Biochemical Characterization of the Equine Arteritis Virus Helicase Suggests a Close Functional Relationship between Arterivirus and Coronavirus Helicases

Abstract

The arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a Zn finger structure linked to a superfamily 1 (SF1) helicase domain. A recombinant form of nsp10, MBP-nsp10, was produced in Escherichia coli as a fusion protein with the maltose-binding protein. The protein was partially purified by affinity chromatography and shown to have ATPase activity that was strongly stimulated by poly(dT), poly(U), and poly(dA) but not by poly(G). The protein also had both RNA and DNA duplex-unwinding activities that required the presence of 5 single-stranded regions on the partial-duplex substrates, indicating a 5-to-3 polarity in the unwinding reaction. Results of this study suggest a close functional relationship between the arterivirus nsp10 and the coronavirus helicase, for which NTPase and duplex-unwinding activities were recently demonstrated. In a number of biochemical properties, both arterivirus and coronavirus SF1 helicases differ significantly from the previously characterized RNA virus SF1 and SF2 enzymes. Thus, the combined data strongly support the idea that nidovirus helicases may represent a separate group of RNA virus-encoded helicases with distinct properties.Equine arteritis virus (EAV) is the prototype of the Arteriviridae, a family of positive-stranded, enveloped RNA viruses which also includes Lactate dehydrogenase-elevating virus, Porcine reproductive and respiratory syndrome virus, and Simian haemorrhagic fever virus (for a review, see 47). A common ancestry of the Arteriviridae and Coronaviridae seems probable (6), and, consequently, the two families have been united in the order Nidovirales (3). The phylogenetic relationship between arteri-and coronaviruses is most evident from the organization and expression of their replicase genes. Thus, for example, both arteri-and coronaviruses (i) encode a very similar array of functional domains in their replicase genes, (ii) use ribosomal frameshifting to express key replicative functions, (iii) control the activity of the individual subunits of the viral replication and transcription machinery by extensive proteolytic processing of large protein precursors, and (iv) use a discontinuous transcription mechanism to produce a nested set of subgenomic (sg) mRNAs for structural gene expression (3,8).The EAV replicase gene comprises the 5Ј-terminal threefourths of the 12.7-kb genome and is composed of two open reading frames (ORFs), ORF1a and ORF1b (6). The upstream ORF1a encodes the ORF1a protein (187 kDa), and ORF1a and ORF1b together encode the ORF1ab protein (345 kDa). Expression of the ORF1b-encoded part of the ORFlab protein involves a ribosomal frameshift in the ORF1a-1b overlap region during translation of the genomic RNA (6). The primary translation products, which are also called replicase polyproteins, are extensively processed by three virus-encoded proteinases to produce 12 mature proteins (nonstructural protein 1 [nsp1] to nsp12), as well as multiple processing intermediates (for a recent review, s...

Understanding Helicases as a Means of Virus Control

“…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. Zinc binding domains have been found linked to other helicases and they are frequently found in proteins that interact intimately with nucleic acids.…”

The in Vitro RNA Synthesizing Activity of the Isolated Arterivirus Replication/Transcription Complex Is Dependent on a Host Factor

“…Analysis of the distribution of replicase subunits between the cytoplasmic S10 fraction and the RTC-containing P10 fraction revealed that proteins previously implicated in viral RNA synthesis (nsp9-RdRp and nsp10-helicase) (28,29) and DMV formation (nsp2 and nsp3) (30) exclusively cosedimented with the active RTC. In contrast, the main protease nsp4 was found predominantly in the cytosolic S10 fraction and not cosedimenting with RTC activity, suggesting that its presence is not (or, rather, no longer) required for RTC activity.…”

“…Most of these replicase subunits appear to become associated with intracellular membranes in the perinuclear region of the infected cell (11)(12)(13), where they are thought to assemble into RTCs. The ORF1b-encoded subunits contain the core enzymatic activities that are involved in viral RNA synthesis, like the RNA-dependent RNA polymerase (RdRp) and RNA helicase (28,29), whereas ORF1a encodes, in addition to the three protease domains, several putative transmembrane subunits. The latter appear to play a more "structural" role by inducing DMV formation (30) and presumably anchoring the RTC to intracellular membranes (11).…”

The Autocatalytic Release of a Putative RNA Virus Transcription Factor from Its Polyprotein Precursor Involves Two Paralogous Papain-like Proteases That Cleave the Same Peptide Bond

“…The ORF1b-encoded polyprotein, which includes the putative RdRp activity and a recently established RNA helicase activity that is associated with a unique zinc finger structure (9,10), is only produced if a ribosomal frameshift from ORF1a into ORF1b takes place during translation (11). This translational strategy is expected to yield two extremely large polyproteins, pp1a and pp1ab, of about 450 and 750 kDa, respectively.…”