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The crystal structure of the Hazara virus nucleocapsid protein.

Surtees R, Ariza A, Punch EK, Trinh CH, Dowall SD, Hewson R, Hiscox JA, Barr JN, Edwards TA - BMC Struct. Biol. (2015)

Bottom Line: To characterise further similarities between HAZV and CCHFV, and support the use of HAZV as a model for CCHFV infection, we investigated the structure of the HAZV nucleocapsid protein (N) and compared it to CCHFV N.The crystal structure of HAZV N reveals a close similarity to CCHFV N, supporting the use of HAZV as a model for CCHFV.Structural similarity between the N proteins should facilitate study of the CCHFV and HAZV replication cycles without the necessity of working under containment level 4 (CL-4) conditions.

View Article: PubMed Central - PubMed

Affiliation: Public Health England, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK. bs06ras@leeds.ac.uk.

ABSTRACT

Background: Hazara virus (HAZV) is a member of the Bunyaviridae family of segmented negative stranded RNA viruses, and shares the same serogroup as Crimean-Congo haemorrhagic fever virus (CCHFV). CCHFV is responsible for fatal human disease with a mortality rate approaching 30 %, which has an increased recent incidence within southern Europe. There are no preventative or therapeutic treatments for CCHFV-mediated disease, and thus CCHFV is classified as a hazard group 4 pathogen. In contrast HAZV is not associated with serious human disease, although infection of interferon receptor knockout mice with either CCHFV or HAZV results in similar disease progression. To characterise further similarities between HAZV and CCHFV, and support the use of HAZV as a model for CCHFV infection, we investigated the structure of the HAZV nucleocapsid protein (N) and compared it to CCHFV N. N performs an essential role in the viral life cycle by encapsidating the viral RNA genome, and thus, N represents a potential therapeutic target.

Results: We present the purification, crystallisation and crystal structure of HAZV N at 2.7 Å resolution. HAZV N was expressed as an N-terminal glutathione S-transferase (GST) fusion protein then purified using glutathione affinity chromatography followed by ion-exchange chromatography. HAZV N crystallised in the P212121 space group with unit cell parameters a = 64.99, b = 76.10, and c = 449.28 Å. HAZV N consists of a globular domain formed mostly of alpha helices derived from both the N- and C-termini, and an arm domain comprising two long alpha helices. HAZV N has a similar overall structure to CCHFV N, with their globular domains superposing with an RMSD = 0.70 Å, over 368 alpha carbons that share 59 % sequence identity. Four HAZV N monomers crystallised in the asymmetric unit, and their head-to-tail assembly reveals a potential interaction site between monomers.

Conclusions: The crystal structure of HAZV N reveals a close similarity to CCHFV N, supporting the use of HAZV as a model for CCHFV. Structural similarity between the N proteins should facilitate study of the CCHFV and HAZV replication cycles without the necessity of working under containment level 4 (CL-4) conditions.

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Surface representation of Apo-HAZV N electrostatic potential. The electrostatic surface potential of HAZV N is shown colour coded from red (negative) to blue (positive) in dimensionless units of kb.T/ec, where kb is Boltzmann’s constant, T is the temperature, and ec is the charge of an electron (generated using the APBS Pymol plug-in [39]). Potential RNA binding sites, the Platform and the Crevice are labeled
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Fig6: Surface representation of Apo-HAZV N electrostatic potential. The electrostatic surface potential of HAZV N is shown colour coded from red (negative) to blue (positive) in dimensionless units of kb.T/ec, where kb is Boltzmann’s constant, T is the temperature, and ec is the charge of an electron (generated using the APBS Pymol plug-in [39]). Potential RNA binding sites, the Platform and the Crevice are labeled

Mentions: As HAZV N is a proposed RNA binding protein, the electrostatic surface potential of HAZV N was analysed to identify potential RNA binding sites. Surface representations of the HAZV N protein colour-coded red to blue according to charge (negative to positive, respectively) are presented in Fig. 6. The surface representation of HAZV N reveals several areas of positive charge (blue), which are often associated with an increase in RNA binding potential. HAZV N has a positively charged ‘platform’ adjacent to the arm domain (Fig. 6, left) in a similar position to a positively charged platform on the surface of the CCHFV N protein. This platform is one of the largest areas of continuous positive charge on the surface of both HAZV N and CCHFV N, and was therefore suggested to be a possible RNA binding site. Another possible RNA binding site is a crevice on the opposite face of the HAZV N monomer to the positively charged platform (rotate HAZV N monomer 180°, Fig. 6, right). This crevice contains positively charged residues and extends across the length of the globular domain from helix α13 at the beginning of the arm domain, to the base of the globular domain. This crevice is also present in the CCHFV N model, and RNA sequestered here could be protected from degradation.Fig. 6


The crystal structure of the Hazara virus nucleocapsid protein.

