Xenopus Xp54 and human RCK/p54 helicases functionally replace yeast Dhh1p in brome mosaic virus RNA replication

Abstract

By using a Brome mosaic virus (BMV)-Saccharomyces cerevisiae system, we previously showed that the cellular Lsm1p-7p/Pat1p/Dhh1p decapping-activator complex functions in BMV RNA translation and replication. As a first approach in investigating whether the corresponding human homologues play a similar role, we expressed human Lsm1p (hLsm1p) and RCK/p54 in yeast. Expression of RCK/p54 but not hLsm1p restored the defect in BMV RNA translation and replication observed in the dhh1Delta and lsm1Delta strains, respectively. This functional conservation, together with the common replication strategies of positive-stranded RNA viruses, suggests that RCK/p54 may also play a role in the replication of positive-stranded RNA viruses that infect humans.

FIG. 1.

Overexpression of Xp54 and RCK/p54 complements the temperature-sensitive phenotype of the yeast dhh1Δ strain. By contrast, expression of human Lsm1p in the lsm1Δ strain has no effect. Yeast cells were serially diluted and spotted onto two plates, which were then separately placed at 30°C and 35°C. −, empty plasmid; Xp54 and RCK/p54, 2μm GPD promoter-driven Xenopus Xp54 and human RCK/p54 expression plasmids; Dhh1p, centromeric plasmid expressing yeast Dhh1p from its native promoter; hLsm1p, 2μm GPD promoter-driven human Lsm1p expression plasmid; Lsm1p, centromeric plasmid expressing yeast Lsm1p from its native promoter.

FIG. 2.

Xp54 and RCK/p54 functionally replace yeast Dhh1p in BMV RNA2 translation and RNA3 recruitment to the site of replication. (A) Detection of BMV RNA2 and 2a protein in WT and dhh1Δ yeast strains expressing Xp54, RCK/p54, or Dhh1p. Total RNA was extracted from yeast cells, and the accumulation of RNA2 was detected by Northern blotting, using a ³²P-labeled RNA probe specific to positive-stranded BMV RNA2. A probe specific to 18S rRNA was used to control equal loading of total RNA. Total proteins were extracted from an equal number of yeast cells and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with 2a-specific monoclonal antibodies, Xp54- and RCK/p54-specific antiserum, and 3-phosphoglycerate kinase (PGK)-specific antiserum to control equal loading of total protein. The percentages of WT (% WT) 2a protein are the averages of three or more experiments. Note that antibodies raised against Xp54 cross-react with human RCK/p54 and antibodies raised against RCK/p54 cross-react with Xenopus Xp54. (B) Detection of 1a-dependent RNA3 recruitment in WT and dhh1Δ yeast strains expressing Xp54, RCK/p54, or Dhh1p. Total RNA was extracted from yeast cells, and accumulation of RNA3 was detected by Northern blotting, using a ³²P-labeled RNA probe specific to positive-stranded BMV RNA3. Histograms show averages and standard errors of the means of the relative (Rel.) accumulations of RNA3 from three or more experiments. The average accumulation of RNA3 in WT yeast in the presence of 1a protein was set to 100. −, no expression of 1a protein; 1a_G, GAL1 promoter-driven 1a expression plasmid; Xp54 and RCK/p54, 2μm GPD promoter-driven Xenopus Xp54 and human RCK/p54 expression plasmids; Dhh1p, centromeric plasmid expressing yeast Dhh1p from its native promoter.