Rapid evolution of protein binding interfaces has frequently been observed in viral protein complexes, notably in the virus-host interface, including viral surface glycoproteins as well as ribonuclear proteins and non-structural proteins, with fitness advantages being accomplished, for example, through reshaping the binding interfaces, modulating protein structural dynamics, or altering physicochemical properties (Barozi et al., 2022; Evseev and Magor, 2021; Focosi et al., 2024; Planchais et al., 2022; Rochman et al., 2022). Also, entirely new interactions can arise through the viral mimicry of eukaryotic short linear motifs as a result of frequent mutations in the viral protein intrinsically disordered regions, which can greatly augment the virus-host interface (Davey et al., 2015; Schuck and Zhao, 2023). The mutations we have studied here are of a different category, impacting the interactions among viral proteins that enhance viral multi-protein complexes. Previous examples include the intra-host diversity of polymerase subunit interfaces in H5N1 influenza viruses (Welkers et al., 2019). While such mutations are not directly targeted towards the host, they may still contribute to host adaptation or be balancing other mutation effects in an epistatic network, conceivably involving modulation of local effective protein concentrations (Li et al., 2023b). Irrespective of their complete context, they can provide valuable insights into viral protein mechanisms.
Specifically, we have described three different mutations of SARS-CoV-2 N-protein that, in convergent evolution, strengthen the formation of RNPs and enhance viral assembly. N:G214C, N:G215C, and N:P13L have been independently introduced (as highlighted in the phylogenetic trees Figure 9; Figure 10; Figure 11), and persisted in the defining set of mutations in their respective variants of concern (Lambda, Delta, and Omicron, respectively). We have shown here that N:P13L confers a fitness advantage in cell lines, and similarly, N:G215C was shown by Kubinski et al., 2024 to impart improved viral growth. This correlates well with our results studying their molecular mechanisms.
Mutations of N:P12 across the phylogenetic tree of SARS-CoV-2.
Shown are all-time global sequence samples with clade labels and color-coded amino acid at position 13, with the ancestral P13 in green and P13L in yellow. The blue arrow points to the Lambda sequences. Additionally, a cluster of P13L mutations occurred in India in clade 19 A. The phylogenetic tree was generated by Nextstrain (Hadfield et al., 2018).
Mutations of N:G215 across the phylogenetic tree of SARS-CoV-2.
Shown are all-time global sequence samples with clade labels and color-coded amino acid at position 215, with the ancestral G215 in green and G215C in yellow. The phylogenetic tree was generated by Nextstrain (Hadfield et al., 2018).
Mutations of N:G214 and N:G215 across the phylogenetic tree of SARS-CoV-2.
Shown are all-time sequence samples in South America with clade labels and color-coded amino acid at position 214 and 215. The combination of 214 C/G215 strain 21 G (Lambda) is shown in blue, whereas the combination G214/215 C of strain 21 J (Delta) is shown in yellow. The phylogenetic tree was generated by Nextstrain (Hadfield et al., 2018).
For both of the cysteine mutants, molecular dynamics simulations and biophysical studies show how cysteines augment self-association interfaces by extending and redirecting the transiently formed helical coiled-coils in the intrinsically disordered LRS, which play a central role in the assembly of RNPs. By contrast, for N:P13L, unexpectedly, the evolution of RNP stability goes beyond modulation of a previously existing binding interface, and instead, we observe the de novo formation of an additional dynamic self-association interface in the distant disordered N-arm through the stabilization and stacking of transient β-sheets, that we hypothesize cooperatively contributes to the stability of RNPs. Even though the solution affinity of the N-arm P13L interface is ultra-weak, the average local concentration of N-arm chains across the RNP volume (in a back-of-the-envelope calculation assuming a ≈14 nm cube Klein et al., 2020 with a dodecameric N cluster) is ≈7.4 mM, such that disordered N-arm peptides could well create populations of N-arm clusters stabilizing RNPs through this interface.
