The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

Conservation of substrate specificities among coronavirus main proteases.

The key enzyme in coronavirus replicase polyprotein processing is the coronavirus main protease, 3CL(pro). The substrate specificities of five coronavirus main proteases, including the prototypic enzymes from the coronavirus groups I, II and III, were characterized. Recombinant main proteases of human coronavirus (HCoV), transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus, avian infectious bronchitis virus and mouse hepatitis virus (MHV) were tested in peptide-based trans-cleavage assays. The determination of relative rate constants for a set of corresponding HCoV, TGEV and MHV 3CL(pro) cleavage sites revealed a conserved ranking of these sites. Furthermore, a synthetic peptide representing the N-terminal HCoV 3CL(pro) cleavage site was shown to be effectively hydrolysed by noncognate main proteases. The data show that the differential cleavage kinetics of sites within pp1a/pp1ab are a conserved feature of coronavirus main proteases and lead us to predict similar processing kinetics for the replicase polyproteins of all coronaviruses.

Biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases.

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.

Mutational analysis of the active centre of coronavirus 3C-like proteases.

Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined. Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues. Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.

Major genetic marker of nidoviruses encodes a replicative endoribonuclease

Coronaviruses are important pathogens that cause acute respiratory diseases in humans. Replication of the 30-kb positive-strand RNA genome of coronaviruses and discontinuous synthesis of an extensive set of subgenome-length RNAs (transcription) are mediated by the replicase-transcriptase, a barely characterized protein complex that comprises several cellular proteins and up to 16 viral subunits. The coronavirus replicase-transcriptase was recently predicted to contain RNA-processing enzymes that are extremely rare or absent in other RNA viruses. Here, we established and characterized the activity of one of these enzymes, replicative nidoviral uridylate-specific endoribonuclease (NendoU). It is considered a major genetic marker that discriminates nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) from all other RNA virus families. Bacterially expressed forms of NendoU of severe acute respiratory syndrome coronavirus and human coronavirus 229E were revealed to cleave single-stranded and double-stranded RNA in a Mn2+-dependent manner. Single-stranded RNA was cleaved less specifically and effectively, suggesting that double-stranded RNA is the biologically relevant NendoU substrate. Double-stranded RNA substrates were cleaved upstream and downstream of uridylates at GUU or GU sequences to produce molecules with 2'-3' cyclic phosphate ends. 2'-O-ribose-methylated RNA substrates proved to be resistant to cleavage by NendoU, indicating a functional link with the 2'-O-ribose methyltransferase located adjacent to NendoU in the coronavirus replicative polyprotein. A mutagenesis study verified potential active-site residues and allowed us to inactivate NendoU in the full-length human coronavirus 229E clone. Substitution of D6408 by Ala was shown to abolish viral RNA synthesis, demonstrating that NendoU has critical functions in viral replication and transcription.

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.