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1 Institute of Virology and Immunology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany
2 Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
3 Institute for Medical Virology, Johann Wolfgang Goethe University, Frankfurt (Main), Germany
Correspondence
John Ziebuhr
j.ziebuhr{at}mail.uni-wuerzburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Present address: Research Department, Cantonal Hospital, St Gallen, Switzerland. 
Published ahead of print on 19 June 2003 as DOI 10.1099/vir.0.19424-0.
The nucleotide sequence of SARS-CoV, isolate Frankfurt 1, has been deposited in GenBank, accession no. AY291315.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In the course of a few months in 2003, an epidemic emerged that has spread from its likely origin in Guangdong Province, China, to 32 countries. By 11 June 2003 more than 8400 cases and 789 deaths had been recorded by the World Health Organization. The rapid transmission by aerosols (and probably also the faecal–oral route) and the high mortality rate make SARS a global threat for which no efficacious therapy is available. There is now clear evidence that SARS is caused by a previously unknown coronavirus, provisionally termed SARS coronavirus (SARS-CoV) (Peiris et al., 2003b; Drosten et al., 2003; Ksiazek et al., 2003; Fouchier et al., 2003). Genome sequences of SARS-CoV isolates obtained from a number of index patients have been published recently and provide important information on the organization, phylogeny and variability of the 29·7 kb positive-strand RNA genome of SARS-CoV (Rota et al., 2003; Marra et al., 2003; Ruan et al., 2003). By analogy with other coronaviruses (Lai & Holmes, 2001; Siddell, 1995; Gorbalenya, 2001), SARS-CoV gene expression is expected to involve complex transcriptional, translational and post-translational regulatory mechanisms, whose molecular details remain to be determined. SARS-CoV genome expression starts with the translation of two large replicative polyproteins, pp1a (486 kDa) and pp1ab (790 kDa), which are encoded by the viral replicase gene (21 221 nt) that comprises ORFs 1a and 1b (Fig. 1). Expression of the ORF1b-encoded region of pp1ab is predicted to involve ribosomal frameshifting into the -1 frame just upstream of the ORF1a translation termination codon (Brierley et al., 1989). The pp1a and pp1ab polyproteins are processed by viral proteinases to yield the functional components of the membrane-bound replicase complex (Ziebuhr et al., 2000). In contrast to most other coronaviruses, which use three proteinase activities for replicase polyprotein processing (Ziebuhr et al., 2000; Gorbalenya, 2001), SARS-CoV is predicted to encode only two proteinases (Rota et al., 2003; Snijder et al., 2003). The replicase complex mediates both genome replication and transcription of a ‘nested’ set of subgenomic mRNAs. These mRNAs encode the structural proteins, S, E, M and N, and a set of accessory proteins whose number and sequence vary among different coronavirus species (Siddell, 1995). The extraordinary size of the coronavirus replicase (poly)proteins, their generally large phylogenetic distance from those of other RNA viruses, and the presence of several predicted RNA processing activities which are not found in other positive-strand RNA viruses (Gorbalenya, 2001; Snijder et al., 2003), indicate that coronavirus replicases are of an unparalleled complexity. The underlying biological mechanisms and functional constraints that determine the evolution and conservation of these unique activities remain to be elucidated.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) (isolate Frankfurt 1, fifth passage in cell culture) at an m.o.i. of 0·01. Two days after infection, intracellular poly(A)-containing RNA was prepared as described by Thiel et al. (2001). RNA isolated from respiratory tract and stool specimens was prepared using the QIAamp and QIAamp stool kit (Qiagen), respectively, according to the manufacturer's instructions.
Sequencing of the SARS-CoV (Frankfurt 1) RNA genome.
To determine the SARS-CoV genomic sequence, a set of overlapping RT-PCR products with an average size of 2 kb encompassing the entire genome was generated as described by Thiel et al. (1997). To generate RT-PCR products containing the exact 3'-terminal sequence of SARS-CoV genomic RNA, reverse transcription was primed using oligonucleotide OLV1/57 (5'-GCCGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCTTTTTTTTTTTTTTTTTT-3') and PCR was done using oligonucleotides PCR-L (5'-23GGAAAAGCCAACCAACCTCGATCTC47-3') and OLV1/58 (5'-ACGTTCTAGAGCCCAGCCGGCGCCAGCGAGGAGGCT-3'). To generate RT-PCR products containing the exact 5'-terminal sequence the FirstChoice RLM-RACE Kit (Ambion) was used according to the manufacturer's instructions with the following modifications. A synthetic RNA corresponding to human coronavirus 229E (HCoV-229E) nt 1–600 was used as RNA adapter and reverse transcription was primed using oligonucleotide S65 (5'-439CTTTTTCCAGCTCTACTAGACCAC416-3'). Outer PCR was done using oligonucleotides 240up (5'-CCTTACTCGAGGTTCCGTCTCGTG-3') and S132 (5'-341ACGTCTCTAACCTGAAGGACAGGC318-3'); inner PCR was done using oligonucleotides Oli5 (5'-GCGAGGCCGCTAGCAATGG-3') and S133 (5'-235CTAGGTATGCTGATGATCGACTGC212-3'). All RT-PCR products served as template for sequencing analysis using a total number of 149 sequencing primers and the BigDye Terminator v3.1 Cycle Sequencing Kit. Sequencing products were detected using an ABI PRISM 3100 Genetic Analyser (Applied Biosystems) and computer-assisted analysis of sequencing data was facilitated by the Lasergene bio-computing software (DNASTAR).
Analysis of SARS-CoV mRNAs.
