The motif V of plum pox potyvirus CI RNA helicase is involved in NTP hydrolysis and is essential for virus RNA replication.
The plum pox potyvirus (PPV) protein CI is an RNA helicase whose function in the viral life cycle is still unknown. The CI protein contains seven conserved sequence motifs typical of RNA helicases of the superfamily SF2. We have introduced several individual point mutations into the region coding for motif V of the PPV CI protein and expressed these proteins in Escherichia coli as maltose binding protein fusions. Mutations that abolished RNA helicase activity also disturbed NTP hydrolysis. No mutations affected the RNA binding capacity of the CI protein. These mutations were also introduced in the PPV genome making use of a full-length cDNA clone. Mutant viruses carrying CI proteins with reduced RNA helicase activity replicated very poorly in protoplasts and were unable to infect whole plants without rapid pseudoreversion to wild-type. These results indicate that motif V is involved in the NTP hydrolysis step required for potyvirus RNA helicase activity, and that this activity plays an essential role in virus RNA replication inside the infected cell.
Fernández
A
A
Centro Nacional de Biotecnología (CSIC), Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain.
Guo
H S
HS
Sáenz
P
P
Simón-Buela
L
L
Gómez de Cedrón
M
M
García
J A
JA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D017801:Plum Pox Virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D017354:Point Mutation; D011523:Protoplasts / Q000378:metabolism / Q000821:virology; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D015347:RNA Probes / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism / Q000821:virology; D014764:Viral Proteins; D014779:Virus Replication
D015347:RNA Probes; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; C109634:protein CI, plum pox potyvirus; D000251:Adenosine Triphosphatases; D020365:RNA Helicases