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 tem plate RNA recognition and initiation of the synthesis of a complementa ry strand by the viral RNA-dependent RNA polymerase (RdRp) are crucial early steps. Using a template-dependent in vitro RNA synthesizing sys tem of alfalfa mosaic Virus (AlMV) we were able to study the effect of small chemical modifications of the 3' end of the template RNAs on pr oduct 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). Bloc king 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 tem plate competition experiments the RdRp of AIMV displays a high specifi city 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 prot ein, which forms a part of the Viral polymerase (R. Quadt et al., Viro logy 182, 309-315, 1991), was not the main target involved in the inhi bition of RNA synthesis. (C) 1995 Academic Press, Inc