Viral genome-capsid core senses host environments to prime RNA release

Viruses are metastable macromolecular assemblies containing a nucleic acid core packaged by capsid proteins that are primed to disassemble in host-specific environments leading to genome release and replication. The mechanism of how viruses sense environmental changes associated with host entry to prime them for disassembly is unknown. We have applied a combination of mass spectrometry, cryo-EM, and simulation-assisted structure refinement to Turnip crinkle virus (TCV), which serves as a model non-enveloped icosahedral virus (Triangulation number = 3, 180 copies/icosahedron). Our results reveal genomic RNA tightly binds a subset of viral coat proteins to form a stable RNA-capsid core which undergoes conformational switching in response to host-specific environmental changes. These changes include: i) Depletion of Ca2+ which triggers viral particle expansion ii) Increase in osmolytes further disrupt interactions of outer coat proteins from the RNA-capsid core to promote complete viral disassembly. A cryo-EM structure of the expanded particle shows that RNA is asymmetrically extruded from a single 5-fold axis during disassembly. The genomic RNA:capsid protein interactions confer metastability to the TCV capsid and drive release of RNA from the disassembling virion within the plant host cell. AUTHOR SUMMARY RNA viruses including coronaviruses, dengue, influenza, and HIV are a significant threat to human health. These viral particles are finely tuned to undergo complex conformational changes that allow for response to varied environments. Turnip crinkle virus (TCV) serves as an excellent model for studying RNA virus dynamics. Since TCV is non-enveloped and has no post-translational modifications, we can specifically investigate the contributions of RNA to viral dynamics. Genomic RNA is not a passive entity but plays a crucial and previously uncharacterized role in viral disassembly. Our results reveal that the genomic RNA-capsid core serves as an environmental sensor and undergoes conformational switching in response to host cell conditions.

[4,5] of the virus particle, and largely report on more structured elements of the virus particle and 57 offer only limited insights into the interior of the viral particle.  Currently, viral recognition and responses to host environments is poorly understood. Here, we 63 describe the intrinsic dynamics of virus particles and changes in conformation associated with 64 host-specific environments in vitro. 65 We selected Turnip crinkle virus (TCV), a plant RNA icosahedral virus model for mapping  previously been solved at low resolution (11 and 18 Å respectively). These structures show a 83 disordered arm in the A and B conformations and more folded structure in the C conformation. 84 The A subunits form the 5-fold axis, while the B and C subunits form the 3-fold (or quasi 6-fold) 85 axis. The arms of the C subunit form a beta annulus structure at the 3-fold axis that has been 86 postulated to be involved in RNA binding [13]. 87 To obtain additional insights into the interior of the virus particle, we have carried out 88 orthogonal cryo-EM, simulation-assisted structure refinement, and amide hydrogen/deuterium 89 exchange mass spectrometry (HDXMS) analysis of the different states of TCV. HDXMS is a 90 powerful technique that can be used to identify regions involved in conformational changes within 91 a protein, and within protein-protein, protein-lipid, protein-ligand or protein-nucleic acid 92 interfaces by reporting on hydrogen bonding and solvent accessibility [14,15]. HDXMS also 93 allows detection of multiple conformational populations in solution and has been combined with 94 variable urea denaturation to estimate the relative strengths of the icosahedral viral assembly 95 [16,17]. 96 We report a high resolution (3.2 Å) map that clearly shows the asymmetry in the interior 97 of the TCV particle and captures TCV in the process of releasing its genomic RNA into the host 98 plant cell. While the icosahedral geometry can be clearly described at this resolution, the specific 99 RNA-R domain binding interactions inside the whole virus particle are still unresolved and reflect 100 a highly dynamic interior, corroborated by HDXMS. Our findings further reveal a highly 101 disordered R-domain that is composed of genomic RNA-tightly bound (~5.7%) capsid protein.

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Significantly, a majority of the capsid protein (~94.3%) is only loosely bound to the genomic 103 RNA-capsid core. The basis for the TCV virion assembly lies in a subset of the capsid protein 104 population binding strongly to RNA to generate a ribonucleoprotein core capable of nucleating 105 assembly of many more capsid protein units to generate an icosahedral particle. The intrinsic 106 metastability of native TCV is explained by the interplay between the small subset of strong capsid 107 RNA interactions holding together the RNA-capsid core and weaker capsid interactions 108 maintaining the icosahedral geometry. Far from being a passive entity, the genomic RNA-capsid 109 core complex is the 'controller switch' for virus assembly-disassembly transitions.  Table).

