Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of the viral polymerase-like gene

Abstract

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 host membranes to form the viral RNA replication complex. Yeast expressing 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit BMV-directed gene expression. Three complementation groups, represented by mutants mab1-1, mab2-1, and mab3-1 (for maintenance of BMV functions), were selected for initial study. Each of these mutants has a single, recessive, chromosomal mutation that inhibits accumulation of positive- and negative-strand RNA3 and subgenomic mRNA. BMV-directed gene expression was inhibited when the RNA replication template was introduced by in vivo transcription from DNA or by transfection of yeast with in vitro transcripts, confirming that cytoplasmic RNA replication steps were defective. mab1-1, mab2-1, and mab3-1 slowed yeast growth to varying degrees and were temperature-sensitive, showing that the affected genes contribute to normal cell growth. In wild-type yeast, expression of the helicase-like 1a protein increased the accumulation of 2a mRNA and the polymerase-like 2a protein, revealing a new level of viral regulation. In association with their other effects, mab1-1 and mab2-1 blocked the ability of 1a to stimulate 2a mRNA and protein accumulation, whereas mab3-1 had elevated 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 accumulation of 2a.

Figure 1

Schematic of a cDNA cassette (Left) for in vivo transcription of wt RNA3 or RNA3 derivatives B3URA3 or B3GUS, and the subsequent replication of these RNAs (Right). 3a, the BMV 3a movement protein ORF; and X, the wt BMV coat gene, URA3 gene, or GUS gene, as appropriate for the relevant RNA3 derivative. The 5′-flanking GAL1 promoter and 3′-flanking hepatitis delta virus ribozyme are also shown. The horizontal arrow (Center) represents in vivo, DNA-dependent synthesis of RNA3 transcripts as an inoculum to initiate replication, whereas the vertical arrows (Right) depict BMV 1a- and 2a-directed, RNA-dependent RNA3 replication and subgenomic mRNA synthesis. The solid box near the center of negative-strand RNA3 represents the subgenomic mRNA promoter. The coat protein gene or its replacements are not translated from their internal position in RNA3 (dotted box marked X, Upper Right), but only from their 5′-proximal position in the subgenomic mRNA, RNA4 (solid box marked X, Lower Right), making their expression dependent on BMV RNA replication and subgenomic mRNA synthesis (15, 16).

Figure 2

(A) BMV-directed GUS expression in 1a- and 2a-expressing yeast containing a chromosomally integrated B3GUS expression cassette. Parental strain YMI04 and mutant strains mab1, mab2 and mab3 derived from it were grown in gal-containing liquid medium for 48 h, protein was extracted, and GUS activity per mg of total protein was measured. Averages and standard deviations from four independent cultures of each strain are shown. (B) BMV-directed CAT expression in 1a- and 2a-expressing yeast transfected with B3CAT in vitro transcripts. Parental strain YMI04 and mab1, mab2 and mab3 mutant strains were cotransfected with in vitro transcripts of B3CAT and luciferase mRNA (transcribed from pGEM-luc, Promega), incubated 21 h in glc media, and assayed for CAT and luciferase activity. Luciferase mRNA was included as an internal standard since luciferase was translated directly from the transfected mRNA, requiring no BMV-directed RNA synthesis, and showed no strain-specific variation between YMI04 and mab mutant strains. Thus, in each sample, CAT activity was normalized to luciferase activity to control for any variations in transfection efficiency. Averages and SD from three independent transfections of each strain are shown.

Figure 3

mab1, mab2 and mab3 yeast growth phenotypes, which cosegregate with inhibition of BMV RNA replication and gene expression (see Results). The indicated yeast strains were grown in glc-containing medium and diluted in sterile water to A₆₀₀ of 0.2, 0.025, 0.003, and 0.0004 (8-fold serial dilutions). Two microliters of each dilution was spotted on glc plates and incubated at 30°C or 36°C for 3 days. For each strain, the dilution series corresponds to plating ≈10 cells in the most dilute spot. At 30°C, the growth of mab1 yeast was similar to that of parental strain YMI04, whereas mab3 and especially mab2 yeast grew more slowly. After prolonged incubation at 36°C, mab1 and mab3 yeast showed no growth, whereas mab2 yeast growth was weakly detectable (Right and results not shown).

Figure 4

Complementation tests of mab1, mab2, and mab3. Diploid yeast strains expressing 1a and 2a and containing the lys2-integrated B3GUS expression cassette were generated by the indicated crosses among mab1, mab2, and mab3 strains and strains with wt MAB loci (YMI04, YMI06). The diploids were cultured in gal medium for 48 h and GUS activity per mg of total protein measured as in Fig. 2A. Each set of diploids with a common MATa parent was processed in parallel and GUS activity expressed as a % of the activity in the diploid derived by crossing to MATα YMI04, which has wt MAB loci.

Figure 5

Analysis of RNA3 and RNA4 species in parental strain YPH500 and mab1, mab2, and mab3 yeast expressing 1a, 2a and wt RNA3 after 48 h growth in liquid gal medium. (A) Northern blot analysis of positive-strand RNA3 and RNA4. Total RNA was extracted, glyoxylated, electrophoresed in 1% agarose, transferred to nylon membrane, and probed with a strand-specific, ³²P-labeled RNA probe from the BMV coat gene. (B) Northern blot analysis of negative-strand RNA3 and RNA4, as in A except that a strand-specific coat gene probe of opposite polarity was used. (C) Reverse transcriptase primer extension analysis of the 5′ ends of RNA3 species by using a primer complementary to bases 30–44 of RNA3. vRNA, a reaction using BMV virion RNA as template; wt-1a,2a, YPH500 expressing RNA3 but lacking 1a and 2a expression plasmids. Numbers at the right indicate nucleotide position relative to the wt RNA3 5′ end. The solid and open arrowheads indicate the positions of the two bands arising from natural RNA3 (see Results).

Figure 6

Accumulation of (A) 1a mRNA and protein and (B) 2a mRNA and protein in parental strain YMI04 (wt) and mab1, mab2, and mab3 yeast expressing 1a and 2a alone or together, as indicated. Equal amounts of total yeast RNA or protein were loaded in each lane prior to electrophoresis and Northern blot or immunoblot analysis, respectively, as described in Materials and Methods.