Poly(A)-containing RNA from SARS-CoV- and HCoV-infected cells was separated on a 2·2 M formaldehyde/1 % agarose gel, blotted on nylon membrane and hybridized with 32P-multiprime-labelled DNA probes corresponding to the 3'-terminal 794 nt of the SARS-CoV genome and HCoV nt 26297–27273, respectively. RNAs were analysed by autoradiography. To determine the leader-to-body fusion sites of SARS-CoV subgenomic mRNAs, reverse transcription of poly(A)-containing RNA from SARS-CoV-infected cells was primed using oligonucleotides RT-S (5'-22247ATAGGCTGCAGCTGACGTGCCCCA22224-3'), RT-3 (5'-25805GTTTTGGTGTTGAAATGCCGTCACC25781-3'), RT-E (5'-26304TTAACACGCGAGTAGACGTAAACCG26280-3'), RT-M (5'-26964ATCAGTGCCTACACGCTGCGACGC26941-3'), RT 6-8 (5'-28043ACACCTAGCTATAAGCGCACCACC28020-3') and RT-N (5'-28798TGTCTAGCAGCAATAGCGCGAGGGC28774-3'). PCR amplification was done using the SARS-CoV leader-specific oligonucleotide PCR-L in combination with body-specific oligonucleotides PCR-S (5'-22108ACTACATCTATAGGTTGATAGCCCT22084-3'), PCR-3 (5'-25734TAGTCATAGTTATGTGTGTGCCAGC25710-3'), PCR-E (5'-26243AGTACGCACACAATCGAAGCGCAG26220-3'), PCR-M (5'-26781CACAATTGTCCCCCGGAGAGGCAC26758-3'), PCR-6-8 (5'-27937CCTAGAGCACAAAGCCAAGCAGTGC27913-3') and PCR-N (5'-28608AGGAAGTTGTAGCACGGTGGCAGC28585-3'). Sequence analysis of PCR products was done using primers SEQ-S (5'-21738AAGGTATGACAGGGTTGCCAAACG21715-3'), SEQ-3 (5'-25515AAATTGCAAATGAACTGGAAGCCC25492-3'), SEQ-E (5'-26243AGTACGCACACAATCGAAGCGCAG26220-3'), SEQ-M (5'-26640AATCGCAATCCCGCCAGTCACCC26618-3'), SEQ-6 (5'-27244AGGTTCTTCATCATCTAACTCCGA27221-3'), SEQ-7 (5'-27439AGTGCAAATTTATTGTCAGCAAGA27416-3'), SEQ-8 (5'-27937CCTAGAGCACAAAGCCAAGCAGTGC27913-3') and SEQ-N (5'-28398TCGGGTAGCTCTTCGGTAGTAGCC28375-3').
In vitro transcription and translation.
In vitro transcription reactions were done using the RiboMAX Large Scale RNA Production System–T7 (Promega) and m7G(5')ppp(5')G cap structure analogue as described by Thiel et al. (2001). In vitro translation reactions were done in rabbit reticulocyte lysate (Promega) using in vitro-transcribed RNA (Ziebuhr & Siddell, 1999). Alternatively, DNA templates containing a T7 promoter were transcribed and translated using the TNT T7-Coupled Reticulocyte Lysate System (Promega). To analyse the SARS-CoV frameshifting element, a DNA fragment corresponding to SARS-CoV nt 12955–13961 was amplified by RT-PCR using oligonucleotides S25 (5'-17511CAATTTCAGCAGGACAACGGCGAC17488-3'; RT reaction), JZ464 (5'-AATAATACGACTCACTATAGGGAACCATGGCTGGAAATGCTACAGAAGTACCTGC-3', PCR sense primer) and JZ465 (5'-AAAGAATTCTTAACGCATAGCATCGCAGAATTGTAC-3'; PCR antisense primer). To generate a DNA fragment with mutated ‘slippery’ sequence (13392UGUAGCC13398), two PCRs were done using oligonucleotides S59 (5'-12916ATGGTGCTGGGCAGTTTAGCTGCT12939-3'; PCR 1, sense primer), JZ467 (5'-AAAAGGTCTCCGGCTAGAAACGTTGATGCATCCGCAGAC-3'; PCR 1, antisense primer), JZ466 (5'-AAAAGGTCTCTAGCCGGGTTTGCGGTGTAAGTGCAGCCC-3'; PCR 2, sense primer) and S91 (5'-14039TACGAAATCACCGAAATCGTACCA14016-3'; PCR 2, antisense primer). PCR products 1 and 2 were cleaved with BsaI restriction endonuclease and ligated using T4 DNA ligase. The ligation product was used as template to amplify a DNA fragment corresponding to SARS-CoV nt 12955–13961 (with mutated ‘slippery’ sequence) using oligonucleotides JZ464 and JZ465.
For in vitro transcription–translation of the 3CLpro substrate representing nsp7–nsp10 of SARS-CoV (Ser-3837–Gln-4369), the corresponding coding sequence of SARS-CoV was amplified using primers JZ433 (5'-AATAATACGACTCACTATAGGGCGAACCATGTCTAAAATGTCTGACGTAAAGTGCA-3') and JZ434 (5'-AAAGAATTCTTACTGCATCAAGGGTTCGCGGAGTTG-3'). The upstream primer contained a T7 RNA polymerase promoter. For in vitro transcription–translation of the pp1a/pp1ab sequence Lys-737–Ser-1858, containing the PL2pro domain and the presumed nsp2|3 cleavage site, the corresponding coding sequence was amplified by RT-PCR using primers AP91 (5'-TAATACGACTCACTATAGGGACGGGAACACCATGGCAAAAGAAGTAACCTTTCTTGAAGGT-3') and S77 (5'-5839ACGACACAGGCTTGATGGTTGTAG5816-3'). To introduce a PL2pro active-site mutation (Cys-1651 to Ala) in this sequence, two PCRs were done using (1) primers AP91 and AP94 (5'-ATAGCTCTTCATGCATTGTTATCAGCCCATTTAATTGA-3') and (2) AP 95 (5'-ATAGCTCTTCAGCATATTTGTCTAGTGTTTTATTAGCA-3') and S77. Following digestion of the PCR products obtained with SapI and ligation with T4 DNA ligase, the pp1a/pp1ab coding sequence Lys-737–Ser-1858 was re-amplified using the ligation product as a template and primers AP91 and S77. The sequences of PCR products used as templates for in vitro transcription were confirmed by nucleotide sequencing.
Protein expression, purification, and activities.
