Structural analysis of RCNMV provides additional evidence that viruses in the Tombusviridae family have a conserved morphology despite differences in their amino acid sequences. This suggests that the assembly and disassembly mechanisms of species in the family Tombusviridae are, in general, similar, with possible individual variations on a common theme. The unusually robust virions of TBSV, RCNMV, and CarMV hint at a trigger factor that causes at least weakening, if not complete disassembly, of the capsid, followed by genome leakage in the cytosol and production of new virions. The RCNMV structures with different divalent-cation contents obtained in the present study revealed significant conformational rearrangements upon cation depletion with channels opening in the capsid. These channels could provide conduits for the viral RNA to exit the capsid and become available for replication.
CP sequence comparisons among the diverse members of the Tombusviridae
family revealed significant variation in the number of residues that comprise the arms and R domains, with the longest sequences belonging to TBSV and Tobacco necrosis virus
. The putative R domain and arm regions of the RCNMV CP are 52 residues shorter than the corresponding regions in TBSV (Fig. ) and may correlate with the necessity to package a larger genome (~5.3 to 5.8 kb in RCNMV versus ~4.9 kb in TBSV). This hypothesis for structure conservation has been recently confirmed by the ability of a series of TBSV CP N-terminal deletion mutants to form virus-like particles (16
). Coincidentally, a 52-residue TBSV deletion mutant (CP-NΔ52) produces particles which are morphologically very similar to wild-type TBSV virions, and this resembles the situation with the shorter RCNMV CP. The major structural differences between RCNMV and TBSV can be partially attributed to the shorter CP amino terminus in RCNMV.
One of the most striking features of the RCNMV virion 3D reconstruction is the cage, a structure that is composed of 60 slabs organized as 20 trimers, each with a prominent knob at its center (Fig. ). The cage and capsid shell are separated by a 7- to 9-Å gap. The pseudoatomic model of the RCNMV CP subunit accounts for most of the densities in the capsid shell and also the connecting densities corresponding to the extended arms. Altogether, with the exception of 14 to 16 N-terminal residues, the model accounts for the entire CP sequence. The N-terminal residues may be part of the cage slabs. Each slab is ~16 Å thick and ~25 Å wide in cross-section. A feature this large can easily accommodate a single-stranded RNA (ssRNA) fragment complexed with the N-terminal part corresponding to the R domain of CP. Excluded-volume estimates suggest that the cage accounts for as much as 35 to 40% of the total RCNMV RNA genome; the remaining genomic RNA lies inside the cage and appears to be more loosely packed and is most likely disordered or does not conform to icosahedral symmetry.
The presence of an ordered genome has been described for a number of viruses. Within the family Tombusviridae
, cryoEM reconstructions of TBSV (1
) and Hibiscus chlorotic ringspot virus
) revealed the presence of a layer inside the virion capsid tentatively formed by the CP R domains complexed with genomic RNA. However, these regions in TBSV and Hibiscus chlorotic ringspot virus
appeared diffuse with no characteristic discernible details like the discrete slabs observed in the RCNMV cage. This lack of an ordered inner structure in TBSV does not correlate with infectivity since a TBSV CP deletion mutant lacking the 30 N-proximal residues of the RNA-binding domain forms virus-like particles which vary in size yet are just as infectious as wild-type virions (11
In TBSV, a β-annulus formed by three interlocking extended arms (28
) is located at the threefold axis inside the capsid shell. Each arm extends away from the annulus and connects to a corresponding S domain. Similar density features at the same threefold location are observed in the RCNMVNAT
(Fig. , red arrows and red outlines) and RCNMV−Ca
maps. The β-annuli in the RCNMV density map appear as knobs at the center of the slab trimer, and the arms appear as well-defined, curved tubes of density that run from the knob toward the capsid shell (data not shown). On the basis of the sequence alignment of TBSV and RCNMV (Fig. ), RCNMV appears to lack a globular R domain like that known to be present in TBSV. In RCNMV, the N-terminal sequence is predominantly basic and we believe that up to 16 N-terminal residues interact with ordered RNA and are involved in the formation of the slabs.
