RESUMO
The NS3 protein of hepatitis C virus (HCV) contains protease and RNA helicase activities, both of which are likely to be essential for HCV propagation. An arginine residue present in the arginine-glycine (RG)-rich region of many RNA-binding proteins is posttranslationally methylated by protein arginine methyltransferases (PRMTs). Amino acid sequence analysis revealed that the NS3 protein contains seven RG motifs, including two potential RG motifs in the 1486-QRRGRTGRG-1494 motif IV of the RNA helicase domain, in which arginines are potentially methylated by PRMTs. Indeed, we found that the full-length NS3 protein is arginine methylated in vivo. The full-length NS3 protein and the NS3 RNA helicase domain were methylated by a crude human cell extract. The purified PRMT1 methylated the full-length NS3 and the RNA helicase domain, but not the NS3 protease domain. The NS3 helicase bound specifically and comigrated with PRMT1 in vitro. Mutational analyses indicate that the Arg(1493) in the QRR(1488)GRTGR(1493)G region of the NS3 RNA helicase is essential for NS3 protein methylation and that Arg(1488) is likely methylated. NS3 protein methylation by the PRMT1 was decreased in the presence of homoribopolymers, suggesting that the arginine-rich motif IV is involved in RNA binding. The results suggest that an arginine residue(s) in QRXGRXGR motif IV conserved in the virus-encoded RNA helicases can be posttranslationally methylated by the PRMT1.
Assuntos
Motivos de Aminoácidos , Arginina/metabolismo , Hepacivirus/enzimologia , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Helicases/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Linhagem Celular , Deleção de Genes , Hepacivirus/química , Humanos , Metilação , Dados de Sequência Molecular , Mutação Puntual , RNA Helicases/metabolismo , Alinhamento de Sequência , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The E1 and E2 proteins of hepatitis C virus (HCV) are believed to be the viral envelope glycoproteins that are major candidate antigens for HCV vaccine development. We reported previously that the replication-competent recombinant adenovirus encoding core-E1-E2 genes of HCV (Ad/HCV) produces serologically reactive E1 and E2 proteins forming a heterodimer in substantial amounts. Here, we examined immunogenicity of the E1E2 proteins copurified from HeLa cells infected with Ad/HCV virus in mice. Furthermore, we constructed a replication-defective recombinant adenovirus encoding the core-E1-E2 genes of HCV (Ad.CMV.HCV) and examined immunogenicity of the virus in mice. The mice immunized intraperitoneally with the copurified E1E2 proteins induced mainly antibodies to E2, but not to E1 by Western blot analysis. The sera of mice immunized with the E1E2 inhibited the binding of E2 protein to the major extracellular loop of human CD81. E2-specific cytotoxic T cells (CTLs), but not antibodies to the E1E2 antigens were induced in the mice intramuscularly immunized with Ad.CMV.HCV virus. When immunized with both Ad.CMV.HCV virus and the E1E2, mice elicited E2-specific CTLs and antibodies to the E1E2 antigens. The results suggest that immunization of Ad.CMV.HCV virus combined with E2 protein is an effective modality to induce humoral as well as cellular immune response to E2 antigen.
Assuntos
Adenoviridae/genética , Hepacivirus/imunologia , Proteínas de Membrana , Proteínas do Envelope Viral/imunologia , Proteínas Estruturais Virais/imunologia , Animais , Antígenos CD/metabolismo , Feminino , Células HeLa , Anticorpos Anti-Hepatite C/sangue , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Linfócitos T Citotóxicos/imunologia , Tetraspanina 28 , Proteínas do Envelope Viral/metabolismoRESUMO
We found that JBP1, known as a human homolog (Skb1Hs) of Skb1 of fission yeast, interacts with NS3 of the hepatitis C virus in a yeast two-hybrid screen. Amino acid sequence analysis revealed that Skb1Hs/JBP1 contains conserved motifs of S-adenosyl-l-methionine-dependent protein-arginine methyltransferases (PRMTs). Here, we demonstrate that Skb1Hs/JBP1, named PRMT5, is a distinct member of the PRMT family. Recombinant PRMT5 protein purified from human cells methylated myelin basic protein, histone, and the amino terminus of fibrillarin fused to glutathione S-transferase. Myelin basic protein methylated by PRMT5 contained monomethylated and dimethylated arginine residues. Recombinant glutathione S-transferase-PRMT5 protein expressed in Escherichia coli also contained the catalytic activity. Sedimentation analysis of purified PRMT5 on a sucrose density gradient indicated that PRMT5 formed distinct homo-oligomeric complexes, including a dimer and tetramer, that comigrated with the enzyme activity. The PRMT5 homo-oligomers were dissociated into a monomer in the presence of a reducing agent, whereas a monomer, dimer, and multimer were detected in the absence or at low concentrations of a reducing agent. The results indicate that both covalent linkage by a disulfide bond and noncovalent association are involved in the formation of PRMT5 homo-oligomers. Western blot analysis of sedimentation fractions suggests that endogenous PRMT5 is present as a homo-oligomer in a 293T cell extract. PRMT5 appears to have lower specific enzyme activity than PRMT1. Although PRMT1 is known to be mainly located in the nucleus, human PRMT5 is predominantly localized in the cytoplasm.