Plasmid construction, expression in Escherichia coli and purification of the maltose-binding protein (MBP) fusion proteins MBP–HCoV 3CLpro and MBP–TGEV (porcine transmissible gastroenteritis virus) 3CLpro, and the corresponding active-site mutants, MBP–HCoV 3CLpro_C3109V and MBP–TGEV 3CLpro_C3022A, have been described previously (Ziebuhr et al., 1995, 1997; Hegyi et al., 2002). The same approach was taken to express the SARS-CoV pp1a/pp1ab amino acids 3241–3545 (i.e. the SARS-CoV 3CLpro domain lacking the two C-terminal residues, Phe-3545 and Gln-3546) and the corresponding active-site Cys-3385-to-Ala mutant. The coronavirus proteinases were released from MBP by factor Xa cleavage and used, according to previously published protocols (Ziebuhr & Siddell, 1999), in trans-cleavage assays with in vitro-translated substrate or 0·5 mM of synthetic 15-mer peptides whose sequences were derived from the N-terminal TGEV and mouse hepatitis virus (MHV) 3CLpro autoprocessing sites (Seybert et al., 1997; Hegyi & Ziebuhr, 2002). The SARS-CoV helicase (SARS-CoV HEL, pp1ab residues Ala-5302–Gln-5902) and a control protein, SARS-CoV HEL_KA, in which the conserved Lys of the Walker A box (SARS-CoV pp1ab K5589) was replaced by Ala, were expressed and purified in a similar way. Briefly, the helicase-coding region was amplified by RT-PCR using primers JZ425 (5'-GCTGTAGGTGCTTGTGTATTGTGC-3') and JZ426 (5'-AAAACTGCAGTTATTGTAATGTAGCCACATTGCGACGTGG-3'). The PCR product was digested with PstI and inserted in XmnI- and PstI-digested pMal-c2 DNA (New England Biolabs). The mutation was introduced using a PCR-in vivo recombination method (Yao et al., 1992). Expression and purification of MBP–HEL and MBP–HEL_KA were done essentially as described for the HCoV-229E helicase (Heusipp et al., 1997). The partially double-stranded DNA substrate used in the unwinding assay was produced by annealing oligonucleotides D2 [5'-GGTGCAGCCGCAGCGGTGCTCG-d(pT)30-3'] and [
-32P]ATP-labelled D3 [5'-d(pT)30-CGAGCACCGCTGCGGCTGCACC-3'] as described by Seybert et al. (2000a). The unwinding reaction was done for 30 min at 25 °C in buffer A (HEPES/KOH, pH 7·4, 10 % glycerol, 5 mM magnesium acetate, 2 mM dithiothreitol and 0·1 mg BSA ml-1) using 10 nM of substrate and various concentrations of MBP–HEL (8, 80 and 800 nM) and MBP–HEL_KA (800 nM), respectively. The reaction products were analysed on polyacrylamide/TBE gels, which were exposed to X-ray film. ATPase reactions were done in buffer A for 5 min at 25 °C using the following concentrations: MBP–HEL and MBP–HEL_KA each at 0·8 µM, 10 µM [-32P]ATP, 1 µM poly(U)250 (when included). The samples were analysed by polyethyleneimine–cellulose thin-layer chromatography with 0·25 M potassium phosphate, pH 4·0, as the liquid phase. The reaction products were quantified by phosphorimaging of the dried chromatographic plates (ImageQuant software, Molecular Dynamics).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The virus was propagated in African green monkey kidney (Vero and Vero E6) cells, poly(A)-containing RNA was isolated and used for reverse transcription and PCR amplification with primers derived from the SARS-CoV TOR2 sequence. The exact genome termini were determined by 5' and 3' RACE methods. The complete sequence encompasses 29 727 nt [excluding the 3' poly(A) tail] and has been deposited with the GenBank database (accession no. AY291315). Comparison of the genomic sequence of SARS-CoV Frankfurt 1 with 17 previously sequenced SARS-CoV isolates (TOR2, AY274119.3; Urbani, AY278741.1; CUHK-W1, AY278554.2; BJ01, AY278488.2; HKU-39849, AY278491.2; BJ02, AY278487.3; BJ03, AY278490.3; BJ04, AY279354.2; GZ01, AY278489.2; SIN2679, AY283796.1; SIN2500, AY283794.1; ZJ01, AY297028.1; SIN2677, AY283795.1; TW1, AY291451.1; CUHK-Su10, AY282752.1; SIN2748, AY283797.1; SIN2774, AY283798.1) revealed four nucleotide exchanges that have not been described previously for other SARS-CoV isolates and thus seem to be specifically linked to the Frankfurt 1 isolate (A2557, U11448, U24933, U28268). Three of these (may) lead to amino acid substitutions (assuming all predicted ORFs are expressed). Interestingly, the sequence analysis also revealed that, upon SARS-CoV propagation in cell culture (starting with passage 3), a virus variant emerged in which a 45 nt, in-frame sequence (27670–27714) was deleted from ORF7b, thus reducing the genome size to 29 682 nt. The deletion provides evidence that SARS-CoV may undergo rapid adaptation in cell culture. The biological significance of this observation remains to be investigated in detail.
