Hepatitis C Virus NS5B Protein Is a Membrane-Associated Phosphoprotein with a Predominantly Perinuclear Localization

Hwang, Soon B.; Park, Kyu-Jin; Kim, Yong‐Sun; Sung, Young Chul; Lai, Michael M. C.

doi:10.1006/viro.1996.8357

Cited by 104 publications

(102 citation statements)

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“…The mouse monoclonal antibody against NS5B was made by NS5B protein expressed from a recombinant baculovirus and was a generous gift of Dr. Soon Hwang (Hallym University, Korea) [28,29]. The mouse monoclonal antibody against PTB isoforms 1, 2, and 4 (anti-PTB) was purified from culture medium of the mouse hybridoma cell, BB7, purchased from ATCC (Manassas, VA).…”

Section: Antibodiesmentioning

confidence: 99%

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

et al. 2006

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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 proteinassociated 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.

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Section: Antibodiesmentioning

confidence: 99%

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

et al. 2006

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“…To test the effect of PRK2 knock-down on NS5B phosphorylation, Huh7TR-NS stable cells seeded in a 10-cm-diameter plate were induced with 1 g/ml tetracycline and transfected with 5 g of either pSUPER or pSUPER-PRK2 using FuGENE 6. After further growth for 48 h, cell labeling with 32 P i was performed as described above. Expression levels of PRK2 were determined by immunoblotting with anti-PRK2 antibodies.…”

Section: Establishment Of a Tetracycline-inducible Stable Cell Line Ementioning

confidence: 99%

“…Recently, phosphorylation of the N-terminal 126-amino acid region of cucumber mosaic virus RNA polymerase 2a protein by a 60-kDa tobacco plant origin protein kinase was demonstrated to inhibit interaction of 2a RNA polymerase with the 1a protein, which is essential for replication of cucumber mosaic virus (31). The phosphorylated form of HCV NS5B was observed when expressed in insect cells using a recombinant baculovirus (32). However, phosphorylation of HCV NS5B in human liver cells has not been demonstrated, and the cellular kinase that phosphorylates HCV NS5B has not been yet identified.…”

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Protein Kinase C-related Kinase 2 Regulates Hepatitis C Virus RNA Polymerase Function by Phosphorylation

Kim¹,

Kim²,

Kim

et al. 2004

Journal of Biological Chemistry

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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...

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“…The focal point of such investigations has been NS5B, which possesses an RNA-dependent RNA polymerase (RdRp) activity and is believed to be the key enzyme catalyzing HCV RNA synthesis (7)(8)(9)(10)(11)(12)(13)(14). Its crystal structure reveals that it contains the classical finger, palm, and thumb subdomains of the polymerases with the unique feature of a more fully enclosed active site tunnel (15)(16)(17), and a recent report by Bressanelli and colleagues (18) has provided additional information about the complex of NS5B with ribonucleotides.…”

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Modulation of the Hepatitis C Virus RNA-dependent RNA Polymerase Activity by the Non-Structural (NS) 3 Helicase and the NS4B Membrane Protein

Piccininni¹,

Varaklioti²,

Nardelli³

et al. 2002

Journal of Biological Chemistry

135

124

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The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is believed to be the central catalytic enzyme responsible for HCV replication but there are many unanswered questions about how its activity is controlled. In this study we reveal that two other HCV proteins, NS3 (a protease/helicase) and NS4B (a hydrophobic protein of unknown function), physically and functionally interact with the NS5B polymerase. We describe a new procedure for generating highly pure NS4B, and use this protein in biochemical studies together with NS5B and NS3. To study the functional effects of the protein-protein interactions, we have developed an in vitro replication assay using the natural noncoding 3 regions of the respective positive ((؉)-3 -untranslated region) and negative ((؊)-3 -terminal region) RNA strands of the HCV genome. Our studies show that NS3 dramatically modulates template recognition by NS5B and changes the synthetic products generated by this enzyme. The use of an NTPase-deficient mutant form of NS3 demonstrates that the NTPase activity (and thus helicase activity) of this protein is specifically required for these effects. Moreover, NS4B is found to be a negative regulator of the NS3-NS5B replication complex. Overall, these results reveal that NS3, NS4B, and NS5B can interact to form a regulatory complex that could feature in the process of HCV replication.Hepatitis C virus (HCV) 1 is a major pathogen of parenterally transmitted non-A, non-B hepatitis (1) and often causes the development of malignant chronic disease, including liver cirrhosis and hepatocellular carcinoma (2). With nearly 3% of the population of the world infected with HCV and no protective vaccine available at present, this disease has emerged as a serious global health problem since the virus was first identified (3, 4). HCV is a positive-stranded RNA virus with a genome of ϳ9400 bp. This genomic RNA initially directs the synthesis of at least 10 structural and nonstructural viral proteins (5). Following that, it is utilized by the viral RNA-dependent RNA polymerase (the nonstructural protein 5B; NS5B) as template to generate a complementary negative-stranded RNA. Once synthesized, the negative strands are transcribed into new molecules of positive-stranded genomic RNA, which in turn provide additional templates for viral protein synthesis as well as genomic RNA for the production of progeny virus (5, 6). However, the molecular events that mediate this process remain largely unclear.Several attempts to dissect the mechanistic details of the viral replication cycle have been reported to date. The focal point of such investigations has been NS5B, which possesses an RNA-dependent RNA polymerase (RdRp) activity and is believed to be the key enzyme catalyzing HCV RNA synthesis (7-14). Its crystal structure reveals that it contains the classical finger, palm, and thumb subdomains of the polymerases with the unique feature of a more fully enclosed active site tunnel (15)(16)(17), and a recent report by Bressanelli and colleagues (18) has provided add...

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Hepatitis C Virus NS5B Protein Is a Membrane-Associated Phosphoprotein with a Predominantly Perinuclear Localization

Cited by 104 publications

References 0 publications

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

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

Protein Kinase C-related Kinase 2 Regulates Hepatitis C Virus RNA Polymerase Function by Phosphorylation

Modulation of the Hepatitis C Virus RNA-dependent RNA Polymerase Activity by the Non-Structural (NS) 3 Helicase and the NS4B Membrane Protein

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