RNA-Mediated Virus Assembly: Mechanisms and Consequences for Viral Evolution and Therapy

Abstract

Viruses, entities composed of nucleic acids, proteins, and in some cases lipids lack the ability to replicate outside their target cells. Their components self-assemble at the nanoscale with exquisite precision-a key to their biological success in infection. Recent advances in structure determination and the development of biophysical tools such as single-molecule spectroscopy and noncovalent mass spectrometry allow unprecedented access to the detailed assembly mechanisms of simple virions. Coupling these techniques with mathematical modeling and bioinformatics has uncovered a previously unsuspected role for genomic RNA in regulating formation of viral capsids, revealing multiple, dispersed RNA sequence/structure motifs [packaging signals (PSs)] that bind cognate coat proteins cooperatively. The PS ensemble controls assembly efficiency and accounts for the packaging specificity seen in vivo. The precise modes of action of the PSs vary between viral families, but this common principle applies across many viral families, including major human pathogens. These insights open up the opportunity to block or repurpose PS function in assembly for both novel antiviral therapy and gene/drug/vaccine applications.

Keywords: Gillespie algorithms; Hamiltonian paths; RNA packaging signals; genome organization; mathematical modeling; normal mode analysis; single-molecule FCS; virus assembly.

Figure 1. Asymmetry and packaging signal (PS)-mediated assembly of the male-specific, fraction 2 (MS2) bacteriophage.

(a) RNA stem-loops (SLs) sharing their coat protein (CP) recognition motif with a translational repression operator (TR) (shown) act as allosteric effectors, triggering formation of the asymmetric A/B quasiconformer (blue/green) of the CP dimer from an unliganded, symmetric C/C dimer (magenta) (36, 99, 117). The X-ray structure showing the CP surface lattice (left), and a cross-section of the icosahedrally averaged cryo–electron microscopy (cryo-EM) structure revealing the multiple contacts to genomic RNA (right) (125). (b) Cross-section of the high-resolution asymmetric cryo-EM reconstruction of MS2 at 3.6 Å (29). The CP layer is shown in yellow, RNA in blue, and single-copy maturation protein (MP) in orange, and in a colour gradient in the inset. (c) The RNA backbone model built into the density in panel b with the MP also shown, together with SLs in contact with CPs (i.e., PSs) built as complete models, adapted from the data file associated with Reference . Density is color coded from TR toward the 5′ and 3′ ends (purple and green, respectively). (d) The data in panel c confirm the predicted division of genome organization in MS2 into the distinct-hemispheres-model prediction (35) illustrated, color coded as in panel c.

Figure 2. Packaging-signal (PS)-mediated assembly of Satellite tobacco necrosis virus (STNV) and mathematical modeling.

(a) Single-molecule fluorescence correlation spectroscopy (smFCS) traces of STNV coat protein (CP) interacting with dye-labeled STNV (orange) or MS2 (green) genomes. A drop in hydrodynamic radius occurs only with the cognate genome, demonstrating sequence specificity of this interaction (9). (b) Molecular basis of PS action in STNV. The N-terminal tail of the CP subunit shown in green-magenta (left) becomes more ordered as a PS binds and triggers assembly, as can be seen in the corresponding purple section of a tail in the virus-like particle (right). (c) Cooperativity between PSs is revealed by smFCS with oligonucleotides encompassing one (navy) or five (black) PSs from the 127-mer 5′ genomic fragment. Note the larger fragment undergoes a collapse in hydrodynamic radius (R _h) similar to that seen in the full-length RNA (90). (d) Mathematical modeling of PS-mediated assembly for a dodecahedral model system formed from 12 pentagonal units that can associate with, and disassociate from, any one of 12 binding sites (PSs) on model RNAs (top). These are represented here and in (e) as strings of colored beads indicating positions of PSs with high (green; ≥−12 kcal/M and <−8 kcal/M), intermediate (blue; ≥−8 kcal/M and < −4 kcal/M), and weak (red; ≥−4 kcal/M) affinities for CP (39); here all beads are shown in green as an example for illustration purposes. The assembly process is modeled via a Gillespie algorithm based on a set of reactions (bottom) describing the association and disassociation to and from RNA and between bound CPs on neighboring PSs. (e) If a complete aliquot of CP, able to assemble around each RNA, is added at the start of the simulation, capsid yield (green) is less than two-thirds of the expected yield and is characterized by significant amounts of malformed species, as well as variable nucleation at most PS pairs (bottom; percentages indicating propensity to act as a nucleation site). If CP is added according to a protein ramp (right), capsid yield is almost 100% and nucleation is highly localized.

Figure 3. Packaging-signal (PS)-mediated assembly in human pathogens.

(a, left) The Human parechovirus-1 (HPeV1) capsid seen along a fivefold axis from the outside and (right) a pentamer viewed from the inside. The latter shows density for ordered hexanucleotides (gray) corresponding to its PSs (VP0, VP1 & VP3 correspond to the three structural proteins). (b) Electron density map showing the specific recognition of G1 of the GXUXXX recognition motif (top left) and a view of one hexanucleotide and its interactions with residues in the oligo-binding pocket (top right). The generic PS motif is shown below (left), with the specific PS (lower right) that encodes part of the PS-binding site (110). This coupling of genetic code and assembly instructions perhaps explains how the mechanism persists in error-prone replicators. (c) Single-molecule fluorescence correlation spectroscopy trace of PS-mediated assembly of HBV nucleocapsids in the presence of its most conserved PSs and its immediate flanking 3′ sequence that can fold into distinct secondary structures (black, red, and navy). (d, top) The proposed sequence-specific charge neutralization of the arginine-rich domains by RNA, permitting interaction between CP dimers as the first step of assembly. (bottom) A cryo-EM reconstruction of the principal product of the assembly reaction shown in panel c, a T=4 virus-like particle (VLP); and (lower right) a cross-section of an asymmetric cryo–EM reconstruction of this VLP (91).