Subgenomic mRNA synthesis
Coronaviruses (and arteriviruses) use a unique strategy to synthesize a set of subgenomic RNAs with common 5' and 3' sequences (Fig. 1
) (Lai & Holmes, 2001; Siddell, 1995; Pasternak et al., 2001; Sawicki & Sawicki, 1998). Each mRNA contains a short 5'-terminal ‘leader’ sequence derived from the 5' end of the genome. The fusion of the noncontiguous sequences is currently believed to be achieved by a discontinuous step during minus-strand synthesis and involves transcription regulatory sequences (TRSs). In addition to the TRS at the 3' end of the leader sequence (leader TRS), TRSs are located upstream of the genes in the 3'-proximal part of the genome (body TRSs) (Lai & Holmes, 2001; Siddell, 1995; Sawicki & Sawicki, 1998). To confirm that SARS-CoV also uses this discontinuous transcription strategy and to elucidate the molecular details of the resulting subgenomic RNAs, we analysed intracellular RNA synthesis by Northern blotting and determined the SARS-CoV mRNA sequences at the sites where the common 5' leader is fused to the various 3' ‘body’ sequences, thus generating the subgenomic RNAs identified in SARS-CoV-infected cells. A Northern blot analysis using poly(A)-containing RNA isolated from SARS-CoV-infected cells and a probe specific for the 3'-proximal 794 nt revealed the synthesis of as many as nine RNAs, with RNA 1 representing the viral genome of 29·7 kb. The sizes of the subgenomic mRNAs were assessed using the previously characterized HCoV-229E RNAs (Thiel et al., 2001) as markers. To provide conclusive evidence for the presence of common 5' leader sequences in each of the SARS-CoV mRNAs and to determine the leader-to-body fusion sites precisely, the 5'-proximal regions of mRNAs 2 to 9 were amplified by RT-PCR and sequenced. The amplification strategy used in these experiments is illustrated for subgenomic mRNA 3 in Fig. 1(d). In some cases we obtained, in addition to the expected RT-PCR product for a given mRNA, larger PCR products that corresponded to the expected RT-PCR products for the next largest subgenomic mRNAs. Sequence analysis of these products confirmed their identity unambiguously. The data obtained by RT-PCR amplification, sequence analysis and Northern blotting consistently suggest that SARS-CoV produces eight subgenomic mRNAs. Furthermore, the study revealed that a minimal consensus sequence, 5'-ACGAAC-3', is sufficient to direct the synthesis of SARS-CoV subgenomic mRNAs, most probably by base-pairing of its negative-stranded counterpart to the leader TRS during minus-strand synthesis. The number of identical nucleotides in leader TRS and body TRS regions varies between 6 and 11 (Fig. 1c), but there is no clear correlation between the extent of sequence complementarity and abundance of a given mRNA (Fig. 1b and 1c), indicating that additional factors (such as sequence elements, RNA structures, proteins) are involved in regulating the relative abundance of viral mRNAs. It is tempting to speculate that the transcription mechanism used by coronaviruses, arteriviruses and (in part) toroviruses (van Vliet et al., 2002) has evolved to allow the production of a large set of structural and nonstructural (some of them probably virulence-associated) proteins (de Haan et al., 2002), whose abundance can be regulated at the transcriptional level. Regulation of coronavirus gene expression can be even further extended by the presence of additional, downstream ORFs in the 5' unique regions of some of the subgenomic mRNAs. These are generally expressed by leaky scanning of ribosomes or internal ribosomal entry (Lai & Holmes, 2001; Siddell, 1995; Thiel et al., 1994). As shown in Fig. 1, SARS-CoV also produces four subgenomic RNAs (mRNA 3, 7, 8, 9) with downstream ORFs in their unique regions. The functions of the corresponding SARS-CoV gene products remain to be characterized. The observed 45 nt deletion in the putative ORF7b (see above) appears to suggest that at least one of these gene products is dispensable in cell culture. However, it cannot be excluded that the 15 aa deletion from the ORF7b gene product gives rise to an active (or partially active) protein.
Translation
The structures of SARS-CoV mRNAs (Fig. 1) lead us to suggest that five of the nine SARS-CoV RNAs are functionally bicistronic. For most of them, the mechanisms used to express the downstream ORFs remain to be determined. On the basis of the available data for other coronaviruses (Brierley et al., 1989; Eleouet et al., 1995; Herold et al., 1993; Kocherhans et al., 2001), it seemed likely that ORF1b expression from the genomic RNA would involve -1 ribosomal frameshifting, a process that essentially depends on two elements, known as the ‘slippery’ sequence, i.e. the site where the ribosomes shift into the -1 reading frame, and a complex RNA pseudoknot structure (Brierley et al., 1989, 1995). Analysis of the SARS-CoV sequence flanking the ORF1a termination codon revealed a putative SARS-CoV frameshifting element comprised of a putative ‘slippery’ sequence (13392UUUAAAC13398) and, further downstream, stretches of complementary sequences that can be modelled to form a typical pseudoknot structure (Fig. 2a) (Brierley et al., 1995). To confirm that these elements mediate frameshifting in SARS-CoV and to define the frameshift site precisely, we synthesized RNAs containing the SARS-CoV frameshift region and produced a mutant version of the presumed ‘slippery’ sequence (13392UUUAAAC13398
13392UGUAGCC13398). As shown in Fig. 2(b), efficient ribosomal frameshifting depended on an essentially unmodified 13392UUUAAAC13398 sequence, supporting the prediction that this sequence constitutes the actual slippage site.
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). Except for infectious bronchitis virus (IBV) (Liu et al., 1995; Lim et al., 2000), all previously characterized coronaviruses encode two (paralogous) papain-like cysteine proteinases (PL1pro and PL2pro), which cleave the N-proximal polyprotein regions at three sites (Gorbalenya et al., 1991; Bonilla et al., 1997; Baker et al., 1989; Kanjanahaluethai & Baker, 2000; Ziebuhr et al., 2001; Herold et al., 1998), while the 3C-like cysteine proteinase (3CLpro; also called main proteinase, Mpro) cleaves the central and C-proximal regions at 11 conserved sites (Ziebuhr et al., 2000). The conservation of both the positions and sequences of coronavirus pp1a/pp1ab cleavage sites allows the substrate specificities of proteinases of newly identified coronaviruses to be predicted (Fig. 3). Thus, it has been proposed that SARS-CoV PL2pro cleaves three sites in the N-proximal region of pp1a/pp1ab (Snijder et al., 2003). This is similar to IBV, where one proteinase, PL2pro, cleaves two sites in this region, but stands in contrast to other coronaviruses (e.g. HCoV-229E and MHV), which cleave three sites by using two PLpro activities, PL1pro and PL2pro. To establish the proteolytic activity of the SARS-CoV PL2pro domain at one of the predicted sites, we expressed, by in vitro translation, the SARS-CoV pp1a/pp1ab amino acids 737–1858 along with a mutant construct in which the presumed active-site Cys nucleophile of PL2pro was replaced by Ala (C-1651–A). The data shown in Fig. 4 strongly suggest that rapid, probably co-translational autoprocessing of the wild-type protein occurred. The apparent sizes of the major products of 
125 (C-1651–A) and 115 kDa (wild-type sequence) are consistent with cleavage at the predicted site, 818Gly|Ala819 (Snijder et al., 2003). Probably due to the presence of only two Met residues in the N-terminal cleavage product, the expected N-terminal 9 kDa protein could not be detected convincingly. Comparative analysis of coronavirus PLpro cleavage sites revealed that the (predicted) SARS-CoV PL2pro sites are more conserved than the equivalent HCoV-229E PL1pro and PL2pro sites (Fig. 5). The poor conservation of HCoV-229E PLpro cleavage sites probably results from the overlapping substrate specificities of PL1pro and PL2pro, which we reported previously (Ziebuhr et al., 2001). Consistent with the hypothesis that conservation of only one PLpro domain is linked to an increase in specificity, we also find that the IBV PL2pro cleavage sites are much better conserved than the corresponding HCoV-229E PL1pro/PL2pro sites (Fig. 5). The observed narrow substrate specificity of the SARS-CoV PL2pro will certainly facilitate the development of selective proteinase inhibitors.
