The two-component NS2B-NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase.
Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.
Chernov
Andrei V
AV
Burnham Institute for Medical Research, La Jolla, California 92037, USA.
Shiryaev
Sergey A
SA
Aleshin
Alexander E
AE
Ratnikov
Boris I
BI
Smith
Jeffrey W
JW
Liddington
Robert C
RC
Strongin
Alex Y
AY
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000821:virology; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D012313:RNA / Q000737:chemistry; D020365:RNA Helicases / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D014902:West Nile virus / Q000201:enzymology*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004247:DNA; D012313:RNA; D020365:RNA Helicases; D011994:Recombinant Proteins; 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