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Of the three domains, the R domain showed the greatest magnitude exchange while the S 118 and P domains showed lower relative exchange overall, consistent with the R domain being the 119 least structurally ordered domain (Fig 2A). Mass spectral envelopes of N-terminal peptides 120 spanning the R domain upon deuterium exchange showed two distinct populations corresponding 121 to a low exchanging (blue) and high exchanging (green) population respectively ( Fig 2B). These  To capture conformational changes associated with particle expansion, we next carried out 136 HDXMS of expanded TCV which was generated by chelation of Ca 2+ with EDTA. Differences in 137 exchange between the expanded and native TCV particles are represented as a deuterium exchange 138 difference plot (Fig 3A). Increases in exchange were observed predominantly in the S domain.

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These regions exhibiting increases in exchange greater than 0.5 D are indicated in red ( Fig 3B).    The RNA-bound fraction showed low deuterium exchange for the R domain peptides while 170 the RNA-free fraction corresponded to the higher exchanging population. Figure 4B  in the RNA-capsid core-complex implying that the low exchanging population in the native virus 177 particle represented the proportion of the R domain tightly bound to genomic RNA. Importantly, the higher exchanging population of the native state showed lower deuterium 179 exchange compared to free coat protein from the disassembled virus ( Fig 4B). This indicated that 180 the higher exchanging population represented the fraction of R domain peripherally bound to the 181 genomic RNA in the native particle, which is disrupted by high salt, leading to disassembly of 182 TCV into the RNA-capsid core-complex and free TCV coat proteins. While mass spectra of all the 183 deuterium exchanged R domain peptides allowed an estimate (~13.6%) of the relative abundance 184 of RNA-bound and free coat protein, the mass spectra of deuterium exchanged peptide 66-89 alone 185 allowed the most accurate quantitation to a baseline resolution estimate of the relative abundance 186 of RNA-bound and RNA-free TCV coat protein (S2 Table).    bound to the RNA-capsid protein core in native TCV (Fig 6).

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These results reveal the critical importance of the genomic RNA capsid protein complex 268 to function as a sensor for the host environment. Increased osmolyte and decreased Ca 2+ in the host environment disrupts weak contacts between the capsid and RNA genome during expansion.

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Conformational changes in the genomic RNA-capsid core lead to the detection of a major 271 (~86.4%) unbound population of R domain, and a minor (~15%) strongly bound population of R 272 domain in the expanded particle. This conformational change primes the RNA for future release.

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Release can then occur in vitro in the presence of high osmolality (500 mM NaCl) which leads to 274 continued association of the RNA-capsid core. In vivo, the primed RNA genome is extruded from 275 the expanded particle by cellular ribosomes as was shown previously [13].

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Our cryo-EM analysis has revealed that the genomic RNA-capsid interactions are not 277 equally distributed throughout the expanded particle. Instead, an asymmetric packaging was 278 observed with a protrusion point a one specific 5-fold axis that also likely represents the covalent 279 p80 dimer. Additionally, increased density from the interior of the particle can be seen near this 280 protrusion point. The covalent dimer is also part of the genome capsid core during assembly, 281 suggesting that the genomic RNA acts as a nucleation point for the RNA capsid core during 282 assembly. This RNA-capsid core is then maintained in the whole viral particle and mediates viral 283 metastability and environmental sensing. During the disassembly process, the RNA directs 284 extrusion toward one specific 5-fold axis.

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These results underscore the significance of RNA structure and conformational dynamics 286 of RNA-capsid cores in viral sensing and disassembly. We have tracked dynamics of TCV through 287 the entire particle disassembly process (Fig 6). During disassembly, the TCV particle goes through    . We set a threshold fit value score of 0.9 for the 382 goodness of fit of the experimental mass spectral envelope for deuterium exchanged peptides with the theoretical envelope. If the fit score for a peptide is less than the threshold, the program 384 will fit the spectral envelope to a bimodal distribution. If the score of the bimodal distribution is 385 greater than that of the unimodal distribution, the program assigns a lower exchanging (left 386 population) and a higher exchanging (right population). Results from deconvolution for mass 387 spectra (peptide 66-89) for each of the three states are shown in S. Figure 2 and S. Table 2