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; Ziebuhr et al., 2000). Furthermore, 3CLpro is the only coronavirus protein for which structure information is available (Anand et al., 2002, 2003). Coronavirus 3CLpros are cysteine proteinases with interesting properties. They feature a serine proteinase (chymotrypsin)-like two-
-barrel fold but employ a catalytic Cys–His dyad instead of the classical Ser–His–Asp triad. Furthermore, they possess a third, C-terminal domain composed of five -helices. Our previous work has shown that the 3CLpro substrate specificities are conserved among the three established groups of coronaviruses (Hegyi & Ziebuhr, 2002) and comparison of the previously known coronavirus cleavage sites with those identified in the SARS-CoV polyproteins (Fig. 3; Fig. 6a and b) suggests conservation of the P4 (Ser, Thr, Val, Pro, Ala), P2 (Leu, Ile, Val, Phe, Met), P1 (Gln) and P1' (Ser, Ala, Gly, Asn, Cys) residues, all of which have previously been proposed to define the coronavirus 3CLpro substrate specificity (Ziebuhr et al., 2000). In SARS-CoV, the strong preference for Leu at the P2 positions of substrates is less prominent and the more frequent use of Phe may indicate a slightly larger S2 subsite. There are also conservative changes in the P4 and P1' positions of SARS-CoV 3CLpro substrates. To determine whether these subtle variations result in differences in substrate specificity, we characterized the SARS-CoV 3CLpro specificity in more detail and compared it with that of the well-characterized HCoV-229E and TGEV enzymes. SARS-CoV 3CLpro was expressed as an MBP fusion protein, an approach that had proven suitable for the production of active coronavirus 3CLpros (Seybert et al., 1997; Ziebuhr et al., 1997; Hegyi et al., 2002). Following factor Xa cleavage of the purified fusion protein, the released SARS-CoV 3CLpro was used in trans-cleavage experiments with in vitro-translated substrates (Fig. 6d) or synthetic peptides derived from group 1 (TGEV) and group 2 (MHV) N-terminal 3CLpro autoprocessing sites (Hegyi & Ziebuhr, 2002; Seybert et al., 1997) (data not shown). The substrate derived from the C-terminal pp1a sequence was chosen because it contained as many as three cleavage sites, allowing side-by-side comparison of cleavage efficiencies at different sites. Furthermore, the fact that the proteolytic processing of this region has already been studied extensively in other coronaviruses (Ziebuhr & Siddell, 1999; Bost et al., 2000) allowed safe predictions on SARS-CoV 3CLpro cleavage products in this region. As shown in Fig. 6(d), the cleavage products (and several intermediate products) predicted to be released by 3CLpro cleavage (Fig. 3; Snijder et al., 2003) were readily detectable upon incubation of the translation product with purified SARS-CoV 3CLpro, but not with the active-site Cys-to-Ala mutant. Significantly, nearly identical cleavage kinetics were observed when purified TGEV and HCoV-229E 3CLpros (instead of SARS-CoV 3CLpro) were incubated with the SARS-CoV-derived substrate. Again, as expected, no cleavage was observed with the active-site Cys-to-Ala (Val) mutants. Consistent data were also obtained in peptide cleavage assays, where the SARS-CoV, HCoV-229E and TGEV enzymes were shown to cleave TGEV and MHV substrates with equal efficiencies (data not shown). The data conclusively demonstrate the proteolytic activity of the expressed SARS-CoV protein and, even more importantly, suggest conservation of coronavirus 3CLpro substrate specificities.
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). On the basis of seven conserved sequence motifs, the coronavirus enzyme has been classified as belonging to the helicase superfamily 1 (Gorbalenya et al., 1989b). We previously reported that helicases of HCoV-229E and the arterivirus equine arteritis virus (EAV) possess polynucleotide-stimulated NTPase activity and 5'-to-3' RNA and DNA duplex-unwinding activities (Seybert et al., 2000a, b). Both coronavirus and arterivirus helicases require an N-terminal (putative) metal-binding domain, consisting of at least 12 conserved Cys and His residues, for activity. The domain is essential for replication, transcription and virion morphogenesis (as demonstrated for EAV) (van Dinten et al., 2000). The SARS-CoV helicase is predicted to be released from pp1ab by 3CLpro-mediated cleavages of the 5301Gln|Ala5302 and 5902Gln|Ala5903 dipeptide bonds (Fig. 3; Snijder et al., 2003). We therefore expressed SARS-CoV pp1ab residues 5302–5902, representing the 67 kDa helicase together with its N-terminal metal-binding domain, in a bacterial expression system. A fusion protein, consisting of MBP and the SARS-CoV helicase domain, was purified by amylose affinity chromatography and used in ATPase and duplex-unwinding assays. Fig. 7(a) shows that this fusion protein (MBP–HEL), but not a control protein (MBP–HEL_KA) in which the conserved Lys residue of the Walker A box (Walker et al., 1982) was replaced by Ala, had ATPase activity that could be stimulated by poly(U). The protein also had DNA duplex-unwinding activity (Fig. 7b). This is consistent with the previously characterized HCoV-229E helicase (Seybert et al., 2000a), which also had revealed a high promiscuity with respect to the substrates used. Thus, both DNA and RNA duplexes were found to be unwound by the HCoV-229E helicase with similar efficiency and all types of nucleotide and ribonucleotide cofactors were used (Seybert et al., 2000a; K. A. Ivanov, V. Thiel & J. Ziebuhr, unpublished data).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Anand, K., Ziebuhr, J., Wadhwani, P., Mesters, J. R. & Hilgenfeld, R. (2003). Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs. Science 300, 1763–1767.