Assuntos
Proteína-Arginina N-Metiltransferases/genética , Sequência de Aminoácidos , Sequência Conservada , Dimerização , Humanos , Metilação , Dados de Sequência Molecular , Proteína-Arginina N-Metiltransferases/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
Mutations of the tumor-suppressor gene p53 have been found in 30-50% cases of hepatocellular carcinoma (HCC). In this study, E1-negative adenoviral vector encoding wild-type p53 under the control of the human cytomegalovirus promoter (AdCMV-p53w) was constructed to evaluate its therapeutic efficacy against tumor nodules developing after injection of HuH7 cell lines in ten nude mice. When each nodule had reached 10 mm in perpendicular diameter, 1.5 x 10(8) pfu of AdCMV-p53w per session was injected intratumorally as follows: In group I (n=3), five sessions were injected every other day. In group II (n=3), only one session. Group III (n=4) as negative controls. The mice were sacrificed at 28 days post AdCMV-p53w injection. Tumor growth was significantly suppressed and delayed in group I and II compared to group III as compared by tumor volume at the end of observation. These results suggest that AdCMV-p53w may not only be effective in treating HCCs expressing mutant p53, but also useful as a local injectable gene therapy.
Assuntos
Adenovírus Humanos , Carcinoma Hepatocelular/terapia , Terapia Genética/métodos , Vetores Genéticos , Neoplasias Hepáticas/terapia , Proteína Supressora de Tumor p53/genética , Animais , Apoptose , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células Tumorais CultivadasRESUMO
Human adenoviruses have been used for mammalian expression vectors and recombinant vaccines for heterologous antigens. We constructed and characterized an infectious adenovirus recombinant containing core-E1-E2 genes of hepatitis C virus (HCV). The core protein was produced mainly during the early phase of viral infection. Expression of HCV E1 and E2 envelope proteins was detected by an immunoprecipitation with HCV-positive patient's sera. The purified E1 and E2 proteins appeared to be composed of mainly a heterodimeric form via noncovalent interaction, as previously observed in other mammalian expression systems. A small portion of E1 and E2 monomers as well as E1E2 aggregates by interdisulfide linkage were detected. Apparently heterodimeric E1E2 complexes were serologically reactive. The results suggest that adenovirus is an useful HCV antigen-expression vector.
Assuntos
Adenovírus Humanos , Vetores Genéticos , Hepacivirus/metabolismo , Proteínas do Core Viral/biossíntese , Proteínas do Envelope Viral/biossíntese , Linhagem Celular Transformada , Clonagem Molecular , Expressão Gênica , Células HeLa , Hepacivirus/genética , Humanos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas do Core Viral/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
A full-length and a truncated gene for the core protein of hepatitis C virus (HCV) were linked to the gene for glutathione S-transferase (GST), and the expression of each GST-HCV core fusion protein was analyzed. The truncated GST-HCV core (1-123) fusion protein was expressed as a mostly soluble and partly insoluble form comprising more than 50% of the total protein in Escherichia coli after induction by isopropylthio-beta-D-galactoside (IPTG), while the full length GST-HCV core (1-191) fusion protein was not expressed, suggesting that the hydrophobic carboxy terminal region in the core protein affects its expression. In addition, the GST-HCV core (1-123) fusion protein purified by GST-agarose chromatography reacted specifically with an anti-HCV serum from a patient.