The Coiled-coil Domain Structure of the Sin Nombre Virus Nucleocapsid Protein

doi:10.1016/j.jmb.2006.12.046

Journal of Molecular Biology

Volume 366, Issue 5, 9 March 2007, Pages 1538-1544

https://doi.org/10.1016/j.jmb.2006.12.046 Get rights and content

Abstract

Hantaviruses can cause hemorrhagic fever with a renal syndrome and hantavirus pulmonary syndrome when transmitted to humans. The nucleocapsid protein of hantaviruses encapsidates viral genomic RNA and associates with transcription and replication complexes. Both the amino and carboxy termini of the nucleocapsid protein had been predicted to form trimers prior to the formation of the ribonucleoprotein. Crystal structures of amino-terminal fragments of the nucleocapsid protein showed the formation of intramolecular antiparallel coiled coils, but not intermolecular trimers. Thus, the amino-terminal part of the nucleocapsid protein is probably insufficient to initiate the trimerization of the full-length molecule.

Introduction

The term hantavirus encompasses any of the more than 20 distinct agents within the genus Hantavirus in the family Bunyaviridae. About half of the known members of the genus Hantavirus are pathogenic in rodents and humans. Hantaviruses of the Old World can cause hemorrhagic fever, whereas hantaviruses of the New World can cause pulmonary syndromes (HPS).¹ The major causative agent of HPS in North America is the Sin Nombre virus (SNV) carried by the deer mouse, Peromyscus maniculatus.

All members of the viral family Bunyaviridae consist of enveloped spherical particles with a helical nucleocapsid and use a genome consisting of three negative-stranded or ambisense RNAs (Figure 1). The three negative-sense RNA segments of the hantaviruses are designated L (large; about 6500 nucleotides (nt)), M (middle; about 3600 to 3700 nt), and S (small; 1700 to 2100 nt). The proteins that they encode are an RNA-dependent RNA polymerase from the L segment (L proteins); a glycoprotein precursor that is processed into two transmembrane glycoproteins (Gn and Gc) from the M segment; and a nucleocapsid (N) protein from the S segment. Hantaviral particles have a lipid envelope formed by a membrane derived from the host cell and Gn-Gc heterodimers. Within the envelope, the three genomic segments and the nucleocapsid form three separate, filamentous, 2.5 nm wide ribonucleoproteins.²

Like nucleoproteins of many negative-strand RNA viruses, hantaviral N protein is a multifunctional molecule involved in various interactions during the life cycle of the virus. It has essential functions in viral RNA replication, encapsidation, and also in virus assembly.³ Trimerization of the N protein4., 5., 6. is a crucial step in these processes³ and was found to play a role in the discrimination between viral and non-viral RNA.⁷ The N protein of hantaviruses contains from 428 (as in SNV) to 433 amino acid residues and has a molecular mass of approximately 50 kDa. A comparison of the amino acid sequences of hantavirus N proteins shows that there are three conserved domains separated by two more variable regions between residues 50 to 80 and 230 to 310. Residues 175 and 217 in the central RNA binding domain of Hantaan virus nucleocapsid8., 9. have high affinity for viral RNAs.

Using two-hybrid analyses, the hantavirus homotypic interactions were mapped to the amino-terminal4., 10. and the carboxy-terminal10., 11. regions. A three-dimensional reconstruction from electron microscopy images of recombinant N protein showed that it assembled into trimers in which individual N proteins form a curved structure.¹² Structure prediction algorithms suggested that N protein residues 3–75 form two coiled-coil segments separated by an intervening kink or turn sequence.⁵ The properties of three chemically synthesized peptides covering residues 3–35, 43–75, and 3–75 have been examined. Peptide 3–35 assembled into trimeric coiled coils at high concentrations and low temperature, whereas peptide 43–75 trimerized efficiently at low concentration, implying that it carries a coiled-coil trigger sequence. However, the longer peptide, 3–75, assembled into dimers and/or trimers at high concentration, although at low concentration it appeared to adopt an intramolecular antiparallel coiled-coil configuration. Based on these results, a 3D structure prediction of an antiparallel coiled-coil domain spanning residues 1 to 77 of the N protein of Tula hantavirus was done very recently.¹³

Here, we report the first atomic resolution structure of a Bunyavirus protein. The structure shows that trimerization of the N protein is not initiated by its amino-terminal region.