In addition to determining the structure of the RCNMVNAT capsid, this study has focused on the role of Ca2+ and Mg2+ ions in RCNMV 3D organization and assembly. Atomic absorption spectroscopy showed that each virion contains significant amounts of divalent cations. When native virions were exposed to low Ca2+ or Mg2+ ion conditions, such as exhaustive dialysis against deionized water, these cations were leached from the virions. To probe their role in RCNMV capsid stability, structural and functional studies were performed with divalent cations selectively removed from virions by the chelating agents EGTA (Ca2+) and EDTA (Ca2+ and Mg2+). Comparative analysis of the structures obtained has shown that removal of Ca2+ ions induces movements of the S and P domains (Fig. and ). The removal of both Ca2+ and Mg2+ ions triggers more global rearrangement in the RCNMV structure (Fig. and ). These data indicate that Ca2+ ions are not solely responsible for structural dynamics of RCNMV virions; Mg2+ ions also significantly contribute to the rearrangements.
Upon loss of divalent cations, each of the 180 P domains exhibited increased flexibility and they reoriented by 30 or more degrees (Fig. ). As a result, the P-P interfaces are changed significantly. Increased mobility of the P domains appears to explain, at least in part, why the resolution of the RCNMV−Ca/−Mg
map was limited to 16.5 Å. Nevertheless, use of the NMFF procedure provided a means to quantitatively model the changes observed in the cryoEM map. The flexible fitting procedure showed dramatic changes in S domains upon divalent-cation depletion that cannot be described in terms of simple rotations and translations. Instead, the S domains appear to adopt a conformation different from that seen in RCNMVNAT
virions. These conformational changes in the P and S domains are responsible for forming the 11- to 13-Å-diameter channels at the quasithreefold axes (Fig. and ). The differences were especially prominent for both α-helices and for the loop (R165 to D174) lining the axis. In its native conformation, loop I158 to G181 lies completely outside of the density envelope in the RCNMV−Ca/−Mg
map when fitted as a rigid body. The NMFF modeling experiments strongly suggest that refolding of this loop occurs (Table ). It is noteworthy in this context that the swollen TBSV structure did not exhibit any significant CP subunit refolding (33
). The conformational changes observed in RCNMV upon loss of divalent cations most likely results from the combined effect of Ca2+
ions. Loss of Ca2+
alone does not significantly alter virion integrity. Removal of Ca2+
seems to initiate structural changes such as small-scale movements of the P and S domains and the slight increase in intersubunit distances at the quasithreefold axes of the capsid, where a channel appears after both cations are lost. This fact alone suggests that the RCNMV cage is tightly linked to the capsid and might exert a force when cations leave the virions, leading to opening of the channels. Additionally, our experiments with cation depletion demonstrated that pH changes used in earlier experiments with TBSV (13
) are not necessary for the channels opening in RCNMV.
Depletion of both Ca2+ and Mg2+ ions also leads to more significant changes in the structure of RCNMV (Fig. ). We assume that Mg2+ ions bind to the ssRNA genome, neutralizing its charge and aiding in its condensation in virions. Loss of Mg2+ ions could create a charge imbalance and lead to the observed splitting of the cage slabs (Fig. , inset).
The channels that arise at the quasithreefold axes in the RCNMV−Ca/−Mg
virions are too constricted to permit nucleases in their native form (>55 Å in diameter) to penetrate the capsid and to cleave the packaged RNA genome. However, these channels are sufficient in size to permit ssRNA (11 to 13 Å in diameter) to leak from the capsid. Such leakage would correlate with increased nuclease sensitivity of RCNMV−Ca/−Mg
to single-strand-specific RNases (Table ). The inner part of the channel (the entrance) includes a few basic residues but is predominantly lined with negatively charged residues (Fig. ). The termini of the genomic RNAs might be attracted to the entrance of the channel, but repulsive forces within the channel would facilitate transit of the RNA to the cytosol. The changes observed in both the cage and the outer shell of RCNMV−Ca/−Mg
may point to the actual RNA release mechanism that occurs for RCNMVNAT
in vivo. Interestingly, studies of Cowpea chlorotic mottle virus
have similarly suggested that viral RNA release by free diffusion occurs through channels at the quasithreefold axes (17
). Channels similar to those in RCNMV were observed in a recent cryoEM reconstruction of TBSV virions that were depleted of divalent cations, followed by a rise in pH to 7.5 (1
ion concentrations are typically in the millimolar range in soil and groundwater (3
). In contrast, they range between submicromolar and micromolar levels in the cytosol. These low levels are maintained and regulated by several enzymes (22
). Such low cation concentrations could trigger leaching of Ca2+
ions from RCNMV virions as they enter cells and in turn would produce the conformational changes that open channels for RNA to exit. In addition, it is also known that a cellular response to stress and infection is to increase cytosolic calcium (2
). Further, once a cell begins apoptosis, regulation of cytosolic calcium is lost (10
), ensuring an increased supply of calcium for virion formation late in the infection cycle.