviruses encodes a viral replicase comprising three functional domains: a capping enzyme at the N terminus, a putative helicase in the middle, and a polymerase at the C terminus. To verify the enzymatic activities associated with the putative helicase domain, the corresponding cDNA fragment from bamboo mosaic virus (BaMV) was cloned into vector pET32 and the protein was expressed in Escherichia coli and purified by metal affinity chromatography. An activity assay confirmed that the putative helicase domain has nucleoside triphosphatase activity. We found that it also possesses an RNA 5-triphosphatase activity that specifically removes the ␥ phosphate from the 5 end of RNA. Both enzymatic activities were abolished by the mutation of the nucleoside triphosphate-binding motif (GKS), suggesting that they have a common catalytic site. A typical m 7 GpppG cap structure was formed at the 5 end of the RNA substrate when the substrate was treated sequentially with the putative helicase domain and the N-terminal capping enzyme, indicating that the putative helicase domain is truly involved in the process of cap formation by exhibiting its RNA 5-triphosphatase activity.Bamboo mosaic virus (BaMV) is a member of the potexvirus group, which belongs to the alphavirus-like superfamily. The ϳ6.4-kb positive-strand RNA genome of BaMV consists of a 94-nucleotide 5Ј-untranslated region, ORF1 (4,098 nucleotides), a triple gene block (ORF2 to ORF4), coat protein-coding region (ORF5), a 142-nucleotide 3Ј-untranslated region, and a poly(A) tail (20). ORF1 of BaMV encodes a 155-kDa polypeptide (replicase) whose amino acid sequence reveals three functional domains: an N-terminal Sindbis virus-like methyltransferase, a central putative RNA helicase, and a C-terminal RNA-dependent RNA polymerase (RdRp) (9,16,24). Recently, the activities of RdRp (18) and RNA capping (guanylyltransferase and methyltransferase) (19) in the C and N termini, respectively, of the BaMV replicase were verified. The central region of the 155-kDa replicase contains several conserved motifs belonging to superfamily 1 (SF1) of RNA helicases (14). This middle region (designated here the helicaselike domain) has thus been hypothesized to be an RNA helicase that assists RdRp in the RNA replication process by unwinding the duplex RNA structure. Besides the central helicase-like domain encoded by ORF1, the 28-kDa movement protein encoded by ORF2 also harbors nucleoside triphosphate (NTP)-binding helicase motifs. Although the overall homology is no more than 20%, the two BaMV proteins have similar sequences in regions containing putative motifs I, II, and VI of SF1 helicases. Since the products of triple gene block are indispensable for the movement of potexviruses through the plasmodesmata between host cells (4, 5), it is believed that the 28-kDa protein helps the viral genome move by its as yet unidentified helicase activity. Recently, the nucleoside triphosphatase (NTPase) and RNA-binding activities on the 28-kDa protein were corrobora...

Aberrant histone methylation is a frequent event during tumor development and progression. KMT1E (also known as SETDB1) is a histone H3K9 methyltransferase that contributes to epigenetic silencing of both oncogenes and tumor suppressor genes in cancer cells. In this report, we demonstrate that KMT1E acts as a metastasis suppressor that is strongly downregulated in highly metastatic lung cancer cells. Restoring KMT1E expression in this setting suppressed filopodia formation, migration, and invasive behavior. Conversely, loss of KMT1E in lung cancer cells with limited metastatic potential promoted migration in vitro and restored metastatic prowess in vivo. Mechanistic investigations indicated that KMT1E cooperates with the TGFb-regulated complex SMAD2/3 to repress metastasis through ANXA2. Together, our findings defined an essential role for the KMT1E/ SMAD2/3 repressor complex in TGFb-mediated lung cancer metastasis. Cancer Res; 74(24); 7333-43. Ó2014 AACR.

The major capsid protein of human polyomavirus JC virus, VP1, has been cloned into a baculovirus genome and expressed in insect cells. The VP1 protein was expressed in the cytoplasm and transported into the nucleus. It was then purified by a sucrose cushion and CsCl density gradient centrifugation to near homogeneity. Electron microscopy showed that isolated recombinant VP1 protein selfassembled into a capsid-like structure similar to the natural empty capsid. Both chelator (EDTA) and reducing agent (DTT) are required to disrupt the capsid structure into the pentameric capsomeres, as demonstrated by haemagglutination assay and electron microscopy. These results suggest that JC virus VP1 can be transported into the nucleus and self-assembled to form capsid-like particles without the involvement of the viral minor capsid proteins, VP2 and VP3. In addition, metal ions and disulphide bonds appear to be important in maintaining the integrity of the viral capsid structure.

Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5' cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an approximately 10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m(7)GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m(7)GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m(7)GTP sustained the formation of the m(7)GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m(7)GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m(7)GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m(7)GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m(7)GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.