Research ArticleVIROLOGY

Organelle luminal dependence of (+)strand RNA virus replication reveals a hidden druggable target

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Science Advances  24 Jan 2018:
Vol. 4, no. 1, eaap8258
DOI: 10.1126/sciadv.aap8258
  • Fig. 1 Ero1p is a rate-limiting factor for BMV RNA replication.

    (A) Total RNA and protein were extracted from WT and ero1-1 strains at 18 hours after galactose induction at a semipermissive temperature (34°C). Accumulation of virus RNAs and proteins was analyzed by Northern and Western blotting. 25S ribosomal RNA (rRNA) and Pgk1p serve as loading controls. Note that (+)RNA3 signal is derived from DNA-dependent (+)RNA3 transcription, 1a-mediated (+)RNA3 stabilization, and 1a + 2apol–mediated (+)RNA3 replication (19). To allow visualization and measurement of the RNA bands, the blots showing negative-strand accumulation were exposed much longer than those for positive strands. (B) Total RNA and protein were isolated from control and WT ERO1-overexpressing (ERO1ox) cells grown under physiological conditions (30°C) and analyzed as described above. (C) Time-course analysis of (+)RNA3 accumulation in control and ERO1-overexpressing cells. The data are the mean values obtained in experiments performed with duplicate or triplicate samples, and each is representative of two or more independent experiments.

  • Fig. 2 Ero1p stimulates BMV (+)RNA accumulation by enhancing 1a’s disulfide-linked complex formation and capping function.

    (A) Schematic representation of the limited BMV-directed RNA synthesis pathway for the RNA3 derivative 5′ Gal RNA3, which has the WT 5′ untranslated region replaced with that of yeast GAL1 mRNA (red line). MP (movement protein) and CP (capsid protein) designate RNA3 open reading frames. (B) Analysis of positive- and negative-strand 5′ Gal RNA3 and RNA4 from WT yeast cells expressing 1a, 2apol, and either vector (ctrl) or exogenous Ero1p (ERO1ox). (C) Analysis of RNA3 and RNA4 from Δxrn1 yeast cells expressing 1a, 2apol, and either vector or exogenous Ero1p. (D) Analysis of RNA3 and RNA4 from WT yeast cells expressing a capping-defective mutant 1a H80A, 2apol, and either vector or exogenous Ero1p. (E) Analysis of disulfide-linked 1a complexes from WT yeast cells used in Fig. 1B. 1a monomer and dithiothreitol (DTT)–sensitive, high–molecular weight (HMW) complex were analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) using neutral 3 to 8% SDS-PAGE gels under both reducing and nonreducing conditions.

  • Fig. 3 BMV 1a permeabilizes ER membranes and releases oxidative equivalents from ER.

    (A) Hygromycin B test. Yeast cells expressing indicated proteins are shown growing on defined solid media supplemented with the indicated hygromycin concentrations. (B) Assay for ER membrane integrity. Yeast cells expressing ER-APEX alone, or with BMV 1a or poliovirus 2B were subjected to biotin-phenol and H2O2 treatments to generate biotin-phenoxyl radicals in the ER lumen. Biotinylated protein fractions were then purified by using streptavidin-conjugated beads, electrophoresed, and visualized by Western blotting using antibodies against indicated proteins. 1a- or 2B-dependent leakage of biotin-phenoxyl radicals from the ER lumen was assessed by the biotinylation of Pgk1p, a cytosolic marker. (C) Redox status of the cytosol and cytosolic surface of the ER membrane. Two green fluorescent protein (GFP) reporters, roGFP2-Grx1 (Cytosol) and p450-roGFP2-Grx1 (ER surface), were used to monitor local glutathione redox statuses in control and 1a- and 2B-expressing yeast cells. Fluorescence intensities were measured with excitation at 405 nm for oxidized roGFP2 and at 485 nm for reduced roGFP2, and the observed fluorescence ratios were shown. (D) BMV 1a helix B and poliovirus 2B peptide-induced dye release from liposomes. Liposomes that encapsulated the self-quenching fluorescence dye SRB were incubated with indicated peptides (10 μM final). Dye release was monitored by fluorescence dequenching measurements.

  • Fig. 4 Linkage among 1a’s membrane permeabilization, disulfide bond formation, and capping functions.

    (A) Identification of viroporin-defective 1a mutants K424G and R426K, and (B to F) characterization of their phenotypes. (B) Assay for ER membrane integrity in yeast cells expressing WT 1a and 1a viroporin mutants by APEX-catalyzed protein biotinylation. (C) Redox statuses of the cytosolic surface of the ER membrane in yeast cells expressing WT 1a and 1a viroporin mutants. (D) Analysis of dye release from liposomes induced by WT, K424G, and R426K helix B peptides. Results are shown for two indicated molar ratios of peptide to phosphatidylcholine (PC); see Results and Discussion for further comments. (E) Analysis of disulfide-linked complexes in yeast cells expressing WT 1a and 1a viroporin mutants. 1a monomer serves as a loading control. (F) Effects of the viroporin mutations on 1a-mediated RNA3 recruitment in WT cells (upper panel) and 1a + 2apol–mediated RNA3 replication in WT and Δxrn1 cells (middle and lower panels). Note that a portion of the (+)RNA3 signals in the replication assay are derived from the recruitment and stabilization of RNA3 by WT 1a and 1a viroporin mutants. (G) Model for BMV RNA replication complex functionalization via membrane permeabilization and protein oxidation.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/1/eaap8258/DC1

    fig. S1. Model for BMV RNA replication complex assembly and function.

    fig. S2. Ero1p-FLAG stimulates BMV RNA replication and is glycosylated in control and 1a-expressing cells.

    fig. S3. Effect of ERO1 overexpression on 1a-increased RNA3 accumulation.

    fig. S4. Subcellular fractionation analysis of ER-APEX.

    fig. S5. Evolutionarily conserved helix A and B sequences.

    References (4952)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Model for BMV RNA replication complex assembly and function.
    • fig. S2. Ero1p-FLAG stimulates BMV RNA replication and is glycosylated in control and 1a-expressing cells.
    • fig. S3. Effect of ERO1 overexpression on 1a-increased RNA3 accumulation.
    • fig. S4. Subcellular fractionation analysis of ER-APEX.
    • fig. S5. Evolutionarily conserved helix A and B sequences.
    • References (49–52)

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