Volume 9, Issue 3, Pages 505-514 (March 2002) A Positive-Strand RNA Virus Replication Complex Parallels Form and Function of Retrovirus Capsids  Michael Schwartz, Jianbo Chen, Michael Janda, Michael Sullivan, Johan den Boon, Paul Ahlquist  Molecular Cell  Volume 9, Issue 3, Pages 505-514 (March 2002) DOI: 10.1016/S1097-2765(02)00474-4 Copyright © 2002 Cell Press Terms and Conditions

Figure 1 1a Transfers RNA3 into a Membrane-Associated, RNase-Resistant State (A) cDNA and flanking regions in RNA expression plasmids. The arrow depicts the yeast GAL1 promoter and boxes denote open reading frames. A self-cleaving ribozyme (Rz) defined the RNA3 3′ end. RNA3ΔRE contains a deletion of the RE cis element. In globin mRNA, the human β-globin gene was flanked by the GAL1 5′ untranslated region and yeast ADH1 poly(A) site. In globinRE, the BMV RE was inserted after the β-globin gene. GlobinREΔB contains the RE with a deletion of RNA3 nt 1100–1113, including box B. (B) Northern and Western blot analysis of the distributions of RNA3, 1a, and integral ER membrane protein Dpm1 in total (T), 20,000 × g supernatant (S) and pellet (P) fractions from lysates of yeast expressing RNA3 with or without 1a. (C) Distribution of RNA3, 1a, and Dpm1 after sucrose gradient flotation analysis of lysate from yeast expressing 1a+RNA3. (D) Distribution of RNA3 in supernatant and pellet fractions as in (B) using lysates of yeast expressing RNA3 with or without 1a, fractionated after no additional treatment (None) or treatment with 0.5% NP-40 for 15 min at 0°C (Det), 0.01 U/μl micrococcal nuclease for 15 min at 30°C (RNase), or 0.5% NP-40 followed by micrococcal nuclease (Det/RNase). (E) Analysis as in (D) of RNA3, RNA3ΔRE, and globin mRNA derivatives expressed with 1a. (F) Analysis as in (D) of (−)RNA3 and (+)RNA in lysates of yeast expressing 1a+2a+RNA3 and replicating RNA3. To facilitate comparison, a light exposure of the (+)RNA3 blot is shown. See Figure 4A, lanes 1–4 to contrast amplified and unamplified (+)RNA3. Molecular Cell 2002 9, 505-514DOI: (10.1016/S1097-2765(02)00474-4) Copyright © 2002 Cell Press Terms and Conditions

Figure 2 1a Induces Perinuclear, ER Lumenal Spherules Representative electron micrographs of yeast cells expressing (A) no BMV components, (B) 1a, or (C–E) 1a+2a+RNA3 are shown. Labels denote cell wall (CW), plasma membrane (PM), nucleoplasm (Nuc), cytoplasm (Cyto), vacuole (V), and ER lumen (ERL). Arrowheads indicate splitting/rejoining of the outer and inner nuclear envelope. Samples were postfixed with osmium except for (D) which was postfixed with tannic acid. For (E), yeast were spheroplasted prior to fixation. Molecular Cell 2002 9, 505-514DOI: (10.1016/S1097-2765(02)00474-4) Copyright © 2002 Cell Press Terms and Conditions

Figure 3 Details of Spherules Copurified with Nuclei Representative electron micrographs of nuclei from yeast expressing (A) no BMV components or (B–E) 1a+2a+RNA3 are shown. Open arrowheads indicate potential connections between spherules and the outer nuclear envelope. ERL, ER lumen; CF, cytoplasmic face of outer ER membrane. Molecular Cell 2002 9, 505-514DOI: (10.1016/S1097-2765(02)00474-4) Copyright © 2002 Cell Press Terms and Conditions

Figure 4 Association of BMV RNA Synthesis with Spherules (A) Western, Northern, and RdRp analyses of nuclear preparations from yeast expressing the indicated BMV components. (B) Excess of (+)RNA over (−)RNA in RdRp products synthesized by nuclear preparations from yeast expressing 1a+2a+RNA3. Equal amounts of unlabeled (+)RNA3 (left lane) and (−)RNA3 (right lane) in vitro transcripts were electrophoresed on an agarose gel, transferred to membrane, and hybridized to 32P-labeled RdRp products. As a calibration standard, equal amounts of 32P-labeled (+) and (−) in vitro transcript probes were hybridized to a parallel blot. (C) Agarose gel electrophoresis of BMV RdRp products before and after denaturation by brief boiling. (D) RNase and detergent susceptibility assays, as in Figure 1D, of membrane-associated RdRp products in nuclear preparations from yeast expressing 1a+2a+RNA3. (E and F) Examples of incorporated BrUTP immunogold labeling of spherules from cells expressing 1a+2a+RNA3 and replicating RNA3 are shown. Molecular Cell 2002 9, 505-514DOI: (10.1016/S1097-2765(02)00474-4) Copyright © 2002 Cell Press Terms and Conditions

Figure 5 Multiple 1a Proteins and 2a Protein Are Localized in Spherules Examples of (A) anti-1a and (B) anti-2a immunogold labeling of nuclear preparations from yeast expressing 1a+2a+RNA3 are shown. Cell components are labeled as in Figures 2 and 3. Reduced membrane definition was due to the need to (A) omit or (B) 10-fold reduce osmium fixation to preserve antigenicity. Molecular Cell 2002 9, 505-514DOI: (10.1016/S1097-2765(02)00474-4) Copyright © 2002 Cell Press Terms and Conditions

Figure 6 Levels of 1a, 2a, and RNA3 in RdRp-Competent Nuclear Membrane Preparations (A) Total protein, Western, Northern, and RdRp analyses of nuclear membranes from yeast expressing the indicated BMV components. (B and C) EM analysis of nuclear membrane preparations from cells expressing (B) no BMV components or (C) 1a+2a+RNA3 are shown. Open arrowheads indicate potential connections between spherules and the cytoplasm. (D) Ratios between BMV components in nuclear membranes from cells expressing 1a+2a+RNA3 (see Results). Molecular Cell 2002 9, 505-514DOI: (10.1016/S1097-2765(02)00474-4) Copyright © 2002 Cell Press Terms and Conditions

Figure 7 Model for BMV RNA Replication Complex Assembly and Function Please see Discussion for details. Molecular Cell 2002 9, 505-514DOI: (10.1016/S1097-2765(02)00474-4) Copyright © 2002 Cell Press Terms and Conditions