However, besides the RNP-stabilizing mutants, we have also observed unexpected RNP destabilization by the ubiquitous R203K/G204R double mutation, which may be caused by the introduction of additional charges close to the self-association interface in the LRS. In our experiments, this destabilization is more than compensated for by the P13L mutation. (Another scenario where ultra-weak interactions can have a critical impact is in molecular condensates. We previously reported the suppression of LLPS by the R203K/G204R mutation, which is rescued by the additional P13L/Δ31–33 mutation (Nguyen et al., 2024). This is consistent with compensatory weak stabilizing and destabilizing impacts of weak interactions on the RNP observed here.)
We arrive at a picture of SARS-CoV-2 RNPs that is far from structurally well defined, matching the concept of fuzzy complexes (Wu and Fuxreiter, 2016). On a molecular level, large portions of the SARS-CoV-2 N-protein (the N-arm, C-arm, and linker) are intrinsically disordered and highly flexible (Cubuk et al., 2021; Różycki and Boura, 2022), which persists in the presence of bound nucleic acid (Cubuk et al., 2024; Guseva et al., 2021; Schiavina et al., 2022). It appears that conformational freedom is also retained to a significant degree in the RNPs. This flexibility could be advantageous for accommodating various RNA secondary structures (Carlson et al., 2022; Landeras-Bueno et al., 2025) and favorably balance the energetic cost of RNP disassembly that is required immediately after viral entry. Also, this serves to accommodate significant sequence variation (Davey et al., 2011; Duro et al., 2015; Schuck and Zhao, 2023). SARS-CoV-2 RNPs appear highly heterogeneous in EM (Carlson et al., 2022; Landeras-Bueno et al., 2025; Yao et al., 2020), and this is reflected in the polymorphic oligomeric states of RNP species we observe in SV-AUC and MP, that we believe is driven by promiscuous self-association or clustering of transient LRS helices (Zhao et al., 2022). Extending previously described characteristics of fuzziness in protein complexes (Duro et al., 2015; Fuxreiter, 2018; Tompa and Fuxreiter, 2008), plasticity seems to involve even basic architectural principles, considering not only the emergence of new distant stabilizing interfaces such as described here in the N-arm, but also the possibility of RNP assembly of truncated N210-419* lacking one of the major nucleic acid binding interfaces (Adly et al., 2023; Bouhaddou et al., 2023; Mears et al., 2025; Mulloy et al., 2025; Syed et al., 2024) (see below).
Unfortunately, this intrinsic heterogeneity poses significant methodological hurdles. Nonetheless, salient structural features and assembly principles may be derived from constraints of known binding interfaces and oligomeric states of the RNP and its subunits, as observed in SV-AUC and MP. While the arrangement sketched in Figure 1C satisfies these requirements, alternate less symmetrical configurations can be conceived that seem at least equally likely and may coexist in polydisperse mixtures of RNPs. For example, there is no evidence to exclude the possibility of anti-parallel LRS helices pointing the folded nucleic acid -binding domains in different relative orientations, or of mixed co-assemblies with N210-419* subunits lacking the NTD (Figure 1—figure supplement 1). Uniformity of N-protein/RNA clusters may not be relevant for adequate gRNA condensation.
Beyond the structural model, to study the effect of a larger number of N-protein mutations derived from variants of concern side-by-side in the context of virus assembly, we have carried out experiments using a VLP assay (Syed et al., 2021; Figure 7). In these experiments, all four structural proteins are transfected into 293T cells to package a reporter RNA into VLPs, and their infection of receiver cells can be compared. While this assay has been widely used for rapid assessment of spike protein and N variants (Syed et al., 2021), it has limitations due to the addition of non-genomic RNA and the lack of double membrane vesicles from which gRNA emerges through the NSP3/NSP4 pore complex potentially poised for packaging (Bessa et al., 2022; Ke et al., 2024; Ni et al., 2023). It should also be recognized that the results do not directly reflect the relative efficiency of RNP assembly only, since protein expression levels, their localization, and their posttranslational modifications are not controlled for. Susceptibility to such factors might be exacerbated with mutations that modulate weak protein interactions. For example, as shown previously (Syed et al., 2024; Zhao et al., 2024), a GSK3 inhibitor inhibiting N-protein phosphorylation significantly enhances VLP formation and eliminates the advantage provided for by the N:G215C mutation relative to the ancestral N – presumably due to an increase in assembly-competent, non-phosphorylated N-protein erasing an affinity advantage. A similar process may be underlying the absent or marginal improvement in VLP readout from the cysteine LRS mutants and P13L at the achieved transfection level in the present work, and the enhanced signal from R203K/G204R and R203M (the latter being consistent with previous reports Li et al., 2025; Syed et al., 2021) modulating protein phosphorylation. Nonetheless, mirroring the results of the biophysical in vitro experiments, the addition of RNP-stabilizing P13L and G214C mutations on top of R203K/G204R led to a significantly larger VLP signal.
