Rewriting nature's assembly manual for a ssRNA virus

Abstract

Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1-127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes.

Keywords: packaging signals; satellite tobacco necrosis virus; synthetic virology; viral assembly.

Fig. 1.

The STNV system. (A) Ribbon diagram of the STNV T = 1 capsid (green) (Left, PDB 3S4G) viewed along a fivefold axis with a trimeric capsomer highlighted (magenta/pink) and (Right) a CP monomer (magenta, PDB 4BCU). Sidechains mutated here are shown and labeled. The disordered N-terminal amino acid sequence is shown as a dashed line, next to the sequence of the first 25 amino acids. (B) Sequence and putative secondary structure of the 127-nt 5′ STNV-1 genomic fragment showing the locations of the PS SLs, named 5′ to 3′ as PS1 to PS5, respectively. Each contains the CP recognition motif, AXXA, in their loops (white circles, black outline). The B3 aptamer is shown similarly above. Nucleotides are color-coded as indicated, here and throughout. (C) Example smFCS assays. Hydrodynamic radius (R_h) values for CP-free, fluorescently labeled RNAs (black line for PS1–PS5 and red line for B3) are determined before and during STNV CP titration at fixed time points (vertical dashed lines). The R_h values were allowed to equilibrate after each step. The PS1–PS5 R_h initially collapses by up to 30% until the CP concentration reaches a threshold, triggering cooperative assembly to T = 1 VLPs (R_h ∼ 11 nm). At the end of each titration, the complexes formed are challenged by addition of RNase A. Largely unchanged R_h values were assumed to indicate that the RNA is in a closed VLP.

Fig. 2.

Defining the CP recognition motif. (A) Ensemble reassembly of variant B3 RNAs, analyzed by sedimentation velocity (variant RNAs are color-coded as given in Inset). The expected T = 1 VLP sediments at ∼42 S, where S is the sedimentation (Sed) coefficient. c(S) is a continuous distribution from the Lamm equation model (SI Appendix). (B) EM images of representative assembly products. (Scale bar, 50 nm, here and throughout.) See also SI Appendix, Fig. S1B. (C) Example variant RNA smFCS displacement assay for results plotted in D. (D) Percent change in R_h following addition of 100-fold molar excess of variant RNAs (color-coded) to a capsomer (R_h ∼ 5 nm) formed with 1-nM AF488-labeled B3. Error bars indicate SEM.

Fig. 3.

Electrostatic interactions and cooperativity of assembly. (A) Wild-type or R8A CPs were titrated into B3 (1 nM, black or red, respectively) or PS1–PS5 (10 nM, cyan or magenta, respectively), and R_h changes were monitored. Titrations points are shown above (B3 in gray) and below (PS1–PS5 in light blue), respectively. (B) Wild-type STNV CP was titrated into 10 nM of each of PS1–PS5 (black), PS1–PS3 (red), PS3–PS5 (blue), or PS2–PS4 (green) (Fig. 4).

Fig. 4.

Assembly of synthetic cassettes. (A) Sequences and putative secondary structures of the wild-type 127-mer, the C2 and C3 cassettes (SI Appendix, Fig. S6). (B) STNV CP titration of all variant PS1–PS5 constructs; conditions as in Fig. 3B. (Inset) EM images of the products with the wild-type 127-mer (black) and C4 (cyan) cassettes.

Fig. 5.

Assembly assays with genomic chimeras: (A) STNV CP was titrated into 1 nM of STNV-1 (black), C1-WT (magenta), or C4-WT (cyan) genomes, and the resulting R_h was monitored using smFCS. The R_h of the recombinant T = 1 particle is indicated in orange. (Inset) Color-coded EM images of the resultant products. (B) STNV CP was titrated into 1 nM of ∆AXXA (blue), PS1–PS5U-WT (red), or C4-∆AXXA (green) genomes, and the resulting R_h was monitored using smFCS. The R_h of the recombinant T = 1 particle is indicated in orange. (Inset) Color-coded EM images of the resultant products.