Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5-to-3 direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5 cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.

Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis

“…SARS-CoV nsp14 was expressed as a maltose-binding protein (MBP) fusion protein in E. coli and purified by amylose-affinity chromatography, an approach that previously has proven suitable for the expression of several SARS-CoV enzymes (15)(16)(17). A significant portion of the overexpressed MBP-nsp14 fusion protein could be obtained in a soluble form and was purified by using a single-step purification protocol (Fig.…”

Understanding Helicases as a Means of Virus Control

“…To fuel strand separation, SARS-CoV helicase hydrolyzes ATP or any of the eight canonical NTPs [53]. SARS-CoV helicase also has the ability to cleave the terminal phosphate from a triphosphate moiety linked to the 5′ end of a RNA molecule [52,54]. Using this RNA 5′-triphosphatase activity (RTPase) activity, SARS-CoV helicase is able to prepare viral RNA to receive a 5′ cap structure.…”

“…Using this RNA 5′-triphosphatase activity (RTPase) activity, SARS-CoV helicase is able to prepare viral RNA to receive a 5′ cap structure. The fact that ATP is a competitive inhibitor of the RTPase reaction catalyzed by SARS helicase suggests that the RNA is hydrolyzed at the same active site used to fuel helicase movement [54]. A similar RTPase that plays a role in CAP formation was also found in the SF1 helicase encoded by bamboo mosaic virus [55] In addition to the SF1 helicase motifs, SARS-CoV nsp13 protein, and its relatives from human coronavirus 229E [56] and equine arteritis virus [57], all contain a cysteine-rich zinc binding domain located at their N-termini.…”

Crystal structure and mechanistic determinants of SARS coronavirus nonstructural protein 15 define an endoribonuclease family

“…Furthermore, crystal structures of five nsps (or subdomains thereof) have been reported: the ADP-ribose-1Љ-phosphatase macro domain of nsp3, the main protease nsp5, the RNA-binding protein nsp9, and the complex of nsp7-nsp8, a proposed processivity factor for the RNA-dependent RNA polymerase nsp12 (8)(9)(10)(11)(12). nsp13 is a superfamily 1 helicase (7, 13), whereas nsp14, nsp15, and nsp16 have been predicted or confirmed to harbor 3Ј-5Ј exonuclease, endonuclease, and 2Ј-O-ribose methyltransferase activities, respectively (7,(13)(14)(15). Among these, the nidovirus uridylate-specific endoribonuclease (NendoU) residing in nsp15 is considered a major genetic marker of nidoviruses because of its nidovirus-wide conservation but complete absence in other RNA viruses (16).…”