Brome mosaic virus RNA replication, capsid assembly and selective RNA encapsidation in Saccharomyces cerevisiae

Brome mosaic virus (BMV) is a well-studied, plant-infecting positive-sense RNA virus. BMV serves as a model system for study of RNA genome replication and gene expression, many of which are shared by other RNA viruses. The yeast Saccharomyces cerevisiae supports BMV-directed RNA replication, so BMV processes may now be studied in an easily manipulated and analyzed host.; To facilitate study of BMV replication in yeast, we designed and tested yeast DNA plasmids that generate infectious BMV RNA transcripts in vivo. Transcript replication depended on BMV 1a and 2a proteins and known cis-acting replication signals. BMV derivatives encoding enzymatic markers for RNA replication were designed and tested. RNA replication increased enzyme activity from one marker >500,000-fold. Similarly, a nutritional marker gene complemented the host defect only when RNA replication occurred. Initiation of replication by in vivo BMV transcripts is highly efficient, and facilitates genetic analysis of BMV RNA replication in yeast. BMV RNAs and proteins made in vivo without BMV RNA replication have additional utility studying other steps of the BMV infection cycle.; To better characterize interactions between BMV RNA and capsid protein (CP) in the absence of RNA replication, BMV RNA2 was co-expressed with CP in yeast. Non-translated RNA2 accumulated when CP was co-expressed in yeast. CP, RNA2 and the CP message co-purified from these yeast in virus-like particles. RNA2-containing particles had the expected 180-subunit structure of plant-derived BMV virions. CP mRNA was encapsidated in an unusual 120-subunit capsid that revealed new CP/CP interactions. The capsid structure was controlled by the packaged RNA. Structural similarities suggest the capsids share assembly steps or intermediates.; Cis-acting requirements for selective packaging RNA2 were investigated in yeast co-expressing the CP. Pairs of non-overlapping, reciprocal RNA2 deletions showed that RNA2 has ≥3, and RNA3 has ≥2, regions sufficient for selective encapsidation by CP. The conserved 3 ' terminal 198 bases of RNA2 directed selective encapsidation of fused non-viral RNA. Such synthetic packaging substrates should facilitate high-resolution mapping of multiple BMV packaging signals.