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. 1999 Nov 23;96(24):13650-5.
doi: 10.1073/pnas.96.24.13650.

RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein

M A Krol  1 , N H Olson, J Tate, J E Johnson, T S Baker, P Ahlquist
Affiliations

RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein

M A Krol et al. Proc Natl Acad Sci U S A. .

Abstract

Repeated, specific interactions between capsid protein (CP) subunits direct virus capsid assembly and exemplify regulated protein-protein interactions. The results presented here reveal a striking in vivo switch in CP assembly. Using cryoelectron microscopy, three-dimensional image reconstruction, and molecular modeling, we show that brome mosaic virus (BMV) CP can assemble in vivo two remarkably distinct capsids that selectively package BMV-derived RNAs in the absence of BMV RNA replication: a 180-subunit capsid indistinguishable from virions produced in natural infections and a previously unobserved BMV capsid type with 120 subunits arranged as 60 CP dimers. Each such dimer contains two CPs in distinct, nonequivalent environments, in contrast to the quasi-equivalent CP environments throughout the 180-subunit capsid. This 120-subunit capsid utilizes most of the CP interactions of the 180-subunit capsid plus nonequivalent CP-CP interactions. Thus, the CP of BMV, and perhaps other viruses, can encode CP-CP interactions that are not apparent from mature virions and may function in assembly or disassembly. Shared structural features suggest that the 120- and 180-subunit capsids share assembly steps and that a common pentamer of CP dimers may be an important assembly intermediate. The ability of a single CP to switch between distinct capsids by means of alternate interactions also implies reduced evolutionary barriers between different capsid structures. The in vivo switch between alternate BMV capsids is controlled by the RNA packaged: a natural BMV genomic RNA was packaged in 180-subunit capsids, whereas an engineered mRNA containing only the BMV CP gene was packaged in 120-subunit capsids. RNA features can thus direct the assembly of a ribonucleoprotein complex between alternate structural pathways.

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Figures

Figure 1
Figure 1
(A) Structure of the BMV RNA genome and plasmid DNA cassettes used to express wild-type BMV RNA2 and CP. GAL1, Rz, and A+ denote the GAL1 promoter, a self-cleaving hepatitis δ virus ribozyme (18), and the yeast ADH1 polyadenylation signal, respectively (14). (B) BMV RNA and protein accumulation in yeast. Equal amounts of total RNA from yeast expressing the indicated BMV components were analyzed by Northern blotting with RNA2 (Top) and CP (Second blot) probes. RNA from plant-derived BMV virions (BMVP) was used as a control. Equal amounts of total protein from yeast expressing the indicated components were analyzed by Western blotting with anti-2a (Third blot) and anti-CP (Bottom) antisera. Controls (lane 5) were baculovirus-expressed 2a protein (19) (Third blot, lane 5) and BMVP CP (Bottom, lane 5).
Figure 2
Figure 2
Analysis of VLPs from yeast expressing BMV RNA2 and CP. (A) RNA analysis. RNA from the indicated fractions (see text) was electrophoresed through 1% agarose, stained for total RNA, and imaged by laser fluorometry. BMVP RNA (lane 5) was included as a control. The indicated bands in lanes 2 and 4 were identified as CP mRNA and RNA2 by hybridization to CP and RNA2 probes. (B) Protein analysis. Total protein was electrophoresed and visualized by silver staining. BMVP CP (rightmost lane) was included as a control. (C) Sucrose gradient sedimentation. The resuspended pellet in lanes 4 of A and B was centrifuged through a 5–30% linear sucrose gradient, and gradient fractions were analyzed by UV absorption at 254 nm. Sedimentation profiles of yeast-derived VLPs alone (solid curve) and with BMVP added (dotted curve) are shown. (D) RNA analysis of sucrose gradient fractions, as in A. BMVP RNA (right lane) was used as a control. RNA2, RNA2 fragments (*), and CP mRNA were identified by hybridization to CP- and RNA2-specific probes. (E) Protein analysis of sucrose gradient fractions, as in B.
Figure 3
Figure 3
Analysis (as in Fig. 2) of VLPs from yeast expressing BMV CP without RNA2. (A) Sucrose gradient sedimentation profiles of yeast-derived VLPs alone (solid curve) and with BMVP added (dotted curve). (B) RNA analysis of sucrose gradient fractions. CP mRNA was identified by hybridization to a CP probe.
Figure 4
Figure 4
CryoEM and three-dimensional image reconstruction of CCMV, BMVP, and yeast-derived VLPs. The 88S and 65S VLPs were purified by sucrose gradient sedimentation from yeast expressing RNA2 + CP and CP only, respectively. (A) Cryoelectron micrographs showing representative fields of the indicated particles. Unstained particles appear dark against the brighter (less dense) supporting layer of vitreous ice. (B) Three-dimensional image reconstructions of CCMV (7), BMVP, and the 88S and 65S VLPs from yeast.
Figure 5
Figure 5
Comparison of 180- and 120-subunit capsids. (A) Space-filling model of CCMV 180-subunit capsid (7). A, B, and C subunits are blue, red, and green, respectively. One A/B dimer and one C/C dimer are each outlined with a thin white line. One of twelve pentamers of A/B dimers is outlined with a thick white line. The ribbon diagram shows B and C subunits surrounding a threefold axis. Arrowheads illustrate head-to-head CP orientation. (B) Modeled 120-subunit BMV capsid displayed as a space-filling model. A and B subunits are blue and red, respectively. One of twelve pentamers-of-dimers is outlined in white. The upper ribbon diagram illustrates the nearly tail-to-tail interaction of A/B dimers surrounding the threefold axis. The lower ribbon diagram illustrates antiparallel interaction of B subunits between threefold axes. Arrowheads are positioned on subunits as in A.
Figure 6
Figure 6
(A) Surface views of modeled (Upper) and reconstructed (Lower) 65S VLP electron density. The density map of the model was computed to 25-Å resolution; the 65S VLP reconstruction is as in Fig. 4B. (B) Stereo image of modeled 65S VLP subunits fitted within the cryoEM density map. Subunits are colored as in Fig. 5. The outer envelope of the cryoEM density map is represented as a purple mesh, and the fivefold axis is indicated by a pentagon. Asymmetric units are indicated by yellow triangles. Note B/B subunit interactions around twofold and threefold axes. (C) Ribbon diagrams of A/B dimers from CCMV (Left) and the modeled 65S VLP dimer (Right). The upper images represent views tangential to the particle surfaces. Blue and red stars indicate exterior loops of A and B subunits, respectively. The lower images represent views from the particle centers.
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