Role of electrostatics in the assembly pathway of a single-stranded RNA virus

Abstract

We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318-3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of all of the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively charged arginine-rich motifs (ARMS) of the CPs with the negatively charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a charge-matched protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally symmetric nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario, we prepare CP mutants with extra and missing (relative to the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA.

Importance: Cowpea chlorotic mottle virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly, i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA driven by electrostatic attraction, followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of such a mechanism by demonstrating that efficient assembly is initiated by the formation of a disordered protocapsid complex whose stoichiometry is governed by electrostatics (charge matching of the anionic RNA and the cationic N termini of the CP).

FIG 3

Sucrose density gradient velocity sedimentation. Different mixtures containing fluorescently labeled CP dimers (CP₂*) were analyzed by sucrose density gradient centrifugation before (a) and after (c) acidification. After centrifugation, the 10 to 40% gradients were fractionated from top (fraction 1) to bottom (fraction 19), and the fluorescence intensity was measured. The sedimentation profile for an assembly reaction consisting of 150 CP₂* per RNA is shown in green; a stoichiometrically equivalent mixture of 60 CP₂* added per CCMV capsid is shown in red, and CCMV virions that were fluorescently labeled (CCMV*) are shown in gray. The peak intensity for CCMV* has been renormalized to match the peak intensity for the assembly reaction. Inlays b and d show representative cryo-EM images of the assembly complexes before and after acidification, respectively. Note that none of the CP₂* present in the assembly mixtures sediments as free dimer (which is found in the top fractions of the gradient), i.e., all of the CP₂* is bound in CP-RNA complexes.

FIG 4

Products of an assembly reaction mixture containing a 500-nt truncation of BMV RNA1 and enough CP to completely package the RNA (25 CP₂/RNA, which corresponds to charge matching between the CP-ARM and the RNA) were imaged by cryo-EM after acidification. A fraction of the T=2 capsids show a partial second shell consisting of the excess of CP required for charge matching. Scale bar, 50 nm.

FIG 1

CP-RNA assembly titration by gel shift assay. A 1% agarose gel was run in low-pH electrophoresis buffer and stained with EtBr. Shown is the titration of a fixed concentration of BMV RNA1 with various amounts of wt capsid protein dimer (CP₂), ranging from 0 in the left-most lane and increasing to the right. Molar CP₂/RNA ratios are reported above each lane. The right-most lane contains wt CCMV. The band positions of assembled capsids (*), naked RNA (†), and incomplete CP-RNA complexes (‡) are noted at the left of the gel. We define complete packaging to occur at the point in the titration that corresponds to the disappearance of incomplete CPRNA complexes (CP₂/RNA ratio, 140). Regions I, II, and III (see the text), i.e., lanes 1 to 6, 7 to 14, and 15 to 19, respectively, correspond to smears of increasingly CP-bound RNAs (I), coexisting incomplete complexes and nucleocapsids (II), and nucleocapsids with increasing amounts of CP bound on their exteriors (III).

FIG 2

CP-RNA assembly titrations and densitometry of gel shift assays. Shown are 1% agarose gels run in low-pH electrophoresis buffer and stained with EtBr. As described for Fig. 1, all gels show the titration of BMV RNA1 with various amounts of mutant capsid protein dimer (mCP₂), beginning at 0 (left-most lane) and increasing to the right; molar mCP₂/RNA ratios are reported above each lane. The right-most lane contains wt CCMV. The band positions of assembled capsids (*), naked RNA (†), and incomplete CP-RNA complexes (‡) are noted to the left of the gels. (a) An assembly titration involving mutant CP₂, which carries a total N-terminal charge of +24 per dimer. (b) A titration using mutant CP₂ carrying a N-terminal charge of +16. Note that the relative position of CP-RNA complexes in the right gel of panel a is different from that in the left gel due to pH changes in the running buffer that occur during electrophoresis. (c) Densitometry profiles generated by plotting the integrated EtBr signal from the capsid band of each lane as a function of mCP₂/RNA. The plot has been normalized so that the band intensity corresponding to the point of complete packaging is equal to one. The lines show best-fit sigmoid curves that are meant to aid the eye in following the extent of packaging as a function of CP₂/RNA.

FIG 5

Negative-stain EM images of BMV RNA1 completely packaged within VLPs assembled from wt CP (a) and mutant R10P CP containing +24 N-terminal charges (b) are indistinguishable. Scale bar shows 50 nm.

FIG 6

RNase A digestion of RNA, wt CCMV, and CP-RNA assembly reactions monitored by native gel electrophoresis and densitometry. Increasing amounts of RNase A were added to samples preequilibrated in neutral pH assembly buffer and left on ice for 30 min before electrophoresis. Electrophoresis was carried out in 1% native agarose gels cast in low-pH gel electrophoresis buffer. After electrophoresis, gels were stained with both EtBr and Coomassie instant blue protein stain. The RNase/RNA mass ratio of increases from left to right and is indicated above each lane. The left-most lane in each gel contains a dsDNA ladder. (a) RNase digestions of BMV RNA1 stained with EtBr. The dsDNA ladder shows (from top to bottom) 2.0, 1.5, 1.0, and 0.5 kbp. (b to d) RNase digestions of CCMV (b), an assembly reaction consisting of 150 CP₂ per RNA (c), and a similarly prepared assembly consisting of 90 CP₂ per RNA (d), also stained with EtBr. The dsDNA ladder shows (from top to bottom) 10.0, 8.0, 6.0, and 5.0 kbp. (e) Densitometry profiles were generated by plotting the integrated protein stain signal from the capsid band of each lane as a function of the RNase/RNA ratio. The plot has been normalized so that the band intensity corresponding to the point containing no RNase is equal to one.

FIG 7

RNA is symbolized as a collection of jointed cylinders (1). Each cylinder represents an amount of (negative) charge equal to the amount of (positive) charge brought by the ARMs of each CP dimer, representing a single binding site. CP dimers are represented by black triangles, which saturate the RNA binding sites during the first stage of assembly, slightly compacting the RNA (2). Upon acidification, lateral interactions between CP dimers increase in strength and drive nucleation of the capsid (represented by dashed lines) as well as further reorganization of the RNA (3). Growth of the capsid displaces the excess of bound CP to the exterior surface, where it is stabilized by electrostatic attraction between its ARMs and the negative surface charge density of the capsid (4).