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Received 13 June 2003;
accepted 19 June 2003.
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B. Yount, R. S. Roberts, A. C. Sims, D. Deming, M. B. Frieman, J. Sparks, M. R. Denison, N. Davis, and R. S. Baric Severe Acute Respiratory Syndrome Coronavirus Group-Specific Open Reading Frames Encode Nonessential Functions for Replication in Cell Cultures and Mice J. Virol., December 1, 2005; 79(23): 14909 - 14922. [Abstract] [Full Text] [PDF]
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R. L. Graham, A. C. Sims, S. M. Brockway, R. S. Baric, and M. R. Denison The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication J. Virol., November 1, 2005; 79(21): 13399 - 13411. [Abstract] [Full Text] [PDF]
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A. Putics, W. Filipowicz, J. Hall, A. E. Gorbalenya, and J. Ziebuhr ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture J. Virol., October 15, 2005; 79(20): 12721 - 12731. [Abstract] [Full Text] [PDF]
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W. Peti, M. A. Johnson, T. Herrmann, B. W. Neuman, M. J. Buchmeier, M. Nelson, J. Joseph, R. Page, R. C. Stevens, P. Kuhn, et al. Structural Genomics of the Severe Acute Respiratory Syndrome Coronavirus: Nuclear Magnetic Resonance Structure of the Protein nsP7 J. Virol., October 15, 2005; 79(20): 12905 - 12913. [Abstract] [Full Text] [PDF]
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B. Kan, M. Wang, H. Jing, H. Xu, X. Jiang, M. Yan, W. Liang, H. Zheng, K. Wan, Q. Liu, et al. Molecular Evolution Analysis and Geographic Investigation of Severe Acute Respiratory Syndrome Coronavirus-Like Virus in Palm Civets at an Animal Market and on Farms J. Virol., September 15, 2005; 79(18): 11892 - 11900. [Abstract] [Full Text] [PDF]
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C. Dye and S. G. Siddell Genomic RNA sequence of Feline coronavirus strain FIPV WSU-79/1146 J. Gen. Virol., August 1, 2005; 86(8): 2249 - 2253. [Abstract] [Full Text] [PDF]
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B. W. Neuman, D. A. Stein, A. D. Kroeker, M. J. Churchill, A. M. Kim, P. Kuhn, P. Dawson, H. M. Moulton, R. K. Bestwick, P. L. Iversen, et al. Inhibition, Escape, and Attenuated Growth of Severe Acute Respiratory Syndrome Coronavirus Treated with Antisense Morpholino Oligomers J. Virol., August 1, 2005; 79(15): 9665 - 9676. [Abstract] [Full Text] [PDF]
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Y.-J. Tan, P.-Y. Tham, D. Z. L. Chan, C.-F. Chou, S. Shen, B. C. Fielding, T. H. P. Tan, S. G. Lim, and W. Hong The Severe Acute Respiratory Syndrome Coronavirus 3a Protein Up-Regulates Expression of Fibrinogen in Lung Epithelial Cells J. Virol., August 1, 2005; 79(15): 10083 - 10087. [Abstract] [Full Text] [PDF]
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 M.-C. Su, C.-T. Chang, C.-H. Chu, C.-H. Tsai, and K.-Y. Chang An atypical RNA pseudoknot stimulator and an upstream attenuation signal for -1 ribosomal frameshifting of SARS coronavirus Nucleic Acids Res., July 29, 2005; 33(13): 4265 - 4275. [Abstract] [Full Text] [PDF]
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R. Casais, M. Davies, D. Cavanagh, and P. Britton Gene 5 of the Avian Coronavirus Infectious Bronchitis Virus Is Not Essential for Replication J. Virol., July 1, 2005; 79(13): 8065 - 8078. [Abstract] [Full Text] [PDF]
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W.-C. Hsu, H.-C. Chang, C.-Y. Chou, P.-J. Tsai, P.-I. Lin, and G.-G. Chang Critical Assessment of Important Regions in the Subunit Association and Catalytic Action of the Severe Acute Respiratory Syndrome Coronavirus Main Protease J. Biol. Chem., June 17, 2005; 280(24): 22741 - 22748. [Abstract] [Full Text] [PDF]
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B. Schelle, N. Karl, B. Ludewig, S. G. Siddell, and V. Thiel Selective Replication of Coronavirus Genomes That Express Nucleocapsid Protein J. Virol., June 1, 2005; 79(11): 6620 - 6630. [Abstract] [Full Text] [PDF]
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L. Chen, C. Gui, X. Luo, Q. Yang, S. Gunther, E. Scandella, C. Drosten, D. Bai, X. He, B. Ludewig, et al. Cinanserin Is an Inhibitor of the 3C-Like Proteinase of Severe Acute Respiratory Syndrome Coronavirus and Strongly Reduces Virus Replication In Vitro J. Virol., June 1, 2005; 79(11): 7095 - 7103. [Abstract] [Full Text] [PDF]
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 Z. R. Yang Mining SARS-CoV protease cleavage data using non-orthogonal decision trees: a novel method for decisive template selection Bioinformatics, June 1, 2005; 21(11): 2644 - 2650. [Abstract] [Full Text] [PDF]
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S. Hussain, J. Pan, Y. Chen, Y. Yang, J. Xu, Y. Peng, Y. Wu, Z. Li, Y. Zhu, P. Tien, et al. Identification of Novel Subgenomic RNAs and Noncanonical Transcription Initiation Signals of Severe Acute Respiratory Syndrome Coronavirus J. Virol., May 1, 2005; 79(9): 5288 - 5295. [Abstract] [Full Text] [PDF]
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D. Endoh, T. Mizutani, R. Kirisawa, Y. Maki, H. Saito, Y. Kon, S. Morikawa, and M. Hayashi Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription Nucleic Acids Res., April 7, 2005; 33(6): e65 - e65. [Abstract] [Full Text] [PDF]
