Viral genomic single-stranded RNA directs the pathway toward a T=3 capsid

Abstract

The molecular mechanisms controlling genome packaging by single-stranded RNA viruses are still largely unknown. It is necessary in most cases for the protein to adopt different conformations at different positions on the capsid lattice in order to form a viral capsid from multiple copies of a single protein. We showed previously that such quasi-equivalent conformers of RNA bacteriophage MS2 coat protein dimers (CP(2)) can be switched by sequence-specific interaction with a short RNA stem-loop (TR) that occurs only once in the wild-type phage genome. In principle, multiple switching events are required to generate the phage T=3 capsid. We have therefore investigated the sequence dependency of this event using two RNA aptamer sequences selected to bind the phage coat protein and an analogous packaging signal from phage Qbeta known to be discriminated against by MS2 coat protein both in vivo and in vitro. All three non-cognate stem-loops support T=3 shell formation, but none shows the kinetic-trapping effect seen when TR is mixed with equimolar CP(2). We show that this reflects the fact that they are poor ligands compared with TR, failing to saturate the coat protein under the assay conditions, ensuring that sufficient amounts of both types of dimer required for efficient assembly are present in these reactions. Increasing the non-cognate RNA concentration restores the kinetic trap, confirming this interpretation. We have also assessed the effects of extending the TR stem-loop at the 5' or 3' end with short genomic sequences. These longer RNAs all show evidence of the kinetic trap, reflecting the fact that they all contain the TR sequence and are more efficient at promoting capsid formation than TR. Mass spectrometry has shown that at least two pathways toward the T=3 shell occur in TR-induced assembly reactions: one via formation of a 3-fold axis and another that creates an extended 5-fold complex. The longer genomic RNAs suppress the 5-fold pathway, presumably as a consequence of steric clashes between multiply bound RNAs. Reversing the orientation of the extension sequences with respect to the TR stem-loop produces RNAs that are poor assembly initiators. The data support the idea that RNA-induced protein conformer switching occurs throughout assembly of the T=3 shell and show that both positional and sequence-specific effects outside the TR stem-loop can have significant impacts on the precise assembly pathway followed.

Fig. 1

The molecular components used in the assembly assays. (a) Coat protein dimer quasi-equivalent conformers are shown as ribbon diagrams, with the FG loops highlighted. Their relationships within a capsid are also shown [Protein Data Bank (PDB) code 2MS2]; the A and C subunits have extended loops, while the B subunit loops fold back toward the globular core of the protein. In the complete capsid, A/B dimers surround the particle 5-fold axes with a ring of B-type loops, while the A- and C-type loops alternate around the 3-fold axes. (b) Sequences and secondary structures of the TR RNA, the aptamer consensus sequences F6 and F7 and the Qβ stem–loop.

Fig. 2

Assembly kinetics of TR and non-cognate stem–loops. (a) Gel filtration–light-scattering assays of capsid assembly. MS2 CP₂ and RNA stem–loops (coloured as indicated) were mixed in 40 mM ammonium acetate, pH 5.2–5.7, to form 1:1 reactions, incubated at 4 °C for 10, 30 or 90 min and then loaded onto a Superose 6 gel-filtration column equilibrated in 50 mM Tris–acetate, pH 7.4, and eluted at 0.4 mL/min. The outflow from the column was analysed simultaneously via UV absorbance and light-scattering, but only the latter traces are shown for clarity. Similar reactions were set up and pre-incubated at a molar ratio of 1:1 for 10 min, and then an additional aliquot of protein was added to create 2:1 reactions. The position at which T=3 capsids eluted is marked with an arrowhead. (b) The extent of capsid formation at each point was estimated using the fitting procedure described in Materials and Methods and is shown here as a histogram. Note that the assembled peak for the Qβ stem–loop includes material eluting in front of the position for T=3 capsid and the capsid peak itself.

