scholarly journals Role of spatial patterning of N-protein interactions in SARS-CoV-2 genome packaging

2021 ◽  
Author(s):  
Ian Seim ◽  
Christine A. Roden ◽  
Amy S. Gladfelter
Keyword(s):  
Nucleic Acids ◽  
Viral Proteins ◽  
Coarse Grained ◽  
Spatial Patterning ◽  
Genomic Rna ◽  
Genome Packaging ◽  
Rna Sequences ◽  
N Protein ◽  
Single Genome ◽  
Cellular Organelles

AbstractViruses 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 compaction and 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. Single genome packaging is possible for diverse arrangements of LLPS-promoting sequences in the genome, but only in limited conditions at the edge of the phase boundary. 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 order.Statement of significanceThe COVID-19 pandemic has motivated research of the basic mechanisms of coronavirus replication. A major challenge faced by viruses such as SARS-CoV-2 is the selective packaging of a large genome in a relatively small capsid while excluding host and subgenomic nucleic acids. Genomic RNA of SARS-CoV-2 can condense with the Nucleocapsid (N)-protein, a protein component critical for packaging of many viruses. Notably, certain regions of the genomic RNA drive condensation of N-protein while other regions solubilize it. Here, we explore how the spatial patterning of these opposing elements promotes single genome compaction, packaging, and cyclization. This model informs future in silico experiments addressing spatial patterning of genomic features that are experimentally intractable because of the extensive size of the genome.

scholarly journals The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties

2021 ◽  
Author(s):  
Aneta Tarczewska ◽  
Marta Kolonko-Adamska ◽  
Mirosław Zarębski ◽  
Jerzy W Dobrucki ◽  
Andrzej Ożyhar ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleocapsid Protein ◽  
Basic Function ◽  
Molecular Properties ◽  
Structural Proteins ◽  
Genomic Rna ◽  
N Protein ◽  
Purification Methods ◽  
Purification Protocol

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.

scholarly journals The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

2020 ◽  
Author(s):  
Jasmine Cubuk ◽  
Jhullian J. Alston ◽  
J. Jeremías Incicco ◽  
Sukrit Singh ◽  
Melissa D. Stuchell-Brereton ◽  
...  
Keyword(s):  
Phase Separation ◽  
Single Molecule ◽  
Protein Function ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Genome Packaging ◽  
Binding Motifs ◽  
N Protein ◽  
Single Genome

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

scholarly journals The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine Cubuk ◽  
Jhullian J. Alston ◽  
J. Jeremías Incicco ◽  
Sukrit Singh ◽  
Melissa D. Stuchell-Brereton ◽  
...  
Keyword(s):  
Phase Separation ◽  
Single Molecule ◽  
Protein Function ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Genome Packaging ◽  
Binding Motifs ◽  
N Protein ◽  
Single Genome

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

scholarly journals Reovirus Low-Density Particles Package Cellular RNA

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1096
Author(s):  
Timothy W. Thoner ◽  
Xiang Ye ◽  
John Karijolich ◽  
Kristen M. Ogden
Keyword(s):  
Rna Polymerase ◽  
Viral Proteins ◽  
Viral Rna ◽  
Low Density ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Double Stranded Rna ◽  
Viral Genomes ◽  
Mammalian Orthoreovirus ◽  
Insight Into

Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and “genomeless” top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.

Coarse-Grained Modeling of Nucleic Acids Using Anisotropic Gay–Berne and Electric Multipole Potentials

2016 ◽  
Vol 12 (2) ◽  
pp. 676-693 ◽  
Author(s):  
Guohui Li ◽  
Hujun Shen ◽  
Dinglin Zhang ◽  
Yan Li ◽  
Honglei Wang
Keyword(s):  
Nucleic Acids ◽  
Coarse Grained

scholarly journals Trimeric Hantavirus Nucleocapsid Protein Binds Specifically to the Viral RNA Panhandle

2004 ◽  
Vol 78 (15) ◽  
pp. 8281-8288 ◽  
Author(s):  
M. A. Mir ◽  
A. T. Panganiban
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Rna Viruses ◽  
Sin Nombre Virus ◽  
Viral Capsid ◽  
Genomic Rna ◽  
Rna Molecules ◽  
N Protein ◽  
High Affinity ◽  
Negative Sense

ABSTRACT Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3′ and 5′ ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

scholarly journals Subcellular localization of nucleocapsid protein of SFTSV and its assembly into the ribonucleoprotein complex with L protein and viral RNA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sithumini M. W. Lokupathirage ◽  
Yoshimi Tsuda ◽  
Kodai Ikegame ◽  
Kisho Noda ◽  
Devinda S. Muthusinghe ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Subcellular Localization ◽  
Critical Role ◽  
Viral Proteins ◽  
Viral Rna ◽  
Host Cells ◽  
N Protein ◽  
L Protein ◽  
Translational Activity ◽  
Antiviral Targets

AbstractSevere fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes novel zoonotic diseases in Asian countries including China, Japan, South Korea, and Vietnam. In phleboviruses, viral proteins play a critical role in viral particle formation inside the host cells. Viral glycoproteins (GPs) and RNA-dependent RNA polymerase (RdRp) are colocalized in the Golgi apparatus and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The nucleocapsid (N) protein was widely expressed in the cytoplasm, even in cells coexpressing GP. However, the role of SFTSV N protein remains unclear. The subcellular localization of SFTSV structural proteins was investigated using a confocal microscope. Subsequently, minigenome and immunoprecipitation assays were carried out. The N protein interacts with viral RNA (vRNA) and further shows translational activity with RdRp which is L protein and localized in the ERGIC and Golgi apparatus when co-expressed with GP. On the other hand, mutant N protein did not interact with vRNA either localized in the ERGIC or Golgi apparatus. The interaction between the N protein of SFTSV and vRNA is important for the localization of viral proteins and viral assembly. This study provides useful insights into the life cycle of SFTSV, which will lead to the detection of antiviral targets.

scholarly journals Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

2021 ◽  
Author(s):  
Christine Roden ◽  
Yifan Dai ◽  
Ian Seim ◽  
Myungwoon Lee ◽  
Rachel Sealfon ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Structure ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Genome Packaging ◽  
Rna Motifs ◽  
Double Stranded Rna ◽  
N Protein ◽  
Binding Domains

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

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