Dengue virus type 2, a member of the family Flaviviridae, encodes a single polyprotein precursor consisting of 3391 amino acid residues that is processed to at least 10 mature proteins by host and viral proteases. The NS3 protein contains a domain commonly found in cellular serine proteinases that in cooperation with NS2B is involved in polyprotein processing. In addition, NS3 and NS5 proteins contain conserved motifs found in several RNA helicases and RNA-dependent RNA polymerases, respectively. Both enzymatic activities have been suggested to be involved in viral RNA replication. In this report, we demonstrate that the NS3 and NS5 proteins interact in vivo in dengue virus type 2-infected monkey kidney (CV-1) cells and in HeLa cells coinfected with recombinant vaccinia viruses encoding these proteins as shown by coimmunoprecipitations and immunoblotting methods. We also show by immunofluorescence, metabolic labeling, and two-dimensional peptide mapping that NS5 is a nuclear phosphoprotein and that phosphorylation occurs on serine residues at multiple sites. Furthermore, NS5 exists in differentially phosphorylated states in the nuclear and the cytoplasmic fractions, and only the cytoplasmic form of NS5 is found to coimmunoprecipitate with NS3, suggesting that differential phosphorylation may control the interaction between these proteins and its function in the viral RNA replicase.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				B.-H. Song, S.-I. Yun, Y.-J. Choi, J.-M. Kim, C.-H. Lee, and Y.-M. Lee A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication RNA, September 1, 2008; 14(9): 1791 - 1813. [Abstract] [Full Text] [PDF]

				H. Kroschewski, S. P. Lim, R. E. Butcher, T. L. Yap, J. Lescar, P. J. Wright, S. G. Vasudevan, and A. D. Davidson Mutagenesis of the Dengue Virus Type 2 NS5 Methyltransferase Domain J. Biol. Chem., July 11, 2008; 283(28): 19410 - 19421. [Abstract] [Full Text] [PDF]

				A. V. Chernov, S. A. Shiryaev, A. E. Aleshin, B. I. Ratnikov, J. W. Smith, R. C. Liddington, and A. Y. Strongin The Two-component NS2B-NS3 Proteinase Represses DNA Unwinding Activity of the West Nile Virus NS3 Helicase J. Biol. Chem., June 20, 2008; 283(25): 17270 - 17278. [Abstract] [Full Text] [PDF]

				D. Luo, T. Xu, C. Hunke, G. Gruber, S. G. Vasudevan, and J. Lescar Crystal Structure of the NS3 Protease-Helicase from Dengue Virus J. Virol., January 1, 2008; 82(1): 173 - 183. [Abstract] [Full Text] [PDF]

				M. M. Emara and M. A. Brinton Interaction of TIA-1/TIAR with West Nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly PNAS, May 22, 2007; 104(21): 9041 - 9046. [Abstract] [Full Text] [PDF]

				T. L. Yap, T. Xu, Y.-L. Chen, H. Malet, M.-P. Egloff, B. Canard, S. G. Vasudevan, and J. Lescar Crystal Structure of the Dengue Virus RNA-Dependent RNA Polymerase Catalytic Domain at 1.85-Angstrom Resolution J. Virol., May 1, 2007; 81(9): 4753 - 4765. [Abstract] [Full Text] [PDF]

				H. Malet, M.-P. Egloff, B. Selisko, R. E. Butcher, P. J. Wright, M. Roberts, A. Gruez, G. Sulzenbacher, C. Vonrhein, G. Bricogne, et al. Crystal Structure of the RNA Polymerase Domain of the West Nile Virus Non-structural Protein 5 J. Biol. Chem., April 6, 2007; 282(14): 10678 - 10689. [Abstract] [Full Text] [PDF]

				J. M. Mackenzie, M. T. Kenney, and E. G. Westaway West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis J. Gen. Virol., April 1, 2007; 88(4): 1163 - 1168. [Abstract] [Full Text] [PDF]

				J. J. H. Chu and P. L. Yang c-Src protein kinase inhibitors block assembly and maturation of dengue virus PNAS, February 27, 2007; 104(9): 3520 - 3525. [Abstract] [Full Text] [PDF]

				K. Clyde, J. L. Kyle, and E. Harris Recent Advances in Deciphering Viral and Host Determinants of Dengue Virus Replication and Pathogenesis J. Virol., December 1, 2006; 80(23): 11418 - 11431. [Full Text] [PDF]