The VLP assay may also be limited in sensitivity to mutation effects due to its restriction to a single round of infection. To avoid this and other potential limitations of the VLP assay for the study of viral packaging, for the key mutation N:P13L, we carried out reverse genetics experiments. These showed the sole N:P13L mutation significantly increases viral fitness (Figure 8).
Regarding the cysteine mutations that have been repeatedly introduced in the LRS prior to the rise of the Omicron variants of concern, it is an open question whether they lead to covalent bonds in vivo or in the VLP assay. While examples of disulfide-linked viral nucleocapsid proteins have been reported (Kubinski et al., 2024; Prokudina et al., 2004; Wootton and Yoo, 2003), a methodological difficulty in their detection is artifactual disulfide bond formation post-lysis of infected cells (Kubinski et al., 2024; Wootton and Yoo, 2003). However, our results clearly show that a major effect of the cysteines already arises in reduced conditions without any covalent bonds, through extension of the LRS helices and concomitant redirection of the disordered N-terminal sequence. While oxidized tetrameric N-proteins of N:G214C and N:G215C can be incorporated into RNPs, the covalent bonds provided only marginally improved RNP stability. Interestingly, the introduction of cysteines imposes preferences of RNP oligomeric states dependent on oxidation state, consistent with our MD simulations highlighting the impact of cysteine orientation of 214 C versus 215 C relative to the hydrophobic surface of the LRS helices. Overall, considering potentially detrimental structural constraints from covalent bonds on LRS clusters seeding RNPs, energetic penalties on RNP disassembly, as well as the required monomeric state of the LRS helix for interaction with the NSP3 Ubl domain (Bessa et al., 2022), at present, it is unclear to what extent the formation of disulfide linkages between LRS helices would be beneficial or detrimental in the viral life cycle.
Recent work by the Soranno laboratory has identified an additional function of the disordered N-arm in transiently interacting with the NTD (Cubuk et al., 2021) and dynamically enhancing the affinity of the NTD for RNA (Cubuk et al., 2024). Using single-molecule Förster Resonance Energy Transfer (smFRET), a fourfold modulatory effect of the P13L/Δ31-33 mutation on the NTD RNA binding affinity was observed in N-arm-NTD constructs. Through MD simulations, the reduced NTD affinity for RNA was attributed to the N-arm Δ31-33 deletion (Cubuk et al., 2024). Superficially, this may seem in slight conflict with our results of similar T10 affinity of full-length ancestral N with and without the P13L/Δ31-33 mutation, but results were obtained in different buffer conditions (50 mM TRIS, pH 7.4 in Cubuk et al., 2024 versus 20 mM HEPES, 150 mM NaCl, pH 7.5 in the present work). In any event, RNA binding of NTD and stabilization of the RNP are different processes; any modulation of N-arm contributions to NTD-RNA interactions through Omicron N-arm mutations Δ31-33 may coexist and be over-compensated for by N-arm self-association interfaces through P13L modulating RNP subunit interactions in the high local N-arm density of the RNP.
