Association between NS3 and NS5 proteins of dengue virus type 2 in the putative RNA replicase is linked to differential phosphorylation of NS5.
Dengue virus type 2, a member of the family Flaviviridae, encodes a single polyprotein precursor consisting of 3391 amino acids 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.
Kapoor
M
M
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160, USA.
Zhang
L
L
Ramachandra
M
M
Kusukawa
J
J
Ebner
K E
KE
Padmanabhan
R
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D003716:Dengue Virus / Q000737:chemistry*; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D020365:RNA Helicases; D012324:RNA-Dependent RNA Polymerase / Q000032:analysis*; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins / Q000032:analysis*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000596:Amino Acids; D020365:RNA Helicases; D012324:RNA-Dependent RNA Polymerase; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases