Oligomerization and RNA binding capacity of Uukuniemi Bunyavirus nucleocapsid protein

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.

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

Frontiers in Immunology ◽

10.3389/fimmu.2021.719037 ◽

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.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1009931 ◽

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.

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

10.1101/2021.06.14.448452 ◽

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004976 ◽

2019 ◽

Vol 294 (13) ◽

pp. 5023-5037 ◽

Cited By ~ 3

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

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Shep RNA-Binding Capacity Is Required for Antagonism of gypsy Chromatin Insulator Activity

G3 Genes|Genome|Genetics ◽

10.1534/g3.118.200923 ◽

2019 ◽

pp. g3.200923.2018 ◽

Cited By ~ 1

Author(s):

Dahong Chen ◽

Margarita Brovkina ◽

Leah H. Matzat ◽

Elissa P. Lei

Keyword(s):

Rna Binding ◽

Binding Capacity ◽

Chromatin Insulator ◽

Insulator Activity

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PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites

Nucleic Acids Research ◽

10.1093/nar/gkaa211 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5511-5526

Author(s):

Tiago R Ferreira ◽

Adam A Dowle ◽

Ewan Parry ◽

Eliza V C Alves-Ferreira ◽

Karen Hogg ◽

...

Keyword(s):

Protein Modification ◽

Transcriptional Control ◽

Leishmania Major ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Capacity ◽

Arginine Methylation ◽

Mrna Metabolism

Abstract RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.

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

Virus Genes ◽

10.1007/s11262-015-1235-4 ◽

2015 ◽

Vol 51 (2) ◽

pp. 276-282 ◽

Cited By ~ 4

Author(s):

Shuling Zhao ◽

Yanan Xue ◽

Jiahui Hao ◽

Changyong Liang

Keyword(s):

Nucleocapsid Protein ◽

Rna Binding ◽

Rice Stripe Virus ◽

Binding Properties

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Functional Domain Structure of Human T-Cell Leukemia Virus Type 2 Rex

Journal of Virology ◽

10.1128/jvi.77.23.12829-12840.2003 ◽

2003 ◽

Vol 77 (23) ◽

pp. 12829-12840 ◽

Cited By ~ 15

Author(s):

Murli Narayan ◽

Ihab Younis ◽

Donna M. D'Agostino ◽

Patrick L. Green

Keyword(s):

Nuclear Export ◽

Rna Binding ◽

Binding Capacity ◽

Leukemia Virus ◽

Functional Domain ◽

Wild Type ◽

Human T Cell ◽

T Cell Leukemia Virus ◽

Carboxy Terminal

ABSTRACT The Rex protein of human T-cell leukemia virus (HTLV) acts posttranscriptionally to induce the cytoplasmic expression of the unspliced and incompletely spliced viral RNAs encoding the viral structural and enzymatic proteins and is therefore essential for efficient viral replication. Rex function requires nuclear import, RNA binding, multimerization, and nuclear export. In addition, it has been demonstrated that the phosphorylation status of HTLV-2 Rex (Rex-2) correlates with RNA binding and inhibition of splicing in vitro. Recent mutational analyses of Rex-2 revealed that the phosphorylation of serine residues 151 and 153 within a novel carboxy-terminal domain is critical for function in vivo. To further define the functional domain structure of Rex-2, we evaluated a panel of Rex-2 mutants for subcellular localization, RNA binding capacity, multimerization and trans-dominant properties, and the ability to shuttle between the nucleus and the cytoplasm. Rex-2 mutant S151A,S153A, which is defective in phosphorylation and function, showed diffuse cytoplasmic staining, whereas mutant S151D,S153D, previously shown to be functional and in a conformation corresponding to constitutive phosphorylation, displayed increased intense speckled staining in the nucleoli. In vivo RNA binding analyses indicated that mutant S151A,S153A failed to efficiently bind target RNA, while its phosphomimetic counterpart, S151D,S153D, bound twofold more RNA than wild-type Rex-2. Taken together, these findings provide direct evidence that the phosphorylation status of Rex-2 is linked to cellular trafficking and RNA binding capacity. Mutants with substitutions in either of the two putative multimerization domains or in the putative activation domain-nuclear export signal displayed a dominant negative phenotype as well as defects in multimerization and nucleocytoplasmic shuttling. Several carboxy-terminal mutants that displayed wild-type levels of phosphorylation and localized to the nucleolus were also partially impaired in shuttling. This is consistent with the hypothesis that the carboxy terminus of Rex-2 contains a novel domain that is required for efficient shuttling. This work thus provides a more detailed functional domain map of Rex-2 and further insight into its regulation of HTLV replication.

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