THE POLYMERASE-LIKE CORE OF BROME MOSAIC-VIRUS 2A PROTEIN, LACKING A REGION INTERACTING WITH VIRAL 1A PROTEIN IN-VITRO, MAINTAINS ACTIVITY AND 1A SELECTIVITY IN RNA REPLICATION

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins required for vira l RNA replication: la and 2a. la contains m(7)G methyltransferase- and helicase-like domains, while 2a contains a polymerase (poll-like core flanked by N- and C-terminal extensions. Genetic studies show that BM V RNA replication requires 1a-2a compatibility, implying direct or ind irect 1a-2a interaction in vivo, In vitro, la interacts with the N-ter minal 125-amino-acid segment of 2a preceding the pol-like core, and pr ior deletion studies suggested that this 2a segment was essential for RNA replication. We have now used protein fusions and deletions to exp lore possible parallels between noncovalent 1a-2a interaction and cova lent fusion of similar protein domains in tobacco mosaic virus and to see whether the N-terminal 2a-1a interaction was the primary basis for 1a-2a compatibility in vivo, We found that 2a can function as part of a tobacco mosaic virus-like 1a-2a fusion and that a 2a segment (amino acids 162 to 697) comprising the pol like core was sufficient to prov ide 2a functions in such a fusion, Unexpectedly, the unfused 2a core s egment also supported RNA replication when it and wild-type la were ex pressed as separate proteins. Moreover, in gene reassortant experiment s with the related cowpea chlorotic mottle virus, the unfused 2a core segment showed the same la compatibility requirements as did wild type BMV 2a. Thus, the pol-like core of 2a must interact with la in a way that is selective and essential for RNA synthesis, and 1a-2a interacti ons are more complex than the single, previously mapped interaction of the N-terminal 2a segment with la.