Flock house virus RNA polymerase initiates RNA synthesis de novo and possesses a terminal nucleotidyl transferase activity.
Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3'-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3' initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication.
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Wenzhe
W
Wu
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Zhaowei
Z
Wang
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Hongjie
H
Xia
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Yongxiang
Y
Liu
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Yang
Y
Qiu
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Yujie
Y
Liu
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Yuanyang
Y
Hu
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Xi
X
Zhou
D015153:Blotting, Western; D016679:Genome, Viral; D030381:Nodaviridae / Q000502:physiology*; D009843:Oligoribonucleotides / Q000235:genetics / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
D009843:Oligoribonucleotides; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D012316:RNA Nucleotidyltransferases; D012324:RNA-Dependent RNA Polymerase