scholarly journals Multiple Nucleic Acid Binding Sites and Intrinsic Disorder of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Implications for Ribonucleocapsid Protein Packaging

2008 ◽  
Vol 83 (5) ◽  
pp. 2255-2264 ◽  
Author(s):  
Chung-Ke Chang ◽  
Yen-Lan Hsu ◽  
Yuan-Hsiang Chang ◽  
Fa-An Chao ◽  
Ming-Chya Wu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Intrinsic Disorder ◽  
Scattering Data ◽  
Nucleic Acid Binding ◽  
Structural Domains ◽  
N Protein ◽  
Disordered Regions

ABSTRACT The nucleocapsid protein (N) of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genomic RNA and is crucial for viability. However, the RNA-binding mechanism is poorly understood. We have shown previously that the N protein contains two structural domains—the N-terminal domain (NTD; residues 45 to 181) and the C-terminal dimerization domain (CTD; residues 248 to 365)—flanked by long stretches of disordered regions accounting for almost half of the entire sequence. Small-angle X-ray scattering data show that the protein is in an extended conformation and that the two structural domains of the SARS-CoV N protein are far apart. Both the NTD and the CTD have been shown to bind RNA. Here we show that all disordered regions are also capable of binding to RNA. Constructs containing multiple RNA-binding regions showed Hill coefficients greater than 1, suggesting that the N protein binds to RNA cooperatively. The effect can be explained by the “coupled-allostery” model, devised to explain the allosteric effect in a multidomain regulatory system. Although the N proteins of different coronaviruses share very low sequence homology, the physicochemical features described above may be conserved across different groups of Coronaviridae. The current results underscore the important roles of multisite nucleic acid binding and intrinsic disorder in N protein function and RNP packaging.

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 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.

scholarly journals Structural basis of nucleic acid binding byNicotiana tabacumglycine-rich RNA-binding protein: implications for its RNA chaperone function

2014 ◽  
Vol 42 (13) ◽  
pp. 8705-8718 ◽  
Author(s):  
Fariha Khan ◽  
Mark A. Daniëls ◽  
Gert E. Folkers ◽  
Rolf Boelens ◽  
S. M. Saqlan Naqvi ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Structural Basis ◽  
Nucleic Acid Binding ◽  
Rna Chaperone ◽  
Chaperone Function

Binding Of Immune Serine Proteases To Nucleic Acids Enhances Their Nuclear Localization and Promotes Their Cleavage Of Nucleic Acid-Binding Protein Substrates

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3471-3471
Author(s):  
Jennifer Whangbo ◽  
Marshall Thomas ◽  
Geoffrey McCrossan ◽  
Aaron Deutsch ◽  
Kimberly Martinod ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nuclear Localization ◽  
Serine Proteases ◽  
Rna Binding ◽  
Nuclear Accumulation ◽  
Target Cells ◽  
Nucleic Acid Binding ◽  
High Affinity ◽  
Nucleic Acid Binding Proteins

Abstract When released from cytotoxic T lymphocytes and natural killer cells, Granzyme (Gzm) serine proteases induce programmed cell death of pathogen-infected cells and tumor cells. The Gzms rapidly accumulate in the target cell nucleus by an unknown mechanism. Many of the known substrates of GzmA and GzmB, the most abundant killer cell proteases, bind to DNA or RNA. Gzm substrates predicted by unbiased proteomics studies are also highly enriched for nucleic acid binding proteins. Here we show by fluorescence polarization assays that Gzms bind DNA and RNA with nanomolar affinity. We hypothesized that Gzm binding to nucleic acids enhances nuclear accumulation in target cells and facilitates their cleavage of nucleic acid-binding substrates. In fact, RNase treatment of cell lysates reduced cleavage of RNA binding protein (RBP) targets by GzmA and GzmB. Moreover, adding RNA to recombinant RBP substrates greatly enhanced in vitro cleavage by GzmB, but adding RNA to non-nucleic acid binding proteins did not. For example, exogenous RNA enhanced GzmB cleavage of recombinant hnRNP C1 (an RBP) but not LMNB1 (a non-RBP). In addition, GzmB cleaved the RNA-binding HuR protein efficiently only when it was bound to an HuR-binding RNA oligonucleotide, but not in the presence of an equal amount of non-binding RNA. Thus, nucleic acids facilitate Gzm cleavage of nucleic acid binding substrates. To evaluate whether nucleic acid binding influences Gzm trafficking in target cells, we incubated fixed target cells with RNase and then added Gzms. RNA degradation in target cells reduced Gzm cytosolic localization and increased nuclear accumulation. Similarly, pre-incubating Gzms with exogenous competitor DNA reduced Gzm nuclear localization. The Gzms form a monophyletic clade with other immune serine proteases including neutrophil elastase (NE) and cathepsin G (CATG). Upon neutrophil activation, NE translocates to the nucleus to drive the formation of neutrophil extracellular traps (NETs). NE and CATG, but not non-immune serine proteases such as trypsin and pancreatic elastase, also bind DNA with high affinity and localize to the nucleus of permeabilized cells. Consistent with this finding, competitor DNA also blocks the nuclear localization of NE. Moreover NE and CATG localization to NETs depends on DNA binding. Thus the antimicrobial activity of NETs may depend in part upon the affinity of these proteases for DNA. Our findings indicate that high affinity nucleic acid binding is a conserved and functionally important property of serine proteases involved in cell-mediated immunity. Disclosures: Lieberman: Alnylam Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Ribonucleocapsid Formation of Severe Acute Respiratory Syndrome Coronavirus through Molecular Action of the N-Terminal Domain of N Protein

2007 ◽  
Vol 81 (8) ◽  
pp. 3913-3921 ◽  
Author(s):  
Kumar Singh Saikatendu ◽  
Jeremiah S. Joseph ◽  
Vanitha Subramanian ◽  
Benjamin W. Neuman ◽  
Michael J. Buchmeier ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Solution Structure ◽  
Rna Binding ◽  
Crystal Packing ◽  
Rna Recognition ◽  
Viral Membrane ◽  
Ribonucleoprotein Complex ◽  
N Protein ◽  
Cubic Form ◽  
Terminal Domain

ABSTRACT Conserved among all coronaviruses are four structural proteins: the matrix (M), small envelope (E), and spike (S) proteins that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in the lumen. The N-terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding, while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C terminus of the N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17 Å (monoclinic) and at 1.85 Å (cubic), respectively, resolved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the β-sheet core. The functionally important positively charged β-hairpin protrudes out of the core, is oriented similarly to that in the IBV N-NTD, and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggests a common mode of RNA recognition, but they probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs suggests that they use different modes of both RNA recognition and oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

scholarly journals Dynamics of Linker Residues Modulate the Nucleic Acid Binding Properties of the HIV-1 Nucleocapsid Protein Zinc Fingers

PLoS ONE ◽  
2014 ◽  
Vol 9 (7) ◽  
pp. e102150 ◽  
Author(s):  
Loussiné Zargarian ◽  
Carine Tisné ◽  
Pierre Barraud ◽  
Xiaoqian Xu ◽  
Nelly Morellet ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleocapsid Protein ◽  
Zinc Fingers ◽  
Nucleic Acid Binding ◽  
Binding Properties ◽  
Hiv 1
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