title: Mutagenesis of D80-82 and G83 residues in West Nile Virus NS2B: effects on NS2B-NS3 activity and viral replication.
abstract: Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D(80)DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D(80)DDG in NS2B on protease activity and viral replication, the negatively charged region D(80)DD and the conserved residue G83 of NS2B were mutated (D(80)DD/E(80)EE, D(80)DD/K(80)KK, D(80)DD/A(80)AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D(80)DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D(80)DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.
authors: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors: Fan
authors: F
authors: Jia
authors: Jingjing
authors: J
authors: Fan
authors: Bo
authors: B
authors: Zhang
authors: Zhiming
authors: Z
authors: Yuan
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D014902:West Nile virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases