Nonstructural 5A Protein of Hepatitis C Virus Modulates Tumor Necrosis Factor α-stimulated Nuclear Factor κB Activation

Park, Kyu Jin; Choi, Sung Hyuk; Lee, Soo Young; Hwang, Soon B.; Lai, Michael M. C.

doi:10.1074/jbc.m111599200

Cited by 58 publications

(60 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The immunoprecipitates were incubated in kinase reaction buffer (20 mM HEPES, pH 7.4, 1 mM MnCl 2 , 5 mM MgCl 2 , 10 mM ␤-glycerolphosphate, 0.1 mM sodium orthovanadate, 2 mM NaF, and 1 mM dithiothreitol) containing 5 Ci of [␥-32 P]ATP at 30°C for 30 min (38). Substrates used in these kinase reactions were either GST-I B␣ (1 g) containing wild type (amino acids 1-54) or mutant sequences that in residues 32 and 36 were substituted with alanine or Hsp27 (1 g).…”

Section: Methodsmentioning

confidence: 99%

Heat Shock Protein 27 Association with the IκB Kinase Complex Regulates Tumor Necrosis Factor α-induced NF-κB Activation

Park

Gaynor

Kwak

2003

Journal of Biological Chemistry

Self Cite

180

132

View full text Add to dashboard Cite

Heat shock protein 27 (Hsp27) is a ubiquitously expressed member of the heat shock protein family that has been implicated in various biological functions including the response to heat shock, oxidative stress, and cytokine treatment. Previous studies have demonstrated that heat shock proteins are involved in regulating signal transduction pathways including the NF-B pathway. In this study, we demonstrated that Hsp27 associates with the I 〉 kinase (IKK) complex and that this interaction was stimulated by tumor necrosis factor ␣ treatment. Phosphorylation of Hsp27 by the kinase mitogen-activated protein kinase-activated protein kinase 2, a downstream substrate of the mitogen-activated protein kinase p38, enhanced the association of Hsp27 with IKK␤ to result in decreased IKK activity. Consistent with these observations, treatment of cells with a p38 inhibitor reduced the association of Hsp27 with IKK␤ and thus resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in down-regulating IKK signaling by reducing its activity following tumor necrosis factor ␣ stimulation.

show abstract

Section: Methodsmentioning

confidence: 99%

Heat Shock Protein 27 Association with the IκB Kinase Complex Regulates Tumor Necrosis Factor α-induced NF-κB Activation

Park

Gaynor

Kwak

2003

Journal of Biological Chemistry

Self Cite

180

132

View full text Add to dashboard Cite

show abstract

“…cDNA corresponding to the NS5B coding sequence of HCV was amplified by PCR using the Korean isolate of HCV (genotype 1b) (11) and subcloned into the BamHI site of pcDNA3 (Invitrogen) or pEF6/Myc-His (Invitrogen). TNFR1, TRADD, TRAF2, MEKK-1, the dominant-negative mutant form of MEKK1 (DN MEKK1), Flag-IKK␣, and Flag-IKK␤ expression vectors were described previously (28,43,44,64). The Flag-IB␣ expression vector was a gift from Dean Ballard (Vanderbilt University).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis C Virus Nonstructural 5B Protein Regulates Tumor Necrosis Factor Alpha Signaling through Effects on Cellular IκB Kinase

Choi

Park

Ahn

et al. 2006

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

Hepatitis C virus (HCV) NS5B protein is a membrane-associated phosphoprotein that possesses an RNAdependent RNA polymerase activity. We recently reported that NS5A protein interacts with TRAF2 and modulates tumor necrosis factor alpha (TNF-␣)-induced NF-B and Jun N-terminal protein kinase (JNK). Since NS5A and NS5B are the essential components of the HCV replication complex, we examined whether NS5B could modulate TNF-␣-induced NF-B and JNK activation. In this study, we have demonstrated that TNF-␣-induced NF-B activation is inhibited by NS5B protein in HEK293 and hepatic cells. Furthermore, NS5B protein inhibited both TRAF2-and IKK-induced NF-B activation. Using coimmunoprecipitation assays, we show that NS5B interacts with IKK␣. Most importantly, NS5B protein in HCV subgenomic replicon cells interacted with endogenous IKK␣, and then TNF-␣-mediated IKK␣ kinase activation was significantly decreased by NS5B. Using in vitro kinase assay, we have further found that NS5B protein synergistically activated TNF-␣-mediated JNK activity in HEK293 and hepatic cells. These data suggest that NS5B protein modulates TNF-␣ signaling pathways and may contribute to HCV pathogenesis.Hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis, which often leads to liver cirrhosis and hepatocellular carcinoma (1, 14). More than 170 million individuals worldwide are infected with HCV. HCV belongs to a member of the Flaviviridae family and contains a single-stranded, positivesense RNA genome of ϳ9,600 nucleotides in length. The HCV genome encodes a single polyprotein precursor of approximately 3,010 amino acids that is cleaved by both cellular signal peptidase and viral protease to generate structural and nonstructural proteins (19,21,33,35). The N-terminally localized core and envelope proteins (E1 and E2) are viral structural proteins, and the remainders of the genome are nonstructural proteins. The nonstructural protein 5B (NS5B) is an RNAdependent RNA polymerase. The NS5B is the key enzyme that catalyzes the replication of HCV. We have previously demonstrated that NS5B is a phosphoprotein that is predominantly localized in the perinuclear region in the cytoplasm (24). Although RNA-dependent RNA polymerase activities have been demonstrated using both bacterially and baculovirus-expressed NS5B proteins (8,16,34,39,40,60), the detailed mechanism of HCV replication is poorly understood by the lack of an efficient cell culture system. The NS5B has been extensively characterized at the biochemical (8, 34) and structural levels (10, 30) and has been a prime target for inhibitors of HCV replication.The nuclear transcription factor NF-B plays a critical role in regulating the expression of many cytokines and immunoregulatory proteins (4-6). The NF-B complex is composed of homodimers or heterodimers of Rel and NF-B proteins, including NF-B1, NF-B2, p65, Rel B, and c-Rel (4). The activity of NF-B can be elevated by various stimuli, including tumor necrosis factor (TNF), interleukin 1 (IL-1), and phorbol esters (55). In most cells,...

