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. 2012 Mar 27;109(13):5046-51.
doi: 10.1073/pnas.1200808109. Epub 2012 Mar 14.

Crimean-Congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyaviruses

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Crimean-Congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyaviruses

Yu Guo et al. Proc Natl Acad Sci U S A. .

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV), a virus with high mortality in humans, is a member of the genus Nairovirus in the family Bunyaviridae, and is a causative agent of severe hemorrhagic fever (HF). It is classified as a biosafety level 4 pathogen and a potential bioterrorism agent due to its aerosol infectivity and its ability to cause HF outbreaks with high case fatality (∼30%). However, little is known about the structural features and function of nucleoproteins (NPs) in the Bunyaviridae, especially in CCHFV. Here we report a 2.3-Å resolution crystal structure of the CCHFV nucleoprotein. The protein has a racket-shaped overall structure with distinct "head" and "stalk" domains and differs significantly with NPs reported so far from other negative-sense single-stranded RNA viruses. Furthermore, CCHFV NP shows a distinct metal-dependent DNA-specific endonuclease activity. Single residue mutations in the predicted active site resulted in a significant reduction in the observed endonuclease activity. Our results present a new folding mechanism and function for a negative-strand RNA virus nucleoprotein, extend our structural insight into bunyavirus NPs, and provide a potential target for antiviral drug development to treat CCHFV infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Size exclusion chromatography (SEC) of CCHFV NP. CCHFV NP samples with or without DNase/RNase treatment (5 mg/mL) were injected, respectively, onto a Superdex 200 10/300 GL column. Retention volume was about 15 mL for the monomeric form of CCHFV NP. (Upper) Retention volumes are shown for the molecular weight standards and SDS/PAGE analysis of the SEC elution fractions corresponding to the two peaks.
Fig. 2.
Fig. 2.
Structure of CCHFV NP. (A) Schematic diagram of the domain organization in the primary sequence of CCHFV NP. The stalk and head domains are colored as light blue and green, respectively. (B) Overall structure in cartoon representation. Missing residues are linked by dotted lines. (C) Topology diagram. Head and stalk domains are colored in green and light blue, respectively.
Fig. 3.
Fig. 3.
Potential RNA-binding region of CCHFV. The electrostatic surface potential of CCHFV NP was calculated using adaptive Poisson-Boltzmann solver (APBS) tools. The positive surface is colored blue, the negative surface, red, with limits ±10 kbT/ec. Positive residues are labeled on CCHFV NP, suggesting the presence of several positively charged grooves that may be involved in RNA binding.
Fig. 4.
Fig. 4.
DNA-specific endonuclease activity of CCHFV NP. (A) Time series of in vitro dsDNA (Upper) and ssDNA (Lower) and (B) dsRNA (Upper) and ssRNA (Lower) degradation assay products. Reaction products of 0.3 μM purified CCHFV NP with 100 ng/μL substrates at 37 °C in a final volume of 10 μL are shown after 5, 10, 20, 40, and 60 min. Reaction products were loaded onto a 20% (wt/vol) polyacrylamide gel and stained with ethidium bromide. (C) Effect of divalent cations on CCHFV NP nuclease activity. Purified CCHFV NP (0.3 μM) and dsDNA substrate were incubated with CoCl2, NiCl2, MgCl2, FeSO4, CaCl2, CuCl2, MnCl2, and ZnCl2 at a concentration of 1 mM. CCHFV NP alone, CCHFV NP and EDTA, or CCHFV NP and EDTA with Mn2+ were used as controls. All reactions were stopped by the addition of 10 mM EDTA. (D) Effect of CCHFV NP endonuclease activity on circular double-stranded plasmids. CCHFV NP was incubated with circular double-stranded plasmids for different lengths of time. All reactions were stopped by the addition of 10 mM EDTA.
Fig. 5.
Fig. 5.
CCHFV NP head domain has endonuclease activity. (A) Structure comparison between the CCHFV NP head domain (from residue K59) and the N-terminal domain of LASV NP. Proteins are shown as cartoon representations in different colors. (B) Potential DNase active site of CCHFV NP head domain. Residues whose hydrophilic side chain is near the potential site for DNA binding are shown as colored sticks. (C) Nuclease activity of relevant mutants at the potential active site of CCHFV NP.

References

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