				R. Takhampunya, R. Padmanabhan, and S. Ubol Antiviral action of nitric oxide on dengue virus type 2 replication. J. Gen. Virol., October 1, 2006; 87(Pt 10): 3003 - 3011. [Abstract] [Full Text] [PDF]

				I. Umareddy, A. Chao, A. Sampath, F. Gu, and S. G. Vasudevan Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA J. Gen. Virol., September 1, 2006; 87(9): 2605 - 2614. [Abstract] [Full Text] [PDF]

				A. Jakubiec, V. Tournier, G. Drugeon, S. Pflieger, L. Camborde, J. Vinh, F. Hericourt, V. Redeker, and I. Jupin Phosphorylation of Viral RNA-dependent RNA Polymerase and Its Role in Replication of a Plus-strand RNA Virus J. Biol. Chem., July 28, 2006; 281(30): 21236 - 21249. [Abstract] [Full Text] [PDF]

				P. D. Uchil, A. V. A. Kumar, and V. Satchidanandam Nuclear localization of flavivirus RNA synthesis in infected cells. J. Virol., June 1, 2006; 80(11): 5451 - 5464. [Abstract] [Full Text] [PDF]

				S. Miller, S. Sparacio, and R. Bartenschlager Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-structural Protein 4B J. Biol. Chem., March 31, 2006; 281(13): 8854 - 8863. [Abstract] [Full Text] [PDF]

				S. M. Best, K. L. Morris, J. G. Shannon, S. J. Robertson, D. N. Mitzel, G. S. Park, E. Boer, J. B. Wolfinbarger, and M. E. Bloom Inhibition of Interferon-Stimulated JAK-STAT Signaling by a Tick-Borne Flavivirus and Identification of NS5 as an Interferon Antagonist J. Virol., October 15, 2005; 79(20): 12828 - 12839. [Abstract] [Full Text] [PDF]

				C. L. Medin, K. A. Fitzgerald, and A. L. Rothman Dengue Virus Nonstructural Protein NS5 Induces Interleukin-8 Transcription and Secretion J. Virol., September 1, 2005; 79(17): 11053 - 11061. [Abstract] [Full Text] [PDF]

				J. Wu, A. K. Bera, R. J. Kuhn, and J. L. Smith Structure of the Flavivirus Helicase: Implications for Catalytic Activity, Protein Interactions, and Proteolytic Processing J. Virol., August 15, 2005; 79(16): 10268 - 10277. [Abstract] [Full Text] [PDF]

				C. Yon, T. Teramoto, N. Mueller, J. Phelan, V. K. Ganesh, K. H. M. Murthy, and R. Padmanabhan Modulation of the Nucleoside Triphosphatase/RNA Helicase and 5'-RNA Triphosphatase Activities of Dengue Virus Type 2 Nonstructural Protein 3 (NS3) by Interaction with NS5, the RNA-dependent RNA Polymerase J. Biol. Chem., July 22, 2005; 280(29): 27412 - 27419. [Abstract] [Full Text] [PDF]

				S.-J. Kim, J.-H. Kim, Y.-G. Kim, H.-S. Lim, and J.-W. Oh Protein Kinase C-related Kinase 2 Regulates Hepatitis C Virus RNA Polymerase Function by Phosphorylation J. Biol. Chem., November 26, 2004; 279(48): 50031 - 50041. [Abstract] [Full Text] [PDF]

				K. H. Choi, J. M. Groarke, D. C. Young, R. J. Kuhn, J. L. Smith, D. C. Pevear, and M. G. Rossmann The structure of the RNA-dependent RNA polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo initiation PNAS, March 30, 2004; 101(13): 4425 - 4430. [Abstract] [Full Text] [PDF]

				M. Nomaguchi, M. Ackermann, C. Yon, S. You, and R. Padmanbhan De Novo Synthesis of Negative-Strand RNA by Dengue Virus RNA-Dependent RNA Polymerase In Vitro: Nucleotide, Primer, and Template Parameters J. Virol., August 15, 2003; 77(16): 8831 - 8842. [Abstract] [Full Text] [PDF]

