Oligomerization of Hantavirus N Protein: C-Terminal α-Helices Interact To Form a Shared Hydrophobic Space

Kaukinen, Pasi; Kumar, Vibhor; Tulimäki, Kirsi; Engelhardt, Peter; Vaheri, Antti; Plyusnin, Alexander

doi:10.1128/jvi.78.24.13669-13677.2004

Cited by 39 publications

(58 citation statements)

References 32 publications

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“…This may reflect significant differences in secondary, tertiary and quaternary structure between these two nucleocapsids. These differences are also highlighted by comparison with the similar-sized nucleocapsid of the hantaviruses, which has been proposed to form trimers as intermediates in RNP assembly (Kaukinen et al, 2004). Our data suggest that the CCHFV N protein may potentially utilize a dimeric intermediate for RNP formation and provides further evidence that bunyaviruses may not utilize a common strategy of RNP formation (Walter & Barr, 2011).…”

Section: Discussionmentioning

confidence: 64%

Expression, purification and crystallization of the Crimean–Congo haemorrhagic fever virus nucleocapsid protein

Carter

Barr

Edwards

2012

Acta Crystallogr F Struct Biol Cryst Commun

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Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Nairovirus genus within the Bunyaviridae family of segmented negative-sense RNA viruses. This paper describes the expression, purification and crystallization of full-length CCHFV nucleocapsid (N) protein and the collection of a 2.1 Å resolution X-ray diffraction data set using synchrotron radiation. Crystals of the CCHFV N protein belonged to space group C2, with unit-cell parameters a = 150.38, b = 72.06, c = 101.23 Å , = 110.70 and two molecules in the asymmetric unit. Circular-dichroism analysis provided insight into the secondary structure, whilst gel-filtration analysis revealed possible oligomeric states of the N protein. Structural determination is ongoing.

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Section: Discussionmentioning

confidence: 64%

Expression, purification and crystallization of the Crimean–Congo haemorrhagic fever virus nucleocapsid protein

Carter

Barr

Edwards

2012

Acta Crystallogr F Struct Biol Cryst Commun

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“…Although the N protein essential for the propagation of the virus adopts a highly conserved structure within a genus, N proteins from different genera differ in their primary sequences and 3D architecture markedly. For example, the Hantaan virus N protein is reported to form trimeric structures using homotypic N-N protein interactions (11)(12)(13). The interaction sites have been mapped principally on the N and C terminals (14).…”

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confidence: 99%

Structure of the Leanyer orthobunyavirus nucleoprotein–RNA complex reveals unique architecture for RNA encapsidation

Niu

Shaw

Wang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Negative-stranded RNA viruses cover their genome with nucleoprotein (N) to protect it from the human innate immune system. Abrogation of the function of N offers a unique opportunity to combat the spread of the viruses. Here, we describe a unique fold of N from Leanyer virus (LEAV, Orthobunyavirus genus, Bunyaviridae family) in complex with single-stranded RNA refined to 2.78 Å resolution as well as a 2.68 Å resolution structure of LEAV N-ssDNA complex. LEAV N is made up of an N-and a C-terminal lobe, with the RNA binding site located at the junction of these lobes. The LEAV N tetramer binds a 44-nucleotide-long single-stranded RNA chain. Hence, oligomerization of N is essential for encapsidation of the entire genome and is accomplished by using extensions at the N and C terminus. Molecular details of the oligomerization of N are illustrated in the structure where a circular ring-like tertiary assembly of a tetramer of LEAV N is observed tethering the RNA in a positively charged cavity running along the inner edge. Hydrogen bonds between N and the C2 hydroxyl group of ribose sugar explain the specificity of LEAV N for RNA over DNA. In addition, base-specific hydrogen bonds suggest that some regions of RNA bind N more tightly than others. Hinge movements around F20 and V125 assist in the reversal of capsidation during transcription and replication of the virus. Electron microscopic images of the ribonucleoprotein complexes of LEAV N reveal a filamentous assembly similar to those found in phleboviruses.crystal structure | nucleoprotein complex with ssRNA | RNP

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“…During infection, the N protein was shown to localize cytoplasmically in the endoplasmic reticulum-Golgi intermediate compartment, presumably as they traffic from the endoplasmic reticulum to the Golgi (22). In addition, immunofluorescence of Cos-7 cells transfected with the N protein alone showed a granular pattern of staining in the perinuclear region (32,34), suggesting colocalization with the Golgi. To test our hypothesis that the conserved surface residues of N 1-74 are important in molecular interaction, we introduced point mutations designed to keep the N 1-74 coiled coil domain intact while altering only specific surface residues and transfected fulllength N protein in mammalian cells to observe the subcellular localization of the N protein.…”