Section snippets

Design and production of recombinant proteins

A set of truncated SNV mutants, 1–75, 1–93, 1–152, 1–170, 1–300, and 1–393, and the 1–428 full-length N protein were designed for crystallization. All mutants were fused with a helper molecule (HT-mf-thromb), where HT is a His tag (MGHHHHHHGSG), mf is a 120-residue mini-fibritin (mf) sequence,14., 15., 16. and a linker with a thrombin cleavage site (GSSGSGLVPRGS) (Figure 2). The helper molecule, which aids trimerization, produces a high level of expression, and facilitates purification, was

Discussion

The structures of the slightly different SNV N protein truncations 1–75 (crystals were grown at pH 5.4) and 1–93 (crystals were grown at 7.5) were essentially identical. The polypeptide consists of two α-helices (residues 1 to 34 and 38 to 75), which form an antiparallel coiled coil with linker residues being 35-DPD-37 (Figure 3). There is no evidence that these proteins have a trimeric association in the crystal structures. Dynamic light scattering shows that the average molecular diameter is

Cloning, protein expression and purification, crystallization, and particle size determination

The gene encoding HT-mf-thromb helper protein was recloned from the plasmid pHisMf¹⁵ into pET23d(+) (Novagen) using restriction sites NcoI and BamHI. The new plasmid (pET23-HisMf) has multiple cloning sites just after the HT-mf-thromb gene.

The plasmid encoding the full-length N of Sin Nombre virus, strain CC107, was a gift from Dr Coleen B. Jonsson (Southern Research Institute, Drug Discovery and Homeland Security Division, and Department of Biochemistry and Molecular Biology, University of

Acknowledgements

We thank Cheryl Towell, Sharon Wilder, and Sheryl Kelly for their help in the preparation of the manuscript. We thank Dr Colleen B. Jonsson for providing the DNA plasmid encoding the N protein of SNV. We thank the staff of beam lines 14 (BioCARS) and 23 (GM/CA CAT) of the Advanced Photon Source for excellent support of our data collections. The work was supported by a National Institutes of Health grant (AI55672) to R.J.K. and M.G.R.

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Cited by (30)

