Interaction between RNA-dependent RNA polymerase of alfalfa mosaic virus and its template: oxidation of vicinal hydroxyl groups blocks in vitro RNA synthesis.

In the life cycle of a (+)-strand RNA plant virus the processes of template RNA recognition and initiation of the synthesis of a complementary strand by the viral RNA-dependent RNA polymerase (RdRp) are crucial early steps. Using a template-dependent in vitro RNA synthesizing system of alfalfa mosaic virus (AIMV) we were able to study the effect of small chemical modifications of the 3' end of the template RNAs on product formation. After oxidation of the 3'-terminal nucleoside of the template no products could be detected. Presumably, RNA synthesis was blocked at the stage of initiation, since the promoter of the RdRp is internal (A. C. Van der Kuyl et al., Virology 176, 346-354, 1990). Blocking was probably due to an irreversible binding of the enzyme to the 3' end of the modified RNA. Using this system it was shown that in template competition experiments the RdRp of AIMV displays a high specificity for its cognate template, either before or after the oxidation of the 3'-terminal nucleoside. From this it was concluded that periodate modification of the 3'-terminal nucleoside has little or no effect on template recognition. Furthermore, we showed that the viral coat protein, which forms a part of the viral polymerase (R. Quadt et al., Virology 182, 309-315, 1991), was not the main target involved in the inhibition of RNA synthesis.