YEAST MUTATIONS IN MULTIPLE COMPLEMENTATION GROUPS INHIBIT BROME MOSAIC-VIRUS RNA REPLICATION AND TRANSCRIPTION AND PERTURB REGULATED EXPRESSION OF THE VIRAL POLYMERASE-LIKE GENE

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with ho st membranes to form the viral RNA replication complex. Yeast expressi ng 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, w e have isolated yeast mutants in multiple pression. Three complementat ion groups, represented by mutants mab1-1, mab2-1, and mab3-1 (for mai ntenance of BMV functions), were selected for initial study. Each of t hese mutants has a single, recessive, chromosomal mutation that inhibi ts accumulation of positive- and negative-strand RNA3 and subgenomic m RNA. BMC-directed gene expression was inhibited when the RNA replicati on template was introduced by in vivo transcription from DNA or by tra nsfection of yeast with in vitro transcripts, confirming that cytoplas mic RNA replication steps were defective. mab1-1, mab2-1, and mab3-1 s ensitive, showing that the affected genes contribute to normal 1a prot ein increased the accumulation of 2a mRNA and the polymerase-like 2a p rotein, revealing a new level of viral mab2-1 blocked the ability of 1 a to stimulate 2a mRNA and protein accumulation, whereas mab3-1 had el evated 2a protein accumulation. Together, these results show that BMV RNA replication in yeast depends on multiple host genes, some of which directly or indirectly affect the regulated expression and accumulati on of 2a.