				W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh Complementation Analysis of the Flavivirus Kunjin NS3 and NS5 Proteins Defines the Minimal Regions Essential for Formation of a Replication Complex and Shows a Requirement of NS3 in cis for Virus Assembly J. Virol., October 2, 2002; 76(21): 10766 - 10775. [Abstract] [Full Text] [PDF]

				A. J. Brooks, M. Johansson, A. V. John, Y. Xu, D. A. Jans, and S. G. Vasudevan The Interdomain Region of Dengue NS5 Protein That Binds to the Viral Helicase NS3 Contains Independently Functional Importin beta 1 and Importin alpha /beta -Recognized Nuclear Localization Signals J. Biol. Chem., September 20, 2002; 277(39): 36399 - 36407. [Abstract] [Full Text] [PDF]

				P. Borowski, M. Lang, A. Haag, H. Schmitz, J. Choe, H.-M. Chen, and R. S. Hosmane Characterization of Imidazo[4,5-d]Pyridazine Nucleosides as Modulators of Unwinding Reaction Mediated by West Nile Virus Nucleoside Triphosphatase/Helicase: Evidence for Activity on the Level of Substrate and/or Enzyme Antimicrob. Agents Chemother., May 1, 2002; 46(5): 1231 - 1239. [Abstract] [Full Text] [PDF]

				K. A. Hanley, J. J. Lee, J. E. Blaney Jr, B. R. Murphy, and S. S. Whitehead Paired Charge-to-Alanine Mutagenesis of Dengue Virus Type 4 NS5 Generates Mutants with Temperature-Sensitive, Host Range, and Mouse Attenuation Phenotypes J. Virol., January 15, 2002; 76(2): 525 - 531. [Abstract] [Full Text] [PDF]

				M. Ackermann and R. Padmanabhan De Novo Synthesis of RNA by the Dengue Virus RNA-dependent RNA Polymerase Exhibits Temperature Dependence at the Initiation but Not Elongation Phase J. Biol. Chem., October 19, 2001; 276(43): 39926 - 39937. [Abstract] [Full Text] [PDF]

				A. E. Matusan, M. J. Pryor, A. D. Davidson, and P. J. Wright Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication J. Virol., October 15, 2001; 75(20): 9633 - 9643. [Abstract] [Full Text] [PDF]

				A. E. Matusan, P. G. Kelley, M. J. Pryor, J. C. Whisstock, A. D. Davidson, and P. J. Wright Mutagenesis of the dengue virus type 2 NS3 proteinase and the production of growth-restricted virus J. Gen. Virol., July 1, 2001; 82(7): 1647 - 1656. [Abstract] [Full Text] [PDF]

				M. Johansson, A. J. Brooks, D. A. Jans, and S. G. Vasudevan A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin-{{beta}} and the viral helicase, NS3 J. Gen. Virol., April 1, 2001; 82(4): 735 - 745. [Abstract] [Full Text]

				P. Borowski, A. Niebuhr, O. Mueller, M. Bretner, K. Felczak, T. Kulikowski, and H. Schmitz Purification and Characterization of West Nile Virus Nucleoside Triphosphatase (NTPase)/Helicase: Evidence for Dissociation of the NTPase and Helicase Activities of the Enzyme J. Virol., April 1, 2001; 75(7): 3220 - 3229. [Abstract] [Full Text]

				R. Banerjee and A. Dasgupta Specific Interaction of Hepatitis C Virus Protease/Helicase NS3 with the 3'-Terminal Sequences of Viral Positive- and Negative-Strand RNA J. Virol., February 15, 2001; 75(4): 1708 - 1721. [Abstract] [Full Text]

				T. Pietschmann, V. Lohmann, G. Rutter, K. Kurpanek, and R. Bartenschlager Characterization of Cell Lines Carrying Self-Replicating Hepatitis C Virus RNAs J. Virol., February 1, 2001; 75(3): 1252 - 1264. [Abstract] [Full Text]

				M. G. JACOBS, P. J. ROBINSON, C. BLETCHLY, J. M. MACKENZIE, and P. R. YOUNG Dengue virus nonstructural protein 1 is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction FASEB J, August 1, 2000; 14(11): 1603 - 1610. [Abstract] [Full Text]

				R. Bartenschlager and V. Lohmann Replication of hepatitis C virus J. Gen. Virol., July 1, 2000; 81(7): 1631 - 1648. [Full Text]

