TRANSCOMPLEMENTATION OF YELLOW-FEVER VIRUS NS1 REVEALS A ROLE IN EARLY RNA REPLICATION

Mutational analysis of the nonstructural protein 1 (NS1) of yellow fev er virus (YF) has implicated it in viral RNA replication. To further e xplore this observation, we sought a method for uncoupling NS1 functio n from NS1 expression and processing as part of the large YF polyprote in. Here we describe a strategy for providing NS1 in trans, utilizing a noncytopathic Sindbis virus vector. Replication of a defective YF ge nome containing a large in-frame deletion of NS1 was dependent on func tional expression of NS1. Recovered mutant virus was shown to contain the deletion and was neutralized by YF-specific antiserum. Complemente d mutant virus increased in titer with kinetics similar to those of pa rental YF 17D but peaked at lower titers. trans complementation has al lowed us to derive high-titer, helper-free stocks of YF defective in N S1 with which to further characterize the role of this gene product in RNA replication. The first cycles of RNA replication were analyzed by using a sensitive strand-specific RNase protection assay. We document these events for mutant and wild-type viruses in the presence or abse nce of complementation. These data strongly suggest a role for NS1 pri or to or at initial minus-strand synthesis.