Fig. 3

Examining the mechanism leading to the kinetic trap. (a) Normalised sedimentation coefficient distributions, C (S), derived from sedimentation velocity analysis of the MS2 capsid re-assembly reactions with TR, Qβ, F6 and F7 RNAs. The two uppermost traces show normalised C(S) plots of individual MS2 capsid re-assembly components. The two remaining C(S) plots show MS2 capsid re-assembly reactions at 1:1 and 2:1 CP₂:RNA molar ratios. The bar chart (alongside) shows the percentage of capsid-like product formed in each reaction at the two reaction ratios. The colours used follow the scheme in Fig. 2 for the different RNAs. All samples were analysed after incubation in 40 mM ammonium acetate at 4 °C for 4 h, and the resulting sedimentation distribution data were analysed using SEDFIT as described in Materials and Methods. (b) Electrospray ionisation mass spectrometry assays of stem–loop induced re-assembly. The spectra were acquired over the m/z range 500–30,000 in ammonium acetate (40 mM). Filled circles represent the initiation complex [CP₂:RNA]. The charge states for selected ions are also labelled. The spectra are for re-assembly reactions at a 1:1 stoichiometry of CP₂:RNA (8 μM:8 μM), t=30 min, for CP₂:TR, CP₂:F6, CP₂:F7 and CP₂:Qβ, respectively. The insets highlight the m/z range 12,000–30,000 and show the broad unresolved higher mass-to-charge signals assigned to the T=3 capsid. Bars below the spectra indicate the magnification factor used for all ions above m/z 12,000 to enhance the clarity. Note the absence of high m/z peaks in the TR reaction even at the highest magnification. (c) Graphs of the competitive binding experiments. CP₂:TR or competitor RNA (F6, F7 or Qβ) was added in a 1:1:1 ratio at a concentration of 10 μM. The reaction was allowed to equilibrate for 5 min, and then spectra were acquired for 1 min. The peak areas of [CP₂:TR] and [CP₂:RNA] were calculated assuming that the initiation complexes with the different RNAs have similar ionisation efficiencies. The peak area of [CP₂:TR] was set at 100%, and the other initiation complexes were normalised to this. The competition experiments were done in triplicate, and error bars shown in the plots represent the ±SD. (d) Light-scattering peaks, as in Fig. 2, for the 10 -min time points of reactions set up with molar excesses of F7 RNA over CP₂, as indicated in the colour key.

Fig. 4

Assembly with extended TR RNAs. (a) Sequences and secondary structures of the extended TR stem–loops. (b) Gel filtration–light-scattering profiles for 1:1 and 2:1 re-assembly reactions with the RNAs shown in (a). Experimental details are as those given in Fig. 2. (c) Quantitation of the extent of capsid formation in each case.

Fig. 5

Mass spectrometry of assembly with S2 RNA. (a) Stoichiometry of the 3-fold intermediate [3(CP₂:RNA)+3CP₂] using TR and S2 to initiate re-assembly in the 2:1 CP₂:RNA stoichiometry at t=30 min. The charge-state distributions corresponding to the intermediates formed during TR and S2 assembly, in the m/z range 6000–12,000 are indicated. The difference in mass between TR and S2 is 4 kDa. The observed masses for the 3-fold intermediates are 182.7 and 194.7 kDa, yielding a mass difference of 12 kDa, as expected. The peaks corresponding to the extended 5-fold intermediate with TR are clearly visible in the lower spectrum with TR (35+ to 38+) but are essentially undetectable in the upper one for S2. (b) Modelling the effect of RNA stem–loop binding to all dimers in a particle 5-fold axis. The yellow RNA stem–loop represents TR (PDB code 1AQ3), and that shown in pink is a 38-nt stem–loop (PDB code 1TXS) that has been truncated to 23 nt and manually positioned over the TR stem–loop.

Fig. 6

The inferred assembly pathway. The cartoon illustrates the inferred assembly pathways consistent with the mass spectrometry, gel-filtration and ultracentrifugation data. A C/C-like (red) coat protein dimer can be switched to an A/B-like dimer (blue/green) by binding an RNA stem–loop (yellow). In principle, such A/B-like species could self-associate, generating initially a pentameric complex that could create a closed shell with T=1 symmetry. This does not occur, ensuring formation of a larger capsid capable of packaging the phage genome. As a result, tightly binding RNA ligands “trap” the coat protein dimer in just one of the two quasi-equivalent conformers required for efficient T=3 shell formation. Addition of RNA-free (C/C-like) coat protein dimers provides the missing assembly component, leading to formation of two higher-order intermediates, corresponding to the 3- and 5-fold assemblies on the pathway to the T=3 shell. A similar outcome is observed with the weakly binding stem–loops because both sorts of coat protein dimer are always present, preventing the kinetic trap. Extended TR oligos provide additional binding sites for incoming protein subunits in the appropriate places to promote conformer switching. From the structures of the 3- and 5-fold intermediates, it is difficult to imagine their assembly pathways intersecting until the final T=3 capsid is completed.

Fig. 7

Assembly with the scrambled extended TR RNAs. (a) Sequences and secondary structures of the Srev-series of RNAs. (b) Gel filtration–light-scattering profiles for 1:1 and 2:1 re-assembly reactions with these RNAs. Experimental details are as those given in Fig. 2.