Interactions between brome mosaic virus RNAs and cytoplasmic processing bodies

Abstract

Cytoplasmic processing bodies are sites where nontranslating mRNAs accumulate for different fates, including decapping and degradation, storage, or returning to translation. Previous work has also shown that the Lsm1-7p complex, Dhh1p, and Pat1p, which are all components of P bodies, are required for translation and subsequent recruitment to replication of the plant virus brome mosaic virus (BMV) genomic RNAs when replication is reproduced in yeast cells. To better understand the role of P bodies in BMV replication, we examined the subcellular locations of BMV RNAs in yeast cells. We observed that BMV genomic RNA2 and RNA3 accumulated in P bodies in a manner dependent on cis-acting RNA replication signals, which also directed nonviral RNAs to P bodies. Furthermore, the viral RNA-dependent RNA polymerase coimmunoprecipitates and shows partial colocalization with the P-body component Lsm1p. These observations suggest that the accumulation of BMV RNAs in P bodies may be an important step in RNA replication complex assembly for BMV, and possibly for other positive-strand RNA viruses.

FIG. 1.

BMV RNA2 and RNA3 accumulate in P bodies, and viral RNA expression increases the size and number of P bodies. (A) Insertion of MS2 sequences into BMV RNA2 or RNA3 does not affect the steady-state abundance of viral RNA. SCR1, loading control. (B) Wild-type strain yRP2065 expressing BMV RNA2 (pRP1402) or RNA3 (pRP1403), MS2-GFP (pRP1094), and Lsm1p-cherry RFP (pRP1400), or no virus control were examined for the accumulation of RNA2 or RNA3 in P bodies. BMV RNA2 and RNA3 steady-state levels were not affected by the addition of MS2 sequences. (C) Strains containing Dhh1p (yRP2081), Pat1p (yRP2082), or Lsm1p (yRP2083) fused to GFP, which can all accumulate in P bodies (35), were analyzed with or without RNA2 or RNA3 expression for the degree of accumulation of these proteins in P bodies.

FIG. 2.

The cis-acting viral element RE is necessary and sufficient for targeting viral transcripts to P bodies. (A) Wild-type strain yRP2065 expressing full-length BMV RNA3 or various BMV RNA3 constructs lacking specific regions {from the top of the column to the bottom, pRP1403(RNA3-MS2), pRP1404 (Δ3′UTR/RNA3-MFA2), pRP1413 (Δ5′UTR/RNA3[GAL]), pRP1406 (RNA3 [GAL-RE-MFA2]), pRP1414 (ΔRE/RNA3[RE]), and MS2-GFP (pRP1094)}. (B) Wild-type strain yRP2065 expressing MS2-GFP (pRP1094) and the heterologous β-globin transcript with or without the RE {from the top of the column to the bottom, pRP1416 (β-globin[MFA2]) and pRP1415 (β-globin [RE-MFA2])}. The numbers located in the bottom right corners indicate the percentages of cells with foci out of 200 cells counted in three to five different experiments.

FIG. 3.

Mutations in the RE that affect RNA replication prevent viral-transcript accumulation in P bodies. Wild-type strain yRP2065 expressing MS2-GFP (pRP1094) and either mutant forms of the RE in RNA3 [left column, pRP1408 (RNA3 Δ32-5′), pRP1409 (RNA3 Δ45-5′), pRP1410 (RNA3 Δ79-5′), pRP1411 (RNA3 Δ52-3′), pRP1412 (RNA3 Δ73-3′), pRP1407 (RNA3 bοx Β)] or β-globin [right column, pRP1418 (β-globin Δ32-5′), pRP1419 (β-globin Δ45-5′), pRP1420 (β-globin Δ79-5′), pRP1421(β-globin Δ52-3′), pRP1422 (β-globin Δ73-3′), pRP1417 (β-globin bοx Β)]. The numbers located in the bottom right corners indicate the percentages of cells with foci out of 200 cells counted in three to five different experiments.

FIG. 4.

Viral RdRp interacts with Lsm1p. Coimmunoprecipitation of RdRp (pRP1402) with HA-tagged Lsm1p (strain yRP1430). Epitope-tagged Lsm1p was immunoprecipitated with anti-HA antibody (12CA5 Roche), and the immunoprecipitate was analyzed by Western blotting for RdRp (29). Wt, wild type.

FIG. 5.

Viral RdRp colocalizes with P bodies. Lsm1p-GFP (strain yRP2083) or Pat1p-GFP (strain yRP2082) expressing RdRp fused to cherry RFP (pRP1399) was analyzed by fluorescence microscopy.