Crystal structure of Middle East respiratory syndrome coronavirus helicase

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) remains a threat to public health worldwide; however, effective vaccine or drug against CoVs remains unavailable. CoV helicase is one of the three evolutionary most conserved proteins in nidoviruses, thus making it an important target for drug development. We report here the first structure of full-length coronavirus helicase, MERS-CoV nsp13. MERS-CoV helicase has multiple domains, including an N-terminal Cys/His rich domain (CH) with three zinc atoms, a beta-barrel domain and a C-terminal SF1 helicase core with two RecA-like subdomains. Our structural analyses show that while the domain organization of nsp13 is conserved throughout nidoviruses, the individual domains of nsp13 are closely related to the equivalent eukaryotic domains of Upf1 helicases. The most distinctive feature differentiating CoV helicases from eukaryotic Upf1 helicases is the interaction between CH domain and helicase core.

In vitro methods for testing antiviral drugs

“…Helicases from several (+)RNA viruses have been characterized, including NS3 helicases from flaviviruses such as dengue virus, HCV, West Nile virus, yellow fever virus, and Japanese encephalitis virus (Cao et al, 2016;Gu and Rice, 2016;Jain et al, 2016;Lin et al, 2017;Nedjadi et al, 2015;Wu et al, 2005). SARS and MERS coronaviruses encode nsp13 with helicase activity (Adedeji and Lazarus, 2016;Hao et al, 2017;Seybert et al, 2000). In Semliki Forest virus, a representative member of the togavirus family, helicase activity is encoded by the N-terminal domain of nsP2 protein.…”

Deducing the Crystal Structure of MERS-CoV Helicase

“…An RNAdependent RNA polymerase nsp12 and a helicase nsp13 are the central components of RTC [8,9]. However, while high-resolution structures of most CoV encoded proteins had been determined soon after SARS-CoV outbreak, the first CoV nsp13 structure, MERS-CoV nsp13, was only solved recently [10]. Nsp13 belongs to helicase superfamily 1 and shares conserved features with the eukaryotic Upf1 helicase [11,12].…”

“…Nsp13 belongs to helicase superfamily 1 and shares conserved features with the eukaryotic Upf1 helicase [11,12]. Nsp13 is a multi-domain protein comprising of an N-terminal Cys/His rich domain (CH domain) and a C-terminal SF1 helicase core [10]. Nsp13 exhibits multiple enzymatic activities, including hydrolysis of NTPs and dNTPs, unwinding of DNA and RNA duplexes with 5 0 -3 0 directionality and the RNA 5 0 -triphosphatase activity [13,14].…”

“…Crystallization of MERS-CoV nsp13 was achieved by adding a synthetic single-stranded 15 poly dT DNA with 5 0 -triphosphate (ppp-15 T) to the protein, which restrains the intrinsic flexibility of nsp13. Benefiting from the presence of an N-terminal zinc-binding domain with three zinc atoms, multi-wavelength anomalous diffraction (MAD) data at the zinc absorption edge was collected, which allowed the determination of the crystal structure of MERS-CoV nsp13 [10].…”

“…3. To remove nucleic acids bound to nsp13, we used the lysis buffer containing high concentrate salt; this is a key step and improves the crystallization of nsp13 [10]. In practice, when sonicated in the buffer containing high concentrate salt, we found that the SUMO-tagged recombinant proteins lead the supernatant of the high-5 cell lysate to be turbid, which finally blocks the affinity columns.…”