An examination of the electrostatic interactions between the N-terminal tail of the Brome Mosaic Virus coat protein and encapsidated RNAs

Abstract

The coat protein of positive-stranded RNA viruses often contains a positively charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction between the tail and the RNA has been postulated as a major force in virus assembly and stabilization. The goal of this work is to examine the correlation between electrostatic interaction and amount of RNA packaged in the tripartite Brome Mosaic Virus (BMV). Nanoindentation experiment using atomic force microscopy showed that the stiffness of BMV virions with different RNAs varied by a range that is 10-fold higher than that would be predicted by electrostatics. BMV mutants with decreased positive charges encapsidated lower amounts of RNA while mutants with increased positive charges packaged additional RNAs up to ∼900 nt. However, the extra RNAs included truncated BMV RNAs, an additional copy of RNA4, potential cellular RNAs, or a combination of the three, indicating that change in the charge of the capsid could result in several different outcomes in RNA encapsidation. In addition, mutant with specific arginines changed to lysines in the capsid also exhibited defects in the specific encapsidation of BMV RNA4. The experimental results indicate that electrostatics is a major component in RNA encapsidation but was unable to account for all of the observed effects on RNA encapsidation. Thermodynamic modeling incorporating the electrostatics was able to predict the approximate length of the RNA to be encapsidated for the majority of mutant virions, but not for a mutant with extreme clustered positive charges. Cryo-electron microscopy of virions that encapsidated an additional copy of RNA4 revealed that, despite the increase in RNA encapsidated, the capsid structure was minimally changed. These results experimentally demonstrated the impact of electrostatics and additional restraints in the encapsidation of BMV RNAs, which could be applicable to other viruses.

Fig. 1. BMV exhibits heterogeneous stiffness among particles packaging different RNAs

A) Joined histograms of particle height and elastic constants measured from 108 three-particle virions and 80 R3/4 virions. The three-particle BMV contains a significant subpopulation of stiffer particles than R3/4 BMV and a few that are softer. B) Boxplot representation of elastic constants distribution of three-particle and R3/4 BMV. The whiskers were set at 5% and 95% of the total range.

Fig. 2. Capsids with an additional 360 positive charges form two subpopulations of virions

A) Sequence of the BMV capsid residues 1–26. All positively-charged residues are in bold and underlined. The triangle indicates the position of extra inserted residues in mutant 1H. B) The R3/4 1H virions separated into two bands in a cesium chloride gradient. The names of the two preparations, 1H-L and 1H-H, are shown to the right of a photograph of the density gradient illuminated with white light. C) Spectrophotometric analysis of the number of nucleotides per BMV capsid. The change in percentage was shown to allow normalization of the spectroscopic results. D) RNAs encapsidated in the 1H-L and 1H-H virions. The left panel shows an ethidium bromide stained glyoxal gel. The right panel shows the RNA from the same gel blotted by the riboprobe specific to BMV 3′UTR. The quantification at the bottom shows the ratio of RNA4 to RNA3 in each sample. R3 and R4 are short for RNA3 and RNA4. E) A demonstration that RNAs from the heated virions generate a noncovalent complex of RNA3 and RNA4. The upper panel shows a native TBE gel and lower panel a glyoxal gel. Both gels were stained with ethidium bromide. RNA from the WT three-particle virion serves as a molecular standard. The 3 kb RNA3-RNA4 complex is denoted by the asterisk. F) Analysis of the RNA complex formed by heating of 1H-L and 1H-H virions. The image shows the ethidium bromide stained native TBE gel.

Fig. 3. Analysis of mutant virions that had an additional 720 positive charges

A) Schematic of the N-terminal sequences of the six mutants characterized. The basic residues are in bold and underlined. The triangles balanced on lines denote the locations of the insertions. Only the substituted residues were typed out in 4R and 4S. B) Spectroscopic analysis of the number of nucleotides per virion. C) RNAs from the six purified BMV mutants. For each set of mutants, the left image was ethidium bromide stained glyoxal gel and the right was Northern blot images that detect BMV RNAs. The ratios of the full-length RNA4 to RNA3 are shown at the bottom of the Northern blot image. The white and black asterisks denote an unknown cellular RNA in the 4R virion. D) RNA complex formed by heating the virions. The RNAs were separated on a TBE gel and stained by ethidium bromide.

Fig. 4. Analysis of mutant virions that had 360 fewer positive charges

A) Schematic of the N-terminal sequences of the four mutants characterized. The basic residues are in bold and underlined. Only the substituted residues were typed out in the mutants. B) Spectroscopic analysis of the number of nucleotides per virion. C) RNAs from the four purified BMV mutants. For each set of mutants, the left image was ethidium bromide stained glyoxal gel and the right was Northern blot image that detect BMV RNAs. The ratios of the full-length RNA4 to RNA3 are shown at the bottom of the Northern blot image. D) RNA complex formed by heating the virions. The RNAs were separated on a TBE gel and stained by ethidium bromide.

Fig. 5. Theoretical prediction of the relationship between the capsid charge and RNA length

A) The free energy is shown as a function of RNA length for the WT and the five mutants in which additional charges were inserted, deleted, or substituted into the N-terminal tails. Results are shown for case 3 described in the text, where there is a concentration c_macro=100 mM of anionic macromolecules excluded from the capsid and the RNA charge is renormalized to 0.7 charges/nucleotide by counterion condensation. B) The same data is plotted to identify the driving force for RNA encapsidation, ΔF_EX, which is the free energy difference between the capsid with the RNA molecules enclosed and the empty capsid. C) The predicted optimal length of packaged RNA is shown as a function of capsid charge for all peptide sequences (1420AA, 1920AA, WT, 1HA, 2HA, 1H, and 2H15) for each of the three sets of assumptions considered in the text: c_macro=0, c_macro=100mM, and c_macro=100 mM with charge renormalization due to counterion condensation. D) Theoretical prediction of the RNA density as a function of radial position within the capsid.

Fig. 6. The cryo-EM structure of 2H₁₅ BMV

A) A quarter cross-section through the 2H₁₅ capsid. The plane of the cross-section includes the 5-fold and quasi 6-fold axes which cut in the centers of the pentameric and hexameric capsomeres. The insert is the averaged 1D radial density profile of the 2H₁₅. B) Models for subunits. Subunit A was shown in red (residues 40–189), subunit B in green (residues 25–189) and subunit C in blue (residues 25–189), respectively. The inserted 8 residues were not included in the numbering of the residues. The density map is shown in transparent grey and displayed at slightly higher isosurface threshold. C) Backbone representation of the 2H₁₅ trimer superimposed on the corresponding part from the crystal model of the wild type BMV (pdb:1js9), which is presented as a dark gray overlay.

Fig. 7. The correlation between the charges in the N-terminal tails and the amount of RNA packaged

The net charges on N-terminal tails per capsid was calculated by multiplying the number of positive charges within the first 49 residues by 180, the number of CP subunits per T=3 capsid. The RNA length for each mutant was estimated by applying the corresponding percentage change from Fig. 2B, 3B and 4B to 2987-nt, the expected size of RNA3 and RNA4 in WT. The mutants with increase, unchanged, and decreased capsid charges were colored in green, yellow and red, respectively.