A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30°C up to 95°C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C 4 to C 16 ) examined was p-nitrophenyl caproate (C 6 ), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.

The hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase required for replication of the HCV RNA genome. We have identified a peptide that most closely resembles a short region of the protein kinase C-related kinase 2 (PRK2) by screening of a random 12-mer peptide library displayed on the surface of the M13 bacteriophage with NS5B proteins immobilized on microwell plates. Competitive phage enzyme-linked immunosorbent assay with a synthetic peptide showed that the phage clone displaying this peptide could bind HCV RNA polymerase with a high affinity. Coimmunoprecipitation and colocalization studies demonstrated in vivo interaction of NS5B with PRK2. In vitro kinase assays demonstrated that PRK2 specifically phosphorylates NS5B by interaction with the N-terminal finger domain of NS5B (amino acids 1-187). Consistent with the in vitro NS5B-phosphorylating activity of PRK2, we detected the phosphorylated form of NS5B by metabolic cell labeling. Furthermore, HCV NS5B immunoprecipitated from HCV subgenomic replicon cells was specifically recognized by an antiphosphoserine antibody. Knock-down of the endogenous PRK2 expression using a PRK2-specific small interfering RNA inhibited HCV RNA replication. In contrast, PRK2 overexpression, which was accompanied by an increase of in the level of its active form, dramatically enhanced HCV RNA replication. Altogether, our results indicate that HCV RNA replication is regulated by NS5B phosphorylation by PRK2. The hepatitis C virus (HCV)1 is a major cause of non-A and non-B hepatitis, leading to liver cirrhosis and hepatocellular carcinoma (1, 2). HCV is an enveloped virus with a positive stranded RNA genome of ϳ9.6 kb belonging to the Hepacivirus genus in the Flaviviridae family (3). The HCV viral genome encodes a single polyprotein of ϳ3,010 amino acids, which is proteolytically processed by a combination of host and viral proteases into at least 10 distinct structural and nonstructural proteins. The structural proteins include C, E1, E2, and p7, and the nonstructural (NS) proteins include NS2, NS3, NS4A, NS4B, NS5A, and NS5B (4, 5). Among the nonstructural proteins, HCV NS5B is an RNA-dependent RNA polymerase (RdRp) that is important for replication of the HCV RNA genome (6 -8). This protein contains motifs shared by all RdRps and possesses the finger, palm, and thumb subdomains (9 -12). HCV NS5B is anchored to the endoplasmic reticulum through the C-terminal domain of 21 hydrophobic amino acids (13-15) and forms a putative HCV RNA replicase complex with other viral NS proteins (16 -19).Many cellular enzymes involved in DNA and RNA metabolism, such as DNA polymerase ␣, topoisomerase II␣, and DNAdependent RNA polymerase I and II, are phosphoproteins, and their functions are known to be regulated by cellular kinase mediated-phosphorylation (20 -26). Several viral RdRps are also modified by phosphorylation. Dengue virus type-2 RNA polymerase is phosphorylated at a serine residue by casein kinase II. Phosphorylation of this polymerase regulates interaction...

Background: Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated.

A bstractThe hepatitis C virus (HCV) RNA-dependent RNA polym erase, NS5B protein, is the key viral enzym e responsible for replication of the HCV viral RNA genom e. Although several full-length and truncated form s of the HCV NS5B proteins have been expressed previously in insect cells, contam ination of host term inal transferase (TNTase) has ham pered analysis of the RNA synthesis initiation m echanism using natural HCV RNA tem plates. W e have expressed the HCV NS5B protein in insect cells using a recom binant baculovirus and purified it to near hom ogeneity w ithout contam inated TNTase. The highly purified recom binant HCV NS5B w as capable of copying 9.6-kb full-length HCV RNA tem plate, and m ini-HCV RNA carrying both 5'-and 3'-untranslated regions (UTRs) of the HCV genom e. In the absence of a prim er, and other cellular and viral factors, the NS5B could elongate over HCV RNA tem plates, but the synthesized products were prim arily in the double stranded form , indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA tem plates representing the 3'-end region of HCV m inus-strand RNA and the X-RNA at the 3'-end of HCV RNA genom e w as also initiated de novo. N o form ation of dim er-size self-prim ed RNA products resulting from extension of the 3'-end hydroxyl group w as observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)20, suggesting that HCV RNA polym erase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genom e.