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Transient oligomerization of the SARS-CoV N protein--implication for virus ribonucleoprotein packaging.

ABSTRACT: The nucleocapsid (N) phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genome into a helical ribonucleocapsid and plays a fundamental role during viral self-assembly. The N protein consists of two structural domains interspersed between intrinsically disordered regions and dimerizes through the C-terminal structural domain (CTD). A key activity of the protein is the ability to oligomerize during capsid formation by utilizing the dimer as a building block, but the structural and mechanistic bases of this activity are not well understood. By disulfide trapping technique we measured the amount of transient oligomers of N protein mutants with strategically located cysteine residues and showed that CTD acts as a primary transient oligomerization domain in solution. The data is consistent with the helical oligomer packing model of N protein observed in crystal. A systematic study of the oligomerization behavior revealed that altering the intermolecular electrostatic repulsion through changes in solution salt concentration or phosphorylation-mimicking mutations affects oligomerization propensity. We propose a biophysical mechanism where electrostatic repulsion acts as a switch to regulate N protein oligomerization.

SUBMITTER: Chang CK

PROVIDER: S-EPMC3662775 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Viruses must efficiently and specifically package their genomes while excluding cellular nucleic acids and viral sub-genomic fragments. Some viruses use specific packaging signals, which are conserved sequence/structure motifs present only in the full-length genome. Recent work has shown that viral proteins important for packaging can undergo liquid-liquid phase separation (LLPS), where one or two viral nucleic acid binding proteins condense with the genome. The compositional simplicity of viral components lends itself well to theoretical modeling compared to more complex cellular organelles. Viral LLPS can be limited to one or two viral proteins and a single genome that is enriched in LLPS-promoting features. In our previous study, we observed that LLPS-promoting sequences of SARS-CoV-2 are located at the 5' and 3' ends of the genome, whereas the middle of the genome is predicted to consist mostly of solubilizing elements. Is this arrangement sufficient to drive single genome packaging, genome compaction, and genome cyclization? We addressed these questions using a coarse-grained polymer model, LASSI, to study the LLPS of nucleocapsid protein with RNA sequences that either promote LLPS or solubilization. With respect to genome cyclization, we find the most optimal arrangement restricts LLPS-promoting elements to the 5' and 3' ends of the genome, consistent with the native spatial patterning. Genome compaction is enhanced by clustered LLPS-promoting binding sites, while single genome packaging is most efficient when binding sites are distributed throughout the genome. These results suggest that many and variably positioned LLPS-promoting signals can support packaging in the absence of a singular packaging signal which argues against necessity of such a feature. We hypothesize that this model should be generalizable to multiple viruses as well as cellular organelles like paraspeckles, which enrich specific, long RNA sequences in a defined arrangement.

Project description:Viruses must efficiently and specifically package their genomes while excluding cellular nucleic acids and viral subgenomic fragments. Some viruses use specific packaging signals, which are conserved sequence or structure motifs present only in the full-length genome. Recent work has shown that viral proteins important for packaging can undergo liquid-liquid phase separation (LLPS), in which one or two viral nucleic acid binding proteins condense with the genome. The compositional simplicity of viral components lends itself well to theoretical modeling compared with more complex cellular organelles. Viral LLPS can be limited to one or two viral proteins and a single genome that is enriched in LLPS-promoting features. In our previous study, we observed that LLPS-promoting sequences of severe acute respiratory syndrome coronavirus 2 are located at the 5' and 3' ends of the genome, whereas the middle of the genome is predicted to consist mostly of solubilizing elements. Is this arrangement sufficient to drive single genome packaging, genome compaction, and genome cyclization? We addressed these questions using a coarse-grained polymer model, LASSI, to study the LLPS of nucleocapsid protein with RNA sequences that either promote LLPS or solubilization. With respect to genome cyclization, we find the most optimal arrangement restricts LLPS-promoting elements to the 5' and 3' ends of the genome, consistent with the native spatial patterning. Genome compaction is enhanced by clustered LLPS-promoting binding sites, whereas single genome packaging is most efficient when binding sites are distributed throughout the genome. These results suggest that many and variably positioned LLPS-promoting signals can support packaging in the absence of a singular packaging signal which argues against necessity of such a feature. We hypothesize that this model should be generalizable to multiple viruses as well as cellular organelles such as paraspeckles, which enrich specific long RNA sequences in a defined arrangement.

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Transient oligomerization of the SARS-CoV N protein--implication for virus ribonucleoprotein packaging.

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