Insights into the oligomerization state-helicase activity relationship of West Nile virus NS3 NTPase/helicase.
West Nile virus (WNV) is a member of the Flaviviridae family of positive-strand RNA viruses. Its viral RNA is translated to produce a polyprotein precursor that is further processed into three structural and seven non-structural proteins. The non-structural protein 3 (NS3) possess both protease and helicase activities. The C-terminal portion of the NS3 contains the ATPase/helicase domain presumably involved in viral replication. This domain has been expressed in Escherichia coli, purified in soluble form and structurally characterized. As judged by analytical centrifugation and size exclusion chromatography, the purified enzyme behaves as a monomer in solution. It has ATPase activity that is stimulated by the presence of RNA and single-stranded DNA molecules (ssDNA). However, we were unable to detect helicase activity at protein concentrations up to 500nM. It has been reported that longer constructions of NS3 helicase domains from other flavivirus, like those which include residues of the linker region between the protease and the helicase domains, have helicase activity. Since all the conformational features of the purified WNV NS3 domain are those of a native protein, it is tempting to assume that the linker region plays a critical role in determining the protein-protein interactions that leads to the formation of the active oligomer.
Feito
Ma José
MJ
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain.
Gómez-Gutiérrez
Julián
J
Ayora
Sylvia
S
Alonso
Juan Carlos
JC
Peterson
Darrell
D
Gavilanes
Francisco
F
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D043583:Nucleoside-Triphosphatase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014902:West Nile virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D017361:Viral Nonstructural Proteins; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases