Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus

Abstract

Positive-strand RNA viruses are the largest virus class and include many pathogens such as hepatitis C virus and the severe acute respiratory syndrome coronavirus (SARS). Brome mosaic virus (BMV) is a representative positive-strand RNA virus whose RNA replication, gene expression, and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. By using traditional yeast genetics, host genes have been identified that function in controlling BMV translation, selecting BMV RNAs as replication templates, activating the replication complex, maintaining a lipid composition required for membrane-associated RNA replication, and other steps. To more globally and systematically identify such host factors, we used engineered BMV derivatives to assay viral RNA replication in each strain of an ordered, genome-wide set of yeast single-gene deletion mutants. Each deletion strain was transformed to express BMV replicase proteins and a BMV RNA replication template with the capsid gene replaced by a luciferase reporter. Luciferase expression, which is dependent on viral RNA replication and RNA-dependent mRNA synthesis, was measured in intact yeast cells. Approximately 4500 yeast deletion strains ( approximately 80% of yeast genes) were screened in duplicate and selected strains analyzed further. This functional genomics approach revealed nearly 100 genes whose absence inhibited or stimulated BMV RNA replication and/or gene expression by 3- to >25-fold. Several of these genes were shown previously to function in BMV replication, validating the approach. Newly identified genes include some in RNA, protein, or membrane modification pathways and genes of unknown function. The results further illuminate virus and cell pathways. Further refinement of virus screening likely will reveal contributions from additional host genes.

Fig. 1.

(A) Viral cDNAs (bold lines) and flanking expression elements in pB12, expressing 1a and 2a, and pB3, expressing RNA3. X, the BMV coat protein gene or any gene replacing it, such as Rluc. (Right) pB3 transcription produces an initial (+)RNA3 inoculum, used by 1a and 2a to direct synthesis of (-)RNA3, which then is used as a template to greatly amplify (+)RNA3 and produce sgRNA4. A_n, poly(A) signal; GAL1/GAL10, yeast promoters; Rz, self-cleaving ribozyme. (B) pB12 supports WT RNA3 replication at a level similar to that of independent plasmids expressing 1a and 2a.

Fig. 2.

(A) Rluc activity assayed on intact WT BY4743 yeast transformed with the indicated combinations of pB12, pB3Rluc, and/or pB3, a plasmid expressing WT RNA3 (20). (B) Northern blot analysis of BMV RNA replication products, RNA3Rluc and sg mRNA RNA4Rluc, in WT YPH500 or isogenic ded1i yeast cells transformed with pB12 and pB3Rluc. (C) Rluc activity assayed on intact WT YPH500 or isogenic ded1i yeast transformed with pB12 and pB3Rluc.

Fig. 3.

Northern and Western analyses of BMV RNAs and proteins from WT BY4743 yeast and selected deletion strains with reduced BMV-directed Rluc expression. Each strain was transformed with pB12 to express 1a and 2a and with pCUP1B3 to express WT RNA3. Percent of WT sgRNA4 accumulation and Rluc expression are indicated. Northern blots of yeast ACT1 and ADH1 mRNAs are shown for comparison. Although not listed in Table 1, SCP160 was included due to its membrane association.

Fig. 4.

Northern and Western analyses of BMV RNAs and proteins from WT BY4743 yeast and selected deletion strains with enhanced BMV-directed Rluc expression. As in Fig. 3, WT RNA3 from pCUP1B3 was used as the replication template. Percent of WT sgRNA4 accumulation and Rluc and GUS expression are indicated. NT, not tested.