Sin Nombre hantavirus nucleocapsid protein exhibits a metal-dependent DNA-specific endonucleolytic activity
2016, Virology
Citation Excerpt :
We further investigated deletion mutants for cleavage activity. For this purpose, we truncated the well described and crystallized N-terminal 74 amino acids (Boudko et al., 2007), which form a coiled-coil structure, from the remainder C-terminal part of the N protein (Fig. 6A). Coiled-coil motifs are responsible not only for intermolecular interactions in proteins but also for interaction with nucleic acids (Lupas, 1996).
We demonstrate that the nucleocapsid protein of Sin Nombre hantavirus (SNV-N) has a DNA-specific endonuclease activity. Upon incubation of SNV-N with DNA in the presence of magnesium or manganese, we observed DNA digestion in sequence-unspecific manner. In contrast, RNA was not affected under the same conditions. Moreover, pre-treatment of SNV-N with RNase before DNA cleavage increased the endonucleolytic activity. Structure-based protein fold prediction using known structures from the PDB database revealed that Asp residues in positions 88 and 103 of SNV-N show sequence similarity with the active site of the restriction endonuclease HindIII. Crystal structure of HindIII predicts that residues Asp93 and Asp108 are essential for coordination of the metal ions required for HindIII DNA cleavage. Therefore, we hypothesized that homologous residues in SNV-N, Asp88 and Asp103, may have a similar function. Replacing Asp88 and Asp103 by alanine led to an SNV-N protein almost completely abrogated for endonuclease activity.
Structure of the Hantavirus Nucleoprotein Provides Insights into the Mechanism of RNA Encapsidation
2016, Cell Reports
Citation Excerpt :
Hantavirus Ns bear no detectable sequence similarity with the Ns of other genera or family. The structures of the 71-amino-acid N-terminal domain of Sin Nombre and Andes hantaviruses show two antiparallel helices that intertwine to form a coiled coil and may contribute to oligomerization (Boudko et al., 2007). The intrinsic flexibility of N and its propensity to oligomerize into polymers at high concentrations has hampered its structural characterization.
Hantaviruses are etiological agents of life-threatening hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. The nucleoprotein (N) of hantavirus is essential for viral transcription and replication, thus representing an attractive target for therapeutic intervention. We have determined the crystal structure of hantavirus N to 3.2 Å resolution. The structure reveals a two-lobed, mostly α-helical structure that is distantly related to that of orthobunyavirus Ns. A basic RNA binding pocket is located at the intersection between the two lobes. We provide evidence that oligomerization is mediated by amino- and C-terminal arms that bind to the adjacent monomers. Based on these findings, we suggest a model for the oligomeric ribonucleoprotein (RNP) complex. Our structure provides mechanistic insights into RNA encapsidation in the genus Hantavirus and constitutes a template for drug discovery efforts aimed at combating hantavirus infections.
The N-terminus of the Montano virus nucleocapsid protein possesses broadly cross-reactive conformation-dependent epitopes conserved in rodent-borne hantaviruses
2012, Virology
Citation Excerpt :
The N protein is a frequently used antigen for detecting hantavirus antibodies, because it is highly conserved and provokes a strong immune response in humans and mice (Bharadwaj et al., 2000; de Carvalho Nicacio et al., 2002). Although antigenic determinants have been detected over the entire length of the N protein, the strongest response is directed toward the N-terminus (Alfadhli et al., 2002; Boudko et al., 2007; Elgh et al., 1996; Jenison et al., 1994; Lundkvist et al., 1996), which contributes substantially to the overall cross-reactive immune response (Lindkvist et al., 2007; Tischler et al., 2008). As such, deletion of the N-terminus from the protein permits the generation of antigens with serotype-specific applications (Koma et al., 2010; Li et al., 2006; Morii et al., 1998; Tischler et al., 2008).
The hantavirus nucleocapsid (N) protein is an important immunogen that stimulates a strong and cross-reactive immune response in humans and rodents. A large proportion of the response to N protein has been found to target its N-terminus. However, the exact nature of this bias towards the N-terminus is not yet fully understood. We characterized six monoclonal antibodies (mAbs) against the N protein of Montano virus (MTNV), a Mexican hantavirus. Five of these mAbs recognized eight American hantaviruses and six European and Asian hantaviruses, but not the Soricomorpha-borne Thottapalayam hantavirus. The N protein-reactive binding regions of the five mAbs were mapped to discontinuous epitopes within the N-terminal 13–51 amino acid residues, while a single serotype-specific mAb was mapped to residues 1–25 and 49–75. Our findings suggest that discontinuous epitopes at the N-terminus are conserved, at least in rodent-borne hantaviruses, and that they contribute considerably to N protein cross-reactivity.
Modulation of apoptosis and immune signaling pathways by the Hantaan virus nucleocapsid protein
2010, Virology
Herein, we show a direct relationship between the Hantaan virus (HTNV) nucleocapsid (N) protein and the modulation of apoptosis. We observed an increase in caspase-7 and -8, but not -9 in cells expressing HTNV N protein mutants lacking amino acids 270–330. Similar results were observed for the New World hantavirus, Andes virus. Nuclear factor kappa B (NF-κB) was sequestered in the cytoplasm after tumor necrosis factor receptor (TNFR) stimulation in cells expressing HTNV N protein. Further, TNFR stimulated cells expressing HTNV N protein inhibited caspase activation. In contrast, cells expressing N protein truncations lacking the region from amino acids 270–330 were unable to inhibit nuclear import of NF-κB and the mutants also triggered caspase activity. These results suggest that the HTNV circumvents host antiviral signaling and apoptotic response mediated by the TNFR pathway through host interactions with the N protein.
Crystal Structure of Human Collagen XVIII Trimerization Domain: A Novel Collagen Trimerization Fold
2009, Journal of Molecular Biology
Collagens contain a unique triple-helical structure with a repeating sequence -G-X-Y-, where proline and hydroxyproline are major constituents in X and Y positions, respectively. Folding of the collagen triple helix requires trimerization domains. Once trimerized, collagen chains are correctly aligned and the folding of the triple helix proceeds in a zipper-like fashion. Here we report the isolation, characterization, and crystal structure of the trimerization domain of human type XVIII collagen, a member of the multiplexin family. This domain differs from all other known trimerization domains in other collagens and exhibits a high trimerization potential at picomolar concentrations. Strong chain association and high specificity of binding are needed for multiplexins, which are present at very low levels.
Trimerization and triple helix stabilization of the collagen XIX NC2 domain
2008, Journal of Biological Chemistry
The mechanisms of chain selection and assembly of fibril-associated collagens with interrupted triple helices (FACITs) must differ from that of fibrillar collagens, since they lack the characteristic C-propeptide. We analyzed two carboxyl-terminal noncollagenous domains, NC2 and NC1, of collagen XIX as potential trimerization units and found that NC2 forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. In contrast, the NC1 domain requires formation of an adjacent collagen triple helix to form interchain disulfide bridges. The NC2 domain of collagen XIX and probably of other FACITs is responsible for chain selection and trimerization.

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¹: Present address: S.P. Boudko, Research Department, Shriners Hospital for Children, 3101 SW Sam Jackson Park Road, Portland, OR 97239, USA.

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Journal of Molecular Biology

The Coiled-coil Domain Structure of the Sin Nombre Virus Nucleocapsid Protein

Abstract

Introduction

Section snippets

Design and production of recombinant proteins

Discussion

Cloning, protein expression and purification, crystallization, and particle size determination

Acknowledgements

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

Methods Enzymol.

Methods Enzymol.

J. Struct. Biol.

Hantaviruses: a global disease problem

Emerg. Infect. Dis.

Hantavirus nucleocapsid protein: a multifunctional molecule with both housekeeping and ambassadorial duties

Arch. Virol.

Hantavirus nucleocapsid protein oligomerization

J. Virol.

Interaction between molecules of hantavirus nucleocapsid protein

J. Gen. Virol.

Trimeric hantavirus nucleocapsid protein binds specifically to the viral RNA panhandle

J. Virol.