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T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard Deubiquitination, a New Function of the Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease? J. Virol., April 1, 2005; 79(7): 4550 - 4551. [Full Text] [PDF]
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E. Manktelow, K. Shigemoto, and I. Brierley Characterization of the frameshift signal of Edr, a mammalian example of programmed -1 ribosomal frameshifting Nucleic Acids Res., March 14, 2005; 33(5): 1553 - 1563. [Abstract] [Full Text] [PDF]
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N. Ito, E. C. Mossel, K. Narayanan, V. L. Popov, C. Huang, T. Inoue, C. J. Peters, and S. Makino Severe Acute Respiratory Syndrome Coronavirus 3a Protein Is a Viral Structural Protein J. Virol., March 1, 2005; 79(5): 3182 - 3186. [Abstract] [Full Text] [PDF]
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A. Seybert, C. C. Posthuma, L. C. van Dinten, E. J. Snijder, A. E. Gorbalenya, and J. Ziebuhr A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases J. Virol., January 15, 2005; 79(2): 696 - 704. [Abstract] [Full Text] [PDF]
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S. Chen, L. Chen, J. Tan, J. Chen, L. Du, T. Sun, J. Shen, K. Chen, H. Jiang, and X. Shen Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase N Terminus Is Indispensable for Proteolytic Activity but Not for Enzyme Dimerization: BIOCHEMICAL AND THERMODYNAMIC INVESTIGATION IN CONJUNCTION WITH MOLECULAR DYNAMICS SIMULATIONS J. Biol. Chem., January 7, 2005; 280(1): 164 - 173. [Abstract] [Full Text] [PDF]
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B. H. Harcourt, D. Jukneliene, A. Kanjanahaluethai, J. Bechill, K. M. Severson, C. M. Smith, P. A. Rota, and S. C. Baker Identification of Severe Acute Respiratory Syndrome Coronavirus Replicase Products and Characterization of Papain-Like Protease Activity J. Virol., December 15, 2004; 78(24): 13600 - 13612. [Abstract] [Full Text] [PDF]
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Y.-J. Tan, B. C. Fielding, P.-Y. Goh, S. Shen, T. H. P. Tan, S. G. Lim, and W. Hong Overexpression of 7a, a Protein Specifically Encoded by the Severe Acute Respiratory Syndrome Coronavirus, Induces Apoptosis via a Caspase-Dependent Pathway J. Virol., December 15, 2004; 78(24): 14043 - 14047. [Abstract] [Full Text] [PDF]
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S. M. Brockway, X. T. Lu, T. R. Peters, T. S. Dermody, and M. R. Denison Intracellular Localization and Protein Interactions of the Gene 1 Protein p28 during Mouse Hepatitis Virus Replication J. Virol., November 1, 2004; 78(21): 11551 - 11562. [Abstract] [Full Text] [PDF]
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R. J. Hogan, G. Gao, T. Rowe, P. Bell, D. Flieder, J. Paragas, G. P. Kobinger, N. A. Wivel, R. G. Crystal, J. Boyer, et al. Resolution of Primary Severe Acute Respiratory Syndrome-Associated Coronavirus Infection Requires Stat1 J. Virol., October 15, 2004; 78(20): 11416 - 11421. [Abstract] [Full Text] [PDF]
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E. Prentice, J. McAuliffe, X. Lu, K. Subbarao, and M. R. Denison Identification and Characterization of Severe Acute Respiratory Syndrome Coronavirus Replicase Proteins J. Virol., September 15, 2004; 78(18): 9977 - 9986. [Abstract] [Full Text] [PDF]
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 W. G. Glass, K. Subbarao, B. Murphy, and P. M. Murphy Mechanisms of Host Defense following Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) Pulmonary Infection of Mice J. Immunol., September 15, 2004; 173(6): 4030 - 4039. [Abstract] [Full Text] [PDF]
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K. A. Ivanov, T. Hertzig, M. Rozanov, S. Bayer, V. Thiel, A. E. Gorbalenya, and J. Ziebuhr Major genetic marker of nidoviruses encodes a replicative endoribonuclease PNAS, August 24, 2004; 101(34): 12694 - 12699. [Abstract] [Full Text] [PDF]
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J. R. St-Jean, H. Jacomy, M. Desforges, A. Vabret, F. Freymuth, and P. J. Talbot Human Respiratory Coronavirus OC43: Genetic Stability and Neuroinvasion J. Virol., August 15, 2004; 78(16): 8824 - 8834. [Abstract] [Full Text] [PDF]
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A. E. Gorbalenya, E. J. Snijder, and W. J. M. Spaan Severe Acute Respiratory Syndrome Coronavirus Phylogeny: toward Consensus J. Virol., August 1, 2004; 78(15): 7863 - 7866. [Full Text] [PDF]
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B. C. Fielding, Y.-J. Tan, S. Shuo, T. H. P. Tan, E.-E. Ooi, S. G. Lim, W. Hong, and P.-Y. Goh Characterization of a Unique Group-Specific Protein (U122) of the Severe Acute Respiratory Syndrome Coronavirus J. Virol., July 15, 2004; 78(14): 7311 - 7318. [Abstract] [Full Text] [PDF]
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K. A. Ivanov and J. Ziebuhr Human Coronavirus 229E Nonstructural Protein 13: Characterization of Duplex-Unwinding, Nucleoside Triphosphatase, and RNA 5'-Triphosphatase Activities J. Virol., July 15, 2004; 78(14): 7833 - 7838. [Abstract] [Full Text] [PDF]
