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. 2000 Apr 11;97(8):3913-8.
doi: 10.1073/pnas.080072997.

Identification and characterization of a host protein required for efficient template selection in viral RNA replication

J Díez  1 , M Ishikawa, M Kaido, P Ahlquist
Affiliations

Identification and characterization of a host protein required for efficient template selection in viral RNA replication

J Díez et al. Proc Natl Acad Sci U S A. .

Abstract

Biochemical studies suggest that positive-strand RNA virus replication involves host as well as viral functions. Brome mosaic virus (BMV) is a member of the alphavirus-like superfamily of animal and plant positive-strand RNA viruses. Yeast expressing the BMV RNA replication proteins 1a and 2a supports BMV RNA replication and mRNA synthesis. Using the ability of BMV to replicate in yeast, we show that efficient BMV RNA replication requires Lsm1p, a yeast protein related to core RNA splicing factors but shown herein to be cytoplasmic. Haploid yeast with an Lsm1p mutation was defective in an early template selection step in BMV RNA replication, involving the helicase-like replication protein 1a and an internal viral RNA element conserved with tRNAs. Lsm1p dependence of this interaction was suppressed by adding 3' poly(A) to the normally unpolyadenylated BMV RNA. Our results show Lsm1p involvement in a specific step of BMV RNA replication and connections between Lsm1p and poly(A) function, possibly through interaction with factors binding mRNA 5' ends.

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Figures

Figure 1
Figure 1
Identification of LSM1. (A) Schematic of the BMV genome, RNA3 replication, and subgenomic mRNA synthesis, showing ORFs (boxed), noncoding regions (single lines), 5′ caps (m7G), intergenic replication enhancer (RE), and tRNA-like 3′ ends (cloverleaf). (B) BMV-directed β-glucuronidase (GUS) expression in 1a- and 2a-expressing wild-type (WT) and mab1 yeast containing a chromosomally integrated B3GUS expression cassette. Total protein was extracted, and GUS activity per milligram of total protein was measured. Averages and SEM from three experiments are shown, except for mab1 + pLSM1, which is the average of two experiments. pLSM1 is a centromeric yeast plasmid containing a 1.5-kilobase (kb) DNA fragment bearing WT LSM1. (C) BMV-directed chloramphenicol acetyl transferase (CAT) expression in mab1 and WT yeast expressing 1a and 2a and electroporated with B3CAT in vitro transcripts. Yeast was incubated in glucose medium and assayed for CAT activity 21 h after electroporation. Averages and SEM from three experiments are shown. (D) Northern blot analysis of positive- and negative-strand BMV RNA3 and subgenomic RNA4 accumulation in WT yeast, the original mab1 mutant strain, the lsm1i isogenic strain, and the lsm1Δ knockout strain expressing 1a, 2a, and WT RNA3. Equal amounts of total yeast RNA were loaded in each lane. The negative-strand PhosphorImager image was printed at higher sensitivity to approximate a 10-fold longer exposure than the positive-strand blot.
Figure 2
Figure 2
(A) Alignment of Lsm1p with selected Sm proteins from Schizosaccharomyces pombe (GenBank Z95620), humans (AF000177), Caenorhabditis elegans (Z69302), and a partial sequence from tomato (AI490867). Identical residues are highlighted in black, and conservative substitutions are highlighted in blue. The solid arrowhead indicates the site of the lsm1 frameshift mutation. Open arrowheads indicate sites of functional HA epitope tag insertion in LSM1-HA2 and LSM1-HA3. Dots indicate spaces included to maximize alignment. (B) Intracellular localization of LSM1p. LSM2-HA3 yeast expressing 1a-, 2a-, and RNA3-derivative B3GUS was processed for confocal immunofluorescence by using antibodies against Lsm1p-HA and 1a and stained with propidium iodide for nuclear DNA. Each image is 6 μm per side.
Figure 3
Figure 3
Accumulation of BMV 1a and 2a proteins and WT RNA3 replication products (positive-strand RNA3 and RNA4) in WT and lsm1i yeast. 1a was expressed from the ADH1 promoter, and 2a was expressed from the ADH1 promoter (2a) or GAL1 promoter [2a(G)] as indicated. Equal amounts of total protein or RNA were electrophoresed in each lane for Western and Northern blot analysis. Averages and SEM from three experiments are shown.
Figure 4
Figure 4
LSM1 is required for efficient 1a-induced RNA3 stabilization. (A) Northern blot analysis of 1a stimulation of RNA3 accumulation in WT and lsm1i yeast in the absence of 2a. Equal amounts of total yeast RNA were loaded in each lane. Averages and SEM from three or more experiments are shown for each sample. Wt RNA3 was expressed from the yeast CUP1 promoter. (B) RNA3 stability analysis in WT and lsm1i yeast in the presence and absence of 1a. Wt RNA3 was expressed from the GAL1 promoter. The indicated yeasts were grown in galactose medium and transferred to glucose medium, which rapidly represses GAL1 transcription (19), and total RNA was extracted at the indicated times. Equal amounts of total yeast RNA were loaded in each lane for Northern analysis. Averages and SEM from three experiments are plotted.
Figure 5
Figure 5
3′ poly(A) suppresses the LSM1 requirement of 1a-induced RNA3 stabilization. (A) Northern blot analysis of 1a stimulation of globin-RE mRNA accumulation in WT and lsm1i yeast. Equal amounts of total yeast RNA were loaded in each lane. (B) Schematic of WT RNA3 and RNA3-poly(A), in which the tRNA-like 3′ end (3′ from a unique StuI site) was replaced by a poly(A) tail generated by the yeast ADH1 polyadenylation site. (C) Northern blot analysis of 1a stimulation of RNA3 and RNA3-poly(A) accumulation in WT and lsm1i yeast. RNA3 and RNA3-poly(A) were expressed from the GAL1 promoter and equal amounts of total yeast RNA were loaded in each lane. Averages and SEM from three or more experiments are shown for each sample.
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