Genetic analysis of the function of the plum pox virus CI RNA helicase in virus movement

The CI protein forms the cylindrical inclusions typical of potyviral infections and is involved in genome replication and virus movement. In this work, we have analyzed the effect of a series of point mutations at the N-terminal region of the CI protein of Plum pox virus (PPV) on the enzymatic activities and the self-interaction ability of the protein, and on virus replication and movement. DD3,4AA mutation, which had no apparent effects on ATPase and RNA helicase activities in vitro, and on virus replication in protoplasts, drastically impaired cell-to-cell spread of the virus. The effect of KK101,102AA mutation was host-specific. While no signals of virus infection were detected in Chenopodium foetidum inoculated with PPV KK101,102AA, the mutation caused a moderate effect on short distance movement in Nicotiana benthamiana and N. clevelandii, which resulted in a more drastic disturbance of systemic spread. None of the mutations analyzed abolished PPV CI self-interaction in the yeast Two-Hybrid system, but they caused a notable reduction in the binding strength, which appears to positively correlate with their effect on virus movement, suggesting that CI–CI interactions required for RNA replication and virus movement could be rather different.