show abstract

“…In addition, the phosphorylation pattern of NS5A plays an important role in HCV genome replication (Evans et al, 2004b;Appel et al, 2005). It also interacts with a multitude of cellular proteins and alters the host cells to support viral RNA replication (Gale et al, 1998;Chung et al, 2000;Park et al, 2002;Evans et al, 2004a;Gao et al, 2004).Transposon-mediated insertion mutagenesis (Goryshin & Reznikoff, 1998) is a powerful tool that allows insertion of short peptides into proteins encoded in cloned DNA to evaluate permissive sites in the protein. It can also be used to assess regions of the protein that are dispensable for its functions.…”

mentioning

confidence: 99%

“…The aminoterminal domain of NS5A appears to interact with several cellular proteins (Gale et al, 1998;Chung et al, 2000;Park et al, 2002;Waris et al, 2003;Evans et al, 2004a;Gao et al, 2004) that may be necessary for replication. In addition, an amphipathic a-helix and a zinc-binding site located at the amino terminus are required for replication (Brass et al, 2002; Elazar et al, 2003;Tellinghuisen et al, 2004Tellinghuisen et al, , 2005 et al, 2000) and the carboxyterminal sequences (residues 399-441) have been demonstrated (Zhu et al, 2003).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Insertion and deletion analyses identify regions of non-structural protein 5A of Hepatitis C virus that are dispensable for viral genome replication

Liu

Ansari

Das

et al. 2006

Journal of General Virology

View full text Add to dashboard Cite

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays an essential role in viral genome replication. A series of transposon-mediated insertion mutants and deletion mutants of NS5A was used to examine the colony-forming ability of HCV subgenomic replicons encoding the mutant proteins. The results reveal that two regions of NS5A can tolerate insertions: one spanning residues 240-314, which contain the interferon sensitivity-determining region (ISDR), and the other spanning residues 349-417 at the carboxy terminus. The majority of these sites also tolerated insertion of enhanced green fluorescent protein. Furthermore, replicons encoding NS5A with deletions in ISDR or in the carboxy-terminal regions were replication-competent, indicating that these regions of NS5A are not necessary for replication. Taken together, the results suggest that the central region spanning the ISDR and the carboxy-terminal region of the molecule are dispensable for the functions of NS5A in viral genome replication.Hepatitis C virus (HCV) is an enveloped virus belonging to the genus Hepacivirus within the family Flaviviridae. The positive-sense,~9?6 kb RNA genome is translated to produce a polyprotein, which is processed proteolytically to generate the mature structural and non-structural (NS) proteins (Major & Feinstone, 1997). The NS proteins are involved in viral genome replication, whereas the structural proteins participate in virion assembly and release (Bartenschlager & Lohmann, 2000;Rosenberg, 2001). Development of cell lines supporting replication of subgenomic replicons of HCV has revealed that adaptive mutations within the NS proteins are required for efficient replication of the replicons derived from genotype 1a and 1b genomes (Lohmann et al., 1999;Blight et al., 2000Blight et al., , 2003Krieger et al., 2001;Yi & Lemon, 2004), although a similarly constructed replicon derived from a genotype 2a virus replicates efficiently with few or no adaptive mutations (Kato et al., 2003).The NS5A protein is multifunctional. It contains an interferon sensitivity-determining region (ISDR) spanning aa 237-276 and may play a role in resistance to alpha interferon (Polyak et al., 1999). It contains an amphipathic a-helix and a zinc-binding domain at the amino terminus, which are required for replication (Brass et al., 2002; Elazar et al., 2003;Tellinghuisen et al., 2004Tellinghuisen et al., , 2005. In addition, the phosphorylation pattern of NS5A plays an important role in HCV genome replication (Evans et al., 2004b;Appel et al., 2005). It also interacts with a multitude of cellular proteins and alters the host cells to support viral RNA replication (Gale et al., 1998;Chung et al., 2000;Park et al., 2002;Evans et al., 2004a;Gao et al., 2004).Transposon-mediated insertion mutagenesis (Goryshin & Reznikoff, 1998) is a powerful tool that allows insertion of short peptides into proteins encoded in cloned DNA to evaluate permissive sites in the protein. It can also be used to assess regions of the protein that are dispensable for its functi...

show abstract

Nonstructural 5A Protein of Hepatitis C Virus Modulates Tumor Necrosis Factor α-stimulated Nuclear Factor κB Activation

Cited by 58 publications

References 59 publications

Heat Shock Protein 27 Association with the IκB Kinase Complex Regulates Tumor Necrosis Factor α-induced NF-κB Activation

Heat Shock Protein 27 Association with the IκB Kinase Complex Regulates Tumor Necrosis Factor α-induced NF-κB Activation

Hepatitis C Virus Nonstructural 5B Protein Regulates Tumor Necrosis Factor Alpha Signaling through Effects on Cellular IκB Kinase

Insertion and deletion analyses identify regions of non-structural protein 5A of Hepatitis C virus that are dispensable for viral genome replication

Contact Info

Product

Resources

About