The double mutant R203K/G204R arose early in the pandemic and was adopted in several variants of concern (including Alpha, Gamma, Lambda, Zeta, and Omicron BA.1) with the triple nucleotide changes G28881A, G28882A, and G28883C (Mears et al., 2025; Syed et al., 2024; Figure 12). As mentioned above, on the protein level, N:R203K/G204R has been shown to alter phosphorylation (although in different ways in in vitro VLP or in vivo reverse genetics experiments; Johnson et al., 2022; Syed et al., 2024; Yun et al., 2022), and phosphorylation in turn reduces nucleic acid binding and promotes viral replication as opposed to assembly functions (Botova et al., 2024; Bouhaddou et al., 2023; Carlson et al., 2020; Syed et al., 2024). Adding to such a switch, in the present work, we observed the loss of RNP stability of N:R203K/G204R relative to the ancestral N, extending the previous observation of reduced LLPS propensity of N:R203K/G204R (Nguyen et al., 2024). Simultaneously, on the RNA level, the N:R203K/G204R mutations also lead to the new formation of a TRS sequence ACGAAC underlying the expression of N210-419* in virus-infected cells (though not expected to occur with N:R203K/G204R in the VLP assay lacking the viral RNA-dependent RNA polymerase). It has been hypothesized that N210-419* confers increased viral fitness through the suppression of the host anti-viral response (Mears et al., 2025; Mulloy et al., 2025), and that it can assist RNP formation (Bouhaddou et al., 2023; Syed et al., 2024). However, the contribution of N210-419* to assembly is still unclear: although it is remarkably capable of forming RNPs in vitro and VLP assays (Adly et al., 2023; Bouhaddou et al., 2023; Syed et al., 2024), in infected cells and virions, N210-419* has been detected only as a minority species (Mears et al., 2025; Mulloy et al., 2025). Also, the recent major Omicron XEC variant (Scarpa et al., 2025; which had close to 60% global frequency at the beginning of 2025; Figure 12) exhibits a fourth consecutive nucleotide change G28884C that maintains a similar RG mutation forming R203K/G204P but ablates the canonical TRS sequence, such that continued expression of N210-419* in XEC is in question. We propose that an alternative or additional mechanism to retain viral assembly functions may be presented by the accompanying P13L mutation, which our data suggest can more than restore loss of RNP stability in the combination of RG mutations with P13L. This combination occurs in No and all Omicron variants so far and was even further stabilized with a cysteine in the LRS in Nλ.
Mutations of N:R203 and N:G204 across the phylogenetic tree of SARS-CoV-2.
Shown are global sequence samples mostly representing sequences of the recent 6 months, with clade labels and color-coded amino acid at positions 203 and 204. The ancestral combination of R203/G215 is shown in green, the mutation 203 M of the Delta VOC in blue, the combination 203 K/204 R common to Alpha and Omicron VOCs in yellow, and the combination 203 K/204 P defining in the Omicron XEC variant in orange. The phylogenetic tree was generated by Nextstrain (Hadfield et al., 2018).
In conclusion, it has been proposed that mutations in SARS-CoV-2 N protein that affect viral assembly can impact infectivity and fitness (Bouhaddou et al., 2023; Syed et al., 2024; Wu et al., 2021; Zhao et al., 2022). We believe the observed modulations of the RNP assembly and stability studied here highlight a key mechanism for this. Although effects on fitness of viruses carrying N mutations are most likely multi-factorial, they have been observed in reverse genetics tissue culture experiments previously for N:R203K/G204R (Johnson et al., 2022; Mears et al., 2025; Wu et al., 2021), N:G215C (Kubinski et al., 2024), and in the present work for N:P13L. On the other hand, the rise of new variants of concern was usually dominated by their spike protein mutations (with the exception of 21I replacement by 21J which has identical spike mutations but acquired N:G215C Marchitelli et al., 2021; Stern et al., 2021; Zhao et al., 2022 in the rise of Delta variant), and in many cases, N mutations of previously dominant variants were completely replaced by another set of N mutations (dramatically exemplified in the displacement of Delta by Omicron variants). This reinforces the view that these N mutations are secondary to alterations in the immune landscape and transmissibility as the primary driver of evolution (Markov et al., 2023). Nonetheless, the remarkable plasticity of RNPs offers multiple avenues to modulate stability and to compensate for potentially RNP-destabilizing effects of mutations that are beneficial in other ways. In convergent evolution, this has been a constant theme of N protein mutations throughout the SARS-CoV-2 pandemics up until today. We hypothesize that the ‘fuzziness’ and pleomorphic ability of RNP assembly, with its variable distribution of overall binding energy into several different weak or ultra-weak protein interfaces, and the poor structural definition ranging from flexible chain configurations to polydisperse oligomeric states, provides an evolutionary advantage of orchestrated disorder to promote epistatic interactions and facilitate host adaptation.