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 L. Gillim-Ross, J. Taylor, D. R. Scholl, J. Ridenour, P. S. Masters, and D. E. Wentworth Discovery of Novel Human and Animal Cells Infected by the Severe Acute Respiratory Syndrome Coronavirus by Replication-Specific Multiplex Reverse Transcription-PCR J. Clin. Microbiol., July 1, 2004; 42(7): 3196 - 3206. [Abstract] [Full Text] [PDF]
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Y.-J. Tan, E. Teng, S. Shen, T. H. P. Tan, P.-Y. Goh, B. C. Fielding, E.-E. Ooi, H.-C. Tan, S. G. Lim, and W. Hong A Novel Severe Acute Respiratory Syndrome Coronavirus Protein, U274, Is Transported to the Cell Surface and Undergoes Endocytosis J. Virol., July 1, 2004; 78(13): 6723 - 6734. [Abstract] [Full Text] [PDF]
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H. Zhang, G. Wang, J. Li, Y. Nie, X. Shi, G. Lian, W. Wang, X. Yin, Y. Zhao, X. Qu, et al. Identification of an Antigenic Determinant on the S2 Domain of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Capable of Inducing Neutralizing Antibodies J. Virol., July 1, 2004; 78(13): 6938 - 6945. [Abstract] [Full Text] [PDF]
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J. Shi, Z. Wei, and J. Song Dissection Study on the Severe Acute Respiratory Syndrome 3C-like Protease Reveals the Critical Role of the Extra Domain in Dimerization of the Enzyme: DEFINING THE EXTRA DOMAIN AS A NEW TARGET FOR DESIGN OF HIGHLY SPECIFIC PROTEASE INHIBITORS J. Biol. Chem., June 4, 2004; 279(23): 24765 - 24773. [Abstract] [Full Text] [PDF]
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T. Hertzig, E. Scandella, B. Schelle, J. Ziebuhr, S. G. Siddell, B. Ludewig, and V. Thiel Rapid identification of coronavirus replicase inhibitors using a selectable replicon RNA J. Gen. Virol., June 1, 2004; 85(6): 1717 - 1725. [Abstract] [Full Text] [PDF]
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K. A. Ivanov, V. Thiel, J. C. Dobbe, Y. van der Meer, E. J. Snijder, and J. Ziebuhr Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase J. Virol., June 1, 2004; 78(11): 5619 - 5632. [Abstract] [Full Text] [PDF]
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M. R. Denison, B. Yount, S. M. Brockway, R. L. Graham, A. C. Sims, X. Lu, and R. S. Baric Cleavage between Replicase Proteins p28 and p65 of Mouse Hepatitis Virus Is Not Required for Virus Replication J. Virol., June 1, 2004; 78(11): 5957 - 5965. [Abstract] [Full Text] [PDF]
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 A. D. L. Sihoe, R. H. L. Wong, A. T. H. Lee, L. S. Lau, N. Y. Y. Leung, K. I. Law, and A. P. C. Yim Severe Acute Respiratory Syndrome Complicated by Spontaneous Pneumothorax Chest, June 1, 2004; 125(6): 2345 - 2351. [Abstract] [Full Text] [PDF]
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 W.-Y. Choy, S.-G. Lin, P. K.-S. Chan, J. S.-L. Tam, Y.M. D. Lo, I. M.-T. Chu, S.-N. Tsai, M.-Q. Zhong, K.-P. Fung, M. M.-Y. Waye, et al. Synthetic Peptide Studies on the Severe Acute Respiratory Syndrome (SARS) Coronavirus Spike Glycoprotein: Perspective for SARS Vaccine Development Clin. Chem., June 1, 2004; 50(6): 1036 - 1042. [Abstract] [Full Text] [PDF]
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M.-P. Egloff, F. Ferron, V. Campanacci, S. Longhi, C. Rancurel, H. Dutartre, E. J. Snijder, A. E. Gorbalenya, C. Cambillau, and B. Canard The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world PNAS, March 16, 2004; 101(11): 3792 - 3796. [Abstract] [Full Text] [PDF]
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A. M. Bressler and F. S. Nolte Preclinical Evaluation of Two Real-Time, Reverse Transcription-PCR Assays for Detection of the Severe Acute Respiratory Syndrome Coronavirus J. Clin. Microbiol., March 1, 2004; 42(3): 987 - 991. [Abstract] [Full Text] [PDF]
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E. VAN DEN BORN, A. P. GULTYAEV, and E. J. SNIJDER Secondary structure and function of the 5'-proximal region of the equine arteritis virus RNA genome RNA, March 1, 2004; 10(3): 424 - 437. [Abstract] [Full Text] [PDF]
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 Y.-J. Tan, P.-Y. Goh, B. C. Fielding, S. Shen, C.-F. Chou, J.-L. Fu, H. N. Leong, Y. S. Leo, E. E. Ooi, A. E. Ling, et al. Profiles of Antibody Responses against Severe Acute Respiratory Syndrome Coronavirus Recombinant Proteins and Their Potential Use as Diagnostic Markers Clin. Vaccine Immunol., March 1, 2004; 11(2): 362 - 371. [Abstract] [Full Text] [PDF]
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X. Xu, Y. Liu, S. Weiss, E. Arnold, S. G. Sarafianos, and J. Ding Molecular model of SARS coronavirus polymerase: implications for biochemical functions and drug design Nucleic Acids Res., December 15, 2003; 31(24): 7117 - 7130. [Abstract] [Full Text] [PDF]
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J. A. Tanner, R. M. Watt, Y.-B. Chai, L.-Y. Lu, M. C. Lin, J. S. M. Peiris, L. L. M. Poon, H.-F. Kung, and J.-D. Huang The Severe Acute Respiratory Syndrome (SARS) Coronavirus NTPase/Helicase Belongs to a Distinct Class of 5' to 3' Viral Helicases J. Biol. Chem., October 10, 2003; 278(41): 39578 - 39582. [Abstract] [Full Text] [PDF]
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