Surtees R, Ariza A, Punch EK, Trinh CH, Dowall SD, Hewson R, Hiscox JA, Barr JN, Edwards TA - BMC Struct. Biol. (2015)

Surface representation of Apo-HAZV N electrostatic potential. The electrostatic surface potential of HAZV N is shown colour coded from red (negative) to blue (positive) in dimensionless units of kb.T/ec, where kb is Boltzmann’s constant, T is the temperature, and ec is the charge of an electron (generated using the APBS Pymol plug-in [39]). Potential RNA binding sites, the Platform and the Crevice are labeled
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696240&req=5

Fig6: Surface representation of Apo-HAZV N electrostatic potential. The electrostatic surface potential of HAZV N is shown colour coded from red (negative) to blue (positive) in dimensionless units of kb.T/ec, where kb is Boltzmann’s constant, T is the temperature, and ec is the charge of an electron (generated using the APBS Pymol plug-in [39]). Potential RNA binding sites, the Platform and the Crevice are labeled
Mentions: As HAZV N is a proposed RNA binding protein, the electrostatic surface potential of HAZV N was analysed to identify potential RNA binding sites. Surface representations of the HAZV N protein colour-coded red to blue according to charge (negative to positive, respectively) are presented in Fig. 6. The surface representation of HAZV N reveals several areas of positive charge (blue), which are often associated with an increase in RNA binding potential. HAZV N has a positively charged ‘platform’ adjacent to the arm domain (Fig. 6, left) in a similar position to a positively charged platform on the surface of the CCHFV N protein. This platform is one of the largest areas of continuous positive charge on the surface of both HAZV N and CCHFV N, and was therefore suggested to be a possible RNA binding site. Another possible RNA binding site is a crevice on the opposite face of the HAZV N monomer to the positively charged platform (rotate HAZV N monomer 180°, Fig. 6, right). This crevice contains positively charged residues and extends across the length of the globular domain from helix α13 at the beginning of the arm domain, to the base of the globular domain. This crevice is also present in the CCHFV N model, and RNA sequestered here could be protected from degradation.Fig. 6

Bottom Line: To characterise further similarities between HAZV and CCHFV, and support the use of HAZV as a model for CCHFV infection, we investigated the structure of the HAZV nucleocapsid protein (N) and compared it to CCHFV N.The crystal structure of HAZV N reveals a close similarity to CCHFV N, supporting the use of HAZV as a model for CCHFV.Structural similarity between the N proteins should facilitate study of the CCHFV and HAZV replication cycles without the necessity of working under containment level 4 (CL-4) conditions.

View Article: PubMed Central - PubMed

Affiliation: Public Health England, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK. bs06ras@leeds.ac.uk.

ABSTRACT

Background: Hazara virus (HAZV) is a member of the Bunyaviridae family of segmented negative stranded RNA viruses, and shares the same serogroup as Crimean-Congo haemorrhagic fever virus (CCHFV). CCHFV is responsible for fatal human disease with a mortality rate approaching 30 %, which has an increased recent incidence within southern Europe. There are no preventative or therapeutic treatments for CCHFV-mediated disease, and thus CCHFV is classified as a hazard group 4 pathogen. In contrast HAZV is not associated with serious human disease, although infection of interferon receptor knockout mice with either CCHFV or HAZV results in similar disease progression. To characterise further similarities between HAZV and CCHFV, and support the use of HAZV as a model for CCHFV infection, we investigated the structure of the HAZV nucleocapsid protein (N) and compared it to CCHFV N. N performs an essential role in the viral life cycle by encapsidating the viral RNA genome, and thus, N represents a potential therapeutic target.

Results: We present the purification, crystallisation and crystal structure of HAZV N at 2.7 Å resolution. HAZV N was expressed as an N-terminal glutathione S-transferase (GST) fusion protein then purified using glutathione affinity chromatography followed by ion-exchange chromatography. HAZV N crystallised in the P212121 space group with unit cell parameters a = 64.99, b = 76.10, and c = 449.28 Å. HAZV N consists of a globular domain formed mostly of alpha helices derived from both the N- and C-termini, and an arm domain comprising two long alpha helices. HAZV N has a similar overall structure to CCHFV N, with their globular domains superposing with an RMSD = 0.70 Å, over 368 alpha carbons that share 59 % sequence identity. Four HAZV N monomers crystallised in the asymmetric unit, and their head-to-tail assembly reveals a potential interaction site between monomers.

Conclusions: The crystal structure of HAZV N reveals a close similarity to CCHFV N, supporting the use of HAZV as a model for CCHFV. Structural similarity between the N proteins should facilitate study of the CCHFV and HAZV replication cycles without the necessity of working under containment level 4 (CL-4) conditions.

Show MeSH
Related in: MedlinePlus