RNA elements within the 5' untranslated region of the West Nile virus genome are critical for RNA synthesis and virus replication.
RNA elements within the flavivirus genome may play essential regulatory roles during virus replication. Here, recombinant West Nile virus (WNV) NS5 protein was used in combination with WNV subgenomic RNA templates to establish in vitro RNA-dependent RNA polymerase and RNA-binding assays. These assays identified mutations in the stem-loop A (SLA) region of the 5' untranslated region (5'UTR) altering NS5 RNA synthesis and RNA-binding capability. These mutations were then introduced into the full-length WNV genome by reverse genetics. Further analysis of the mutant viruses showed that deletion of nt 46-60, which disrupted the stem and side loop of SLA, greatly compromised virus replication, whereas mutations that destroyed the top loop of SLA required for RNA synthesis in vitro did not significantly alter virus replication. These results suggest that SLA present in the 5'UTR of WNV is essential for RNA synthesis in vitro and for virus replication.
Li
Xiao-Feng
XF
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Jiang
Tao
T
Yu
Xue-Dong
XD
Deng
Yong-Qiang
YQ
Zhao
Hui
H
Zhu
Qing-Yu
QY
Qin
E-De
ED
Qin
Cheng-Feng
CF
D020121:5' Untranslated Regions; D000818:Animals; D002522:Chlorocebus aethiops; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D010948:Viral Plaque Assay; D014779:Virus Replication; D014902:West Nile virus / Q000235:genetics / Q000502:physiology*
D020121:5' Untranslated Regions; C096041:NS5 protein, flavivirus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins