Members of the Sindbis-like supergroup of positive strand RNA viruses include the causative agents of yellow fever, St. Louis encephalitis and rubella, as well as a large number of damaging plant viruses. Broad sequences conservation in essential proteins has been observed across the supergroup, suggesting that similar mechanisms for RNA replication exist in the various members. In response to public health concerns, and aided by technical advances, research on the flaviviruses and togaviruses has recently accelerated, but there has been limited progress in characterizing viral RNA replication. Plant virus members of the supergroup, such as turnip yellow mosaic virus (TYMV), have been more amenable to such studies. Results from this laboratory have shown that TYMV RNAs replicate preferentially in cis, and a model is proposed to explain this phenomenon. It is proposed that the essential viral proteins p150 and p70 form a rather tight association, which preferentially assembles into replication complexes in cis. When p70 coding regions are substantially deleted from an RNA that encodes wild type p150, so that p150/p70 complexes are unlikely for form in cis, replication can proceed efficiently in trans. The focus of their proposal is to test he validity of the model in generality and in detail. Theoretical considerations suggest that cis-preferential replication, which amounts to biochemical channelling of newly synthesized viral proteins into replication complexes, is advantageous for monopartite viruses, and that the phenomenon may be widespread; there is strong evidence that at least one essential poliovirus protein acts in cis. The validity of the model for cis-replication will be tested by studying the trans-replication of further constructs. Since the cis-effect is thought to be dependent on p150/p70 complex formation, particular attention will be devoted to testing this notion, to identifying the domains of each protein involved in the interaction and in characterizing the interaction. Deletions and linker insertions will be made to test whether subunits-interaction domains of each protein can be disrupted, resulting in a relaxation of the cis-effect. p150 and p70 will be overexpressed via recombinant baculoviruses, and the purified proteins will be studied in order to duplicate in vitro the postulated mutual interaction and interactions with the template RNA. In order to simplify the mechanistic studies, attempts will be made to establish an in vitro translation/replication system for TYMV RNA, with which it could be tested whether the cis-effect is expressed at the level of (-) strand synthesis, as expected. Finally, transgenic plants that supply either p150 or p70 in trans will be made and used to separate the contribution of p150 an p70 to the cis-effect. These studies may provide opportunities for new approaches to interfering with viral replication.
