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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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HAZV N purification by ion exchange chromatography. HAZV N was separated from GST-HAZV N and GST using ion exchange chromatography. a Protein elution was monitered with an A280 absorbance trace; protein that flowed through the column without binding is represented by Peak 1, whereas protein that bound to the column and was then eluted by an increasing NaCl gradient (dashed grey line) forms Peak 2. b SDS PAGE analysis of Peak 1 and Peak 2. Peak 2 contains primarily HAZV N, whereas Peak 1 contains a mixture of proteins that flowed through the column without binding
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Fig2: HAZV N purification by ion exchange chromatography. HAZV N was separated from GST-HAZV N and GST using ion exchange chromatography. a Protein elution was monitered with an A280 absorbance trace; protein that flowed through the column without binding is represented by Peak 1, whereas protein that bound to the column and was then eluted by an increasing NaCl gradient (dashed grey line) forms Peak 2. b SDS PAGE analysis of Peak 1 and Peak 2. Peak 2 contains primarily HAZV N, whereas Peak 1 contains a mixture of proteins that flowed through the column without binding

Mentions: HAZV N was therefore purified from uncleaved GST-HAZV N and GST by cation exchange chromatography using a Resource S column (Fig. 2). HAZV N has an isoelectric point of 8.73, therefore ion exchange was performed with all buffers equilibrated to pH 7, where HAZV N should be positively charged and bind to the negatively charged Resource S column, and all other proteins should pass through the column without binding (Fig. 2, peak 1). HAZV N was eluted from the column (Fig. 2, peak 2) as the concentration of NaCl was increased from 50 mM to 1 M over 25 column volumes. Cation exchange chromatography removed the majority of protein contaminants leaving only HAZV N, then circular dichroism was used to analyse the thermal stabilities of both HAZV N, and CCHFV N proteins (Fig. 3) as previously described [20]. When monitoring the ellipticity at 222 nm the melting temperature (Tm) of HAZV N was found to be 42.2 °C and of CCHFV N is 46.3 °C. This indicates both proteins are correctly folded and potentially share a similar secondary structure.Fig. 2


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)

HAZV N purification by ion exchange chromatography. HAZV N was separated from GST-HAZV N and GST using ion exchange chromatography. a Protein elution was monitered with an A280 absorbance trace; protein that flowed through the column without binding is represented by Peak 1, whereas protein that bound to the column and was then eluted by an increasing NaCl gradient (dashed grey line) forms Peak 2. b SDS PAGE analysis of Peak 1 and Peak 2. Peak 2 contains primarily HAZV N, whereas Peak 1 contains a mixture of proteins that flowed through the column without binding
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: HAZV N purification by ion exchange chromatography. HAZV N was separated from GST-HAZV N and GST using ion exchange chromatography. a Protein elution was monitered with an A280 absorbance trace; protein that flowed through the column without binding is represented by Peak 1, whereas protein that bound to the column and was then eluted by an increasing NaCl gradient (dashed grey line) forms Peak 2. b SDS PAGE analysis of Peak 1 and Peak 2. Peak 2 contains primarily HAZV N, whereas Peak 1 contains a mixture of proteins that flowed through the column without binding
Mentions: HAZV N was therefore purified from uncleaved GST-HAZV N and GST by cation exchange chromatography using a Resource S column (Fig. 2). HAZV N has an isoelectric point of 8.73, therefore ion exchange was performed with all buffers equilibrated to pH 7, where HAZV N should be positively charged and bind to the negatively charged Resource S column, and all other proteins should pass through the column without binding (Fig. 2, peak 1). HAZV N was eluted from the column (Fig. 2, peak 2) as the concentration of NaCl was increased from 50 mM to 1 M over 25 column volumes. Cation exchange chromatography removed the majority of protein contaminants leaving only HAZV N, then circular dichroism was used to analyse the thermal stabilities of both HAZV N, and CCHFV N proteins (Fig. 3) as previously described [20]. When monitoring the ellipticity at 222 nm the melting temperature (Tm) of HAZV N was found to be 42.2 °C and of CCHFV N is 46.3 °C. This indicates both proteins are correctly folded and potentially share a similar secondary structure.Fig. 2

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