scholarly journals Investigating the specificity and stoichiometry of RNA binding by the nucleocapsid protein of Bunyamwera virus

RNA ◽  
2009 ◽  
Vol 15 (3) ◽  
pp. 391-399 ◽  
Author(s):  
B.-P. Mohl ◽  
J. N. Barr
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Bunyamwera Virus

scholarly journals RNA Binding Properties of Bunyamwera Virus Nucleocapsid Protein and Selective Binding to an Element in the 5′ Terminus of the Negative-Sense S Segment

2000 ◽  
Vol 74 (21) ◽  
pp. 9946-9952 ◽  
Author(s):  
Jane C. Osborne ◽  
Richard M. Elliott
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Metal Chelate ◽  
Rnase A ◽  
Mobility Shift ◽  
N Protein ◽  
Encapsidation Signal ◽  
Negative Sense ◽  
Bunyamwera Virus

ABSTRACT The genome of Bunyamwera virus (BUN) (familyBunyaviridae, genus Bunyavirus) comprises three negative-sense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5′ terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography. An RNA probe containing the 5′-terminal 32 and 3′-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 μg of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5′ terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.

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 Structural Basis for SARS-CoV-2 Nucleocapsid Protein Recognition by Single-Domain Antibodies

2021 ◽  
Vol 12 ◽  
Author(s):  
Qiaozhen Ye ◽  
Shan Lu ◽  
Kevin D. Corbett
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Binding Domain ◽  
Single Domain ◽  
Structural Basis ◽  
Dimerization Domain ◽  
Health Event ◽  
Single Domain Antibodies ◽  
Rna Binding Domain ◽  
Antigen Tests

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.

scholarly journals The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

2021 ◽  
Vol 17 (9) ◽  
pp. e1009931
Author(s):  
Jorge Vera-Otarola ◽  
Estefania Castillo-Vargas ◽  
Jenniffer Angulo ◽  
Francisco M. Barriga ◽  
Eduard Batlle ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Initiation Factor ◽  
Cellular Proteins ◽  
Dependent Manner ◽  
The Andes ◽  
Viral Nucleocapsid

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3’UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3’UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A–eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5’-3’ end interaction, mediated by both viral and cellular proteins.

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 Crimean-Congo hemorrhagic fever virus nucleocapsid protein harbors distinct RNA-binding sites in the stalk and head domains

2019 ◽  
Vol 294 (13) ◽  
pp. 5023-5037 ◽  
Author(s):  
Subbiah Jeeva ◽  
Sheema Mir ◽  
Adrain Velasquez ◽  
Jacquelyn Ragan ◽  
Aljona Leka ◽  
...  
Keyword(s):  
Binding Sites ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Rna Binding Sites

The RNA-binding properties and domain of Rice stripe virus nucleocapsid protein

Virus Genes ◽  
2015 ◽  
Vol 51 (2) ◽  
pp. 276-282 ◽  
Author(s):  
Shuling Zhao ◽  
Yanan Xue ◽  
Jiahui Hao ◽  
Changyong Liang
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Rice Stripe Virus ◽  
Binding Properties

scholarly journals RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly

2015 ◽  
Vol 89 (19) ◽  
pp. 9739-9747 ◽  
Author(s):  
Simone Mattei ◽  
Annica Flemming ◽  
Maria Anders-Össwein ◽  
Hans-Georg Kräusslich ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Electron Tomography ◽  
Significant Proportion ◽  
Dimensional Structure ◽  
Capsid Assembly ◽  
Interaction Domain ◽  
Cryo Electron Tomography ◽  
Rna Genome ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context.IMPORTANCEFormation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA and analyzed their three-dimensional structure by cryo-electron tomography. While most virions displayed an abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein is not required for the formation of a mature, cone-shaped HIV-1 capsid.

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