In vitro quantification of the relative packaging efficiencies of single-stranded RNA molecules by viral capsid protein

Abstract

While most T=3 single-stranded RNA (ssRNA) viruses package in vivo about 3,000 nucleotides (nt), in vitro experiments have demonstrated that a broad range of RNA lengths can be packaged. Under the right solution conditions, for example, cowpea chlorotic mottle virus (CCMV) capsid protein (CP) has been shown to package RNA molecules whose lengths range from 100 to 10,000 nt. Furthermore, in each case it can package the RNA completely, as long as the mass ratio of CP to nucleic acid in the assembly mixture is 6:1 or higher. Yet the packaging efficiencies of the RNAs can differ widely, as we demonstrate by measurements in which two RNAs compete head-to-head for a limited amount of CP. We show that the relative efficiency depends nonmonotonically on the RNA length, with 3,200 nt being optimum for packaging by the T=3 capsids preferred by CCMV CP. When two RNAs of the same length-and hence the same charge-compete for CP, differences in packaging efficiency are necessarily due to differences in their secondary structures and/or three-dimensional (3D) sizes. For example, the heterologous RNA1 of brome mosaic virus (BMV) is packaged three times more efficiently by CCMV CP than is RNA1 of CCMV, even though the two RNAs have virtually identical lengths. Finally, we show that in an assembly mixture at neutral pH, CP binds reversibly to the RNA and there is a reversible equilibrium between all the various RNA/CP complexes. At acidic pH, excess protein unbinds from RNA/CP complexes and nucleocapsids form irreversibly.

Fig 1

Schematic representation of the plasmids and DNA templates used for RNA syntheses. The arrows indicate the T7 transcription promoter, and the cross at the 3′ end represents the highly conserved tRNA-like structure in the corresponding RNA transcripts. The boxes represent the open reading frames (ORFs) of the RNAs, 1a and 2a are viral replicases, and MP is the movement protein. “Δ1a” indicates that the viral replicase ORF was truncated. The representations of the plasmid templates are scaled to their relative lengths.

Fig 2

The incorporation of AF488 into B1 RNA (B1-488) has no effect on the assembly process. (a) The electrophoretic mobility analysis (EMA) shows that the electrophoretic mobilities of VLPs containing different ratios of unlabeled to labeled B1 are identical. (b) The fluorescence intensity of VLPs in which half the B1 is labeled is half that of VLPs containing only labeled B1.

Fig 3

EMA of a competition experiment between B1 and a 1.0-kb RNA. The left panel shows the three main species in a competition experiment: virus-like-particles (VLPs) (*), high-molecular-mass RNA/CP complexes (†), and free RNA (∫). The right panel shows the samples after treatment with RNase A; only the bands corresponding to the VLPs remain. The gel was stained with ethidium bromide.

Fig 4

EMAs of RNAs extracted from competitions between B1 and 0.5-kb RNA (A), 1.0-kb RNA (B), 1.5-kb RNA (C), and 2.0-kb RNA (D). Panels A and D show that both competitor RNAs were packaged in the presence of B1, while panels B and C show that neither the 1.0- nor 1.5-kb RNA was packaged. These EMAs were carried out under denaturing conditions: 8 M urea in TAE.

Fig 5

(a, b, c, and d) Diameter distributions of VLPs containing a single RNA species: RNAs of 1.0 (a), 1.5 (b), 2.0 (c), and 2.5 (d) kb. (e, f, g, and h) Same as panels a to d, but for VLPs from competition experiments between B1 and RNAs of 1.0 (e), 1.5 (f), 2.0 (g), and 2.5 (h) kb. The dotted line in each panel corresponds to the diameter distribution of VLPs containing B1 RNA. Except for the competition between B1 and a 2.0-kb RNA, which contains a higher population of VLPs with a diameter smaller than 26 nm, the diameter distributions for all competition experiments are almost identical to those for VLPs containing only B1 RNA.

Fig 6

(a) The normalized UV-Vis spectra of VLPs from a competition experiment between 0.5-kb RNA and B1 show that the ratio of absorbances at 260 and 280 nm (Abs_{260 nm}/Abs_{280 nm}) is considerably higher than it is for WT CCMV or VLPs containing either 0.5-kb RNA or B1. This ratio reflects the relative concentration of nucleotides in a capsid. (b) The size distribution of VLPs from this competition experiment is almost identical to that for VLPs containing only B1, while the distribution for those containing 0.5-kb RNA is considerably smaller. The higher RNA content in T=3 VLPs indicates that the 0.5-kb RNA was copackaged with B1.

Fig 7

Competition between B1 and C1. (a) This EMA, visualized by exciting Alexa Fluor-546 UTPs, shows that B1 is packaged more efficiently than C1 into VLPs (*). Most of C1 is in the form of high-molecular-mass RNA/CP complexes (†), and there is a small fraction of free RNA (∫). B1-488 (C1-488) and B1-546 (C1-546) correspond to BMV RNA 1 (CCMV RNA 1) labeled with AF488 and AF546. (b) The same gel stained with ethidium bromide showing total RNA content. This EMA also confirms that neither the presence nor the nature of the fluorophore perturbs the assembly process.

Fig 8

Negative-stain electron micrograph of VLPs containing a 4.0-kb-long RNA. There are two different sizes of spherical capsids and elongated particles. In competition experiments between B1 and a 4.0-kb RNA, about 5% of the particles have axial ratios of ≥1.5; in the absence of B1, 25% of the particles have ratios of ≥1.5. The particle at the right end of the white scale bar in the right panel has an axial ratio of 1.63.

Fig 9

Relative packaging efficiency of ssRNAs as a function of length. The vertical bars represent the standard deviations. All measurements are relative to B1 (3,224 nt). The triangles represent competitions where the competitor RNAs form T=3 capsids only. The circles correspond to cases where T=2 and T=3 capsids form, and the square corresponds to the case involving T=3 and T=4. The curve, which is drawn to guide the eye, illustrates that when the RNAs can only form T=3 capsids, the relative packaging efficiency has a maximum at 3.2 kb.

Fig 10

EMAs of competition experiments between B1 and B2 at different times and for different buffer and gel-staining conditions. (a) The signal comes only from AF546-labeled RNA. (b) Same gel stained with ethidium bromide showing the total RNA content. From left to right: the first two lanes show competition experiments in which both RNAs were added at the same time to RAB (pH 7.2); lanes 3 and 4 show competition experiments in which B2 was added after B1, at pH 7.2; and lanes 5 and 6 show the opposite case, again at pH 7.2. The last four lanes show the effect of adding a second RNA after lowering the pH to 4.5 (VSB).