Section: Circular Dichroism Spectroscopy Of N 1-74mentioning

confidence: 96%

mentioning

confidence: 99%

“…The self-association of the N protein into trimers was shown by gradient fractionation and chemical cross-linking (29). Deletion mapping identified that regions at the N and C termini are important in N-N interaction (29 -31), and a model of trimerization was proposed based on the head-to-head and tail-to-tail association of the N-terminal and C-terminal domains, respectively (30,32).The N-terminal region in the Sin Nombre virus N protein (residues 3-73) (33) and the Tula virus (residues 1-77) (34) were predicted to form coiled coil domains. Recently, the structure of the N-terminal coiled coil domain (residues 1-75 and 1-93) of the Sin Nombre virus was determined by crystallography (35).…”

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confidence: 99%

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NMR Structure of the N-terminal Coiled Coil Domain of the Andes Hantavirus Nucleocapsid Protein

Wang

Boudreaux

Estrada

et al. 2008

Journal of Biological Chemistry

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The hantaviruses are emerging infectious viruses that in humans can cause a cardiopulmonary syndrome or a hemorrhagic fever with renal syndrome. The nucleocapsid (N) is the most abundant viral protein, and during viral assembly, the N protein forms trimers and packages the viral RNA genome. Here, we report the NMR structure of the N-terminal domain (residues 1-74, called N 1-74 ) of the Andes hantavirus N protein. N 1-74 forms two long helices (␣ 1 and ␣ 2 ) that intertwine into a coiled coil domain. The conserved hydrophobic residues at the helix ␣ 1 -␣ 2 interface stabilize the coiled coil; however, there are many conserved surface residues whose function is not known. Site-directed mutagenesis, CD spectroscopy, and immunocytochemistry reveal that a point mutation in the conserved basic surface formed by Arg 22 or Lys 26 lead to antibody recognition based on the subcellular localization of the N protein. Thus, Arg 22 and Lys 26 are likely involved in a conformational change or molecular recognition when the N protein is trafficked from the cytoplasm to the Golgi, the site of viral assembly and maturation.Hantaviruses can cause two emerging infectious diseases known as the hantavirus cardiopulmonary syndrome (HCPS) 3 and the hantavirus hemorrhagic fever with renal syndrome (1). Annually, there are over 150,000 cases of hantaviral infections reported world wide (2). Rodents are the primary reservoir of hantaviruses, and humans are normally infected by inhalation of aerosol contaminated with the excreta of infected rodents. The first reported cases of HCPS in North America (3) was caused by a novel hantaviral species (4, 5), the Sin Nombre virus, and had an initial mortality rate of 78%. HCPS has since been reported throughout the United States with a current mortality rate of 35% when correctly diagnosed (6). The major cause of HCPS in South America is the Andes virus, and person-to-person transmission of the Andes virus was reported in Argentina and Chile (7). Hantaviruses are known to invade and replicate primarily in endothelial cells, including the endothelium of vascular tissues lining the heart (8 -10).The genome of hantaviruses consists of three negativestranded RNAs, which encode the nucleocapsid (N) protein, two integral membrane glycoproteins (G1 and G2), and an RNAdependent RNA polymerase (L protein). The N protein is highly immunogenic (11, 12) and elicits a strong immune response, which confers protection in mice (13-15). It is highly conserved and is the most abundant viral protein, and it plays important roles in viral encapsidation, RNA packaging, and host-pathogen interaction (16). The N protein binds to viral proteins (16), host proteins (17-23), and viral RNA (24 -28). The self-association of the N protein into trimers was shown by gradient fractionation and chemical cross-linking (29). Deletion mapping identified that regions at the N and C termini are important in N-N interaction (29 -31), and a model of trimerization was proposed based on the head-to-head and tail-to-tail association of the...

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Oligomerization of Hantavirus N Protein: C-Terminal α-Helices Interact To Form a Shared Hydrophobic Space

Cited by 39 publications

References 32 publications

Expression, purification and crystallization of the Crimean–Congo haemorrhagic fever virus nucleocapsid protein

Expression, purification and crystallization of the Crimean–Congo haemorrhagic fever virus nucleocapsid protein

Structure of the Leanyer orthobunyavirus nucleoprotein–RNA complex reveals unique architecture for RNA encapsidation

NMR Structure of the N-terminal Coiled Coil Domain of the Andes Hantavirus Nucleocapsid Protein

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