Proteolytic processing of turnip yellow mosaic virus replication proteins and functional impact on infectivity.
Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA.
Jakubiec
Anna
A
Institut Jacques Monod, 2 Place Jussieu, 75251 Paris Cedex 05, France.
Drugeon
Gabrièle
G
Camborde
Laurent
L
Jupin
Isabelle
I
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism; D017806:Tymovirus / Q000472:pathogenicity* / Q000502:physiology; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication
D017361:Viral Nonstructural Proteins; D012321:DNA-Directed RNA Polymerases; D010450:Endopeptidases; D020365:RNA Helicases