				A. A. Khromykh, P. L. Sedlak, and E. G. Westaway cis- and trans-Acting Elements in Flavivirus RNA Replication J. Virol., April 1, 2000; 74(7): 3253 - 3263. [Abstract] [Full Text]

				B. Gu, C. Liu, J. Lin-Goerke, D. R. Maley, L. L. Gutshall, C. A. Feltenberger, and A. M. Del Vecchio The RNA Helicase and Nucleotide Triphosphatase Activities of the Bovine Viral Diarrhea Virus NS3 Protein Are Essential for Viral Replication J. Virol., February 15, 2000; 74(4): 1794 - 1800. [Abstract] [Full Text]

				P. Neddermann, A. Clementi, and R. De Francesco Hyperphosphorylation of the Hepatitis C Virus NS5A Protein Requires an Active NS3 Protease, NS4A, NS4B, and NS5A Encoded on the Same Polyprotein J. Virol., December 1, 1999; 73(12): 9984 - 9991. [Abstract] [Full Text]

				A. A. Khromykh, P. L. Sedlak, and E. G. Westaway trans-Complementation Analysis of the Flavivirus Kunjin ns5 Gene Reveals an Essential Role for Translation of Its N-Terminal Half in RNA Replication J. Virol., November 1, 1999; 73(11): 9247 - 9255. [Abstract] [Full Text] [PDF]

				P. Borowski, J. S. zur Wiesch, K. Resch, H. Feucht, R. Laufs, and H. Schmitz Protein Kinase C Recognizes the Protein Kinase A-binding Motif of Nonstructural Protein 3 of Hepatitis C Virus J. Biol. Chem., October 22, 1999; 274(43): 30722 - 30728. [Abstract] [Full Text] [PDF]

				S. Steffens, H.-J. Thiel, and S.-E. Behrens The RNA-dependent RNA polymerases of different members of the family Flaviviridae exhibit similar properties in vitro J. Gen. Virol., October 1, 1999; 80(10): 2583 - 2590. [Abstract] [Full Text]

				J. Oliver Koch and R. Bartenschlager Modulation of Hepatitis C Virus NS5A Hyperphosphorylation by Nonstructural Proteins NS3, NS4A, and NS4B J. Virol., September 1, 1999; 73(9): 7138 - 7146. [Abstract] [Full Text]

				H. M. K. Murthy, S. Clum, and R. Padmanabhan Dengue Virus NS3 Serine Protease. CRYSTAL STRUCTURE AND INSIGHTS INTO INTERACTION OF THE ACTIVE SITE WITH SUBSTRATES BY MOLECULAR MODELING AND STRUCTURAL ANALYSIS OF MUTATIONAL EFFECTS J. Biol. Chem., February 26, 1999; 274(9): 5573 - 5580. [Abstract] [Full Text] [PDF]

				K. E. Reed, A. E. Gorbalenya, and C. M. Rice The NS5A/NS5 Proteins of Viruses from Three Genera of the Family Flaviviridae Are Phosphorylated by Associated Serine/Threonine Kinases J. Virol., July 1, 1998; 72(7): 6199 - 6206. [Abstract] [Full Text] [PDF]

				J.-M. Pawlotsky, G. Germanidis, A. U. Neumann, M. Pellerin, P.-O. Frainais, and D. Dhumeaux Interferon Resistance of Hepatitis C Virus Genotype 1b: Relationship to Nonstructural 5A Gene Quasispecies Mutations J. Virol., April 1, 1998; 72(4): 2795 - 2805. [Abstract] [Full Text] [PDF]

				S. Clum, K. E. Ebner, and R. Padmanabhan Cotranslational Membrane Insertion of the Serine Proteinase Precursor NS2B-NS3(Pro) of Dengue Virus Type 2 Is Required for Efficient in Vitro Processing and Is Mediated through the Hydrophobic Regions of NS2B J. Biol. Chem., December 5, 1997; 272(49): 30715 - 30723. [Abstract] [Full Text] [PDF]

				S. You, B. Falgout, L. Markoff, and R. Padmanabhan In Vitro RNA Synthesis from Exogenous Dengue Viral RNA Templates Requires Long Range Interactions between 5'- and 3'-Terminal Regions That Influence RNA Structure J. Biol. Chem., May 4, 2001; 276(19): 15581 - 15591. [Abstract] [Full Text] [PDF]