The yeast Tet-Promoter Hughes Collection of essential yeast strains was purchased from Open Biosystems (Huntsville, AL). The tet-promoter mutant strains (designated here with the prefix P TET ) were provided in the haploid R1158 background (MATa URA3::CMV-tTA MATa his3-1 leu2-0 met15-0), which was constructed by a one-step integration of the cytomegalovirus (CMV) promoter-driven tTA* transactivator at the URA3 locus [28] . The kanR-tetO7-TATA was then integrated into the promoter of a different essential gene in strain R1158, allowing the repression of essential gene expression upon the addition of dox to growth medium [27] . pB12VG1 expresses BMV 1a and 2a Pol from the GAL1 and GAL10 promoters, respectively [9] . pB3BG29, based on pB3Rluc [9] , uses a truncated GAL1 promoter (GALL [29] ) to express RNA3 with the coat protein ORF replaced by the Renilla luciferase (Rluc) ORF (from pRL-null; Promega, Madison, WI). pB3BG29 also expresses the firefly luciferase (Fluc) ORF (from pGL3-Basic; Promega, Madison, WI) from the GAL10 promoter. To construct pB3BG29, the AgeI-AatII RNA3/Rluc-containing fragment of pB3Rluc [9] replaced the AgeI-AatII FHV RNA1/Rluc-containing fragment of pBDL250-Ren [B. Lindenbach, unpublished] . pB3MS82 expresses a BMV RNA3 derivative in which the coat protein gene has a four-nucleotide insertion and a point mutation, abolishing expression of the coat protein [30] . The use of pB3MS82 in this study, as in many other studies [8, 19, 30, 31, 32, 33] , allows analysis of RNA3 and RNA4 levels while avoiding any possible effects of coat protein expression and RNA encapsidation. pB12VG1 and pB3BG29 were used in reporter gene-based primary screens and pB12VG1 and pB3MS82 were used in secondary validation testing by Northern blotting.