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* Departments of Zoology and
Microbiology, University of Hong Kong, Hong Kong, SAR China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Key Words: SARS • coronavirus • genomic sequence • Subgenomic transcripts • 5'-RACE assay
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Coronaviridae is a viral family that infects birds and mammals and causes a variety of diseases (3). Six species of CoV genomes have been completely sequenced, namely murine hepatitis virus (MHV) (4); avian infectious bronchitis virus (IBV) (5); human CoV 229E (HCV 229E) (6); bovine CoV (BCV) (7); transmissible gastroenteritis virus (TGEV) (8); and porcine epidemic diarrhea virus (PEDV) (9). The size of the genome is about 30 kb, of which more than two-thirds is occupied by open reading frames (ORF) 1a, b. This gene contains two large ORFs, ORF 1a and ORF 1b that are cotranslated with -1 ribosomal frameshifting mechanism (10–13). The gene products of both ORFs are believed to be processed into a number of functional subunits (14, 15). The putative functional domains in ORF1 include two to three papain-like domains, one 3C-like protease domain, one growth factor/receptor-like domain, one polymerase domain, one metal ion-binding domain, and a helicase domain. The remaining one-third of the genome consists mainly of three structural proteins: a surface-spike glycoprotein (S), a transmembrane protein (M), and a nucleocapsid protein (N). Some CoV genomes (Group II) also contain the hemagglutinin-esterase (HE) gene. The spike glycoprotein has major functions in virus-host cell membrane fusion and interaction with host cell surface receptors. Membrane protein is responsible for virus budding, but other viral proteins may also be involved in organizing the virus budding pre-Golgi membranes (16, 17). The nucleocapsid protein has been found to be the central hydrophilic basic domain involved in RNA binding (18). Also, it has recently been suggested that CoVs contain an internal core of helical nucleocapsid, which is composed of both M and N proteins, in which the M protein was supposed to be found only in the envelope protein of the virus (19). Small ORFs are also usually found between these structural genes, depending on the virus species (20–22).
We have extracted the genomic RNA from a tissue culture sample of the SARS CoV strain isolated from one of the earliest Hong Kong patients with SARS. This strain has been given the name HK-39. By using degenerated and specific primer PCR amplification, coupled with cDNA library screening, we were able to obtain the 29-kb complete genomic sequence. In this paper, we report the complete nucleotide sequence of a Hong Kong SARS CoV and compare and analyze its genomic organization and individual genes with those of other known SARS CoV species.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Construction of cDNA Library.
Double-stranded DNA linkers were added to the 3' end of the first-strand cDNA for second strand cDNA synthesis (23). The end of the double-stranded cDNA was modified by T4 polymerase. The processed DNA was than subsequently cloned into pCR2.1 vector (Invitrogen). The clones with the cDNA insert were screened by PCR and were subjected to direct sequencing analysis.
Amplification of Viral Genomic Sequences.
Degenerated primers covering the whole genome of the CoV were designed based on the genomic sequences of other CoVs. With assistance from the results obtained from library screening, degenerated primers amplification, primer walking, and other public sources (e.g., SARS CoV strain Tor2, GenBank accession number NC_004718), the gaps in the genome were finally closed by specific primer PCR and sequencing.
Sequencing.
DNA fragments resulting from PCRs from viral genome and library screening were purified and directly sequenced by BigDye Terminator Cycle Sequencing in an ABI Prism 3100 Genetic Analyzer (Perkin Elmer, Norwalk, CT). CoV sequences were confirmed by searching in NCBI BLAST-X.
5'-RACE and 3'-RACE.
The 5' end of RNA genome and RNA transcripts were identified by using two different 5'-RACE commercial kits: RNA ligase-mediated 5'-RACE (GeneRacer kit; Invitrogen), and end-switching 5'-RACE (SMART RACE cDNA Amplification kit; Clontech, Palo Alto, CA). One microgram of RNA extracted from virus infected cells was used for each 5'-RACE reaction in accordance with the manufacturer’s instructions. Specific primers located near the 5' end of each possible gene were used for PCR, and nested PCR was carried out if needed. For the 3'-RACE, the first-strand cDNA was reverse-transcribed with a 3'-RACE oligo, which had two primers (C1 and C2) annealed sites ahead of oligo dT. A set of specific primers at different sites of the genome were combined with 3'-RACE-anchored primers C1 or C2, respectively, and were used to amplify the possible 3' end of poly(A)+ mRNA transcripts or the RNA genome. All the PCR fragments of 5'- and 3'-RACE were subjected to sequencing directly.
Data Analysis.
The 29.7-kb complete genome sequence was assembled from the sequence contigs using SeqMan of the Lasergene Package (DNASTAR, Madison, WI). The putative ORFs were predicted by EditSeq of the Lasergene Package (DNASTAR). The SARS CoV complete genome sequence was compared with those of other known CoV species. Multiple sequence analysis and optimal alignments were conducted on MegAlign of the Lasergene Package (DNASTAR). Phylogenetic tree construction and bootstrap tests were performed using MEGA 2.1 (Arizona State University, Tempe, AZ). The similarity plots of multiple sequence alignments were performed by SimPlot (Johns Hopkins University School of Medicine, Baltimore, MD). The coiled-coil motif prediction of the S protein was performed by COILS (24). Transmembrane domain topology predictions of the proteins were performed using TMHMM (CBS, Technical University of Denmark, Copenhagen, Lyngby, Denmark).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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). PRRSV is a positive-stranded RNA virus and belongs to the order Nidovirales, the same order as the CoV. Six other fragments totaling about 3 kb covering different regions of the genome were successfully amplified by degenerated primers (Fig. 1A). Specific primers were designed for walking to fill up the gaps. These methods, coupled with cDNA library screening, enabled more than 30% of the genome sequence to be obtained. Work carried out in the BCCA Genome Sciences Center (British Columbia Center for Disease in Canada) enabled us to uncover the complete viral genome in 2 days. When the genome sequence of HK-39 was compared with the sequence of Tor2, a high homology was found. By using the specific primer walking (Fig. 1A), the final sequence of HK-39 was completed.
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). The length and the position of the confirmed ORFs are shown in Figure 1A.
Sequence Alignment and Phylogenetic Analysis.
The Coronaviridae family is classified into three groups according to the structural proteins that affect their antigenicity (25). Phylogenetic analysis of the whole genome and individual ORFs of HK-39 with other known CoV species showed that SARS-CoV only shares a very low level of homology with the other members of the CoV family at the level of nucleotide sequence and forms a separate group (Fig. 2A). The topology of the phylogenetic tree was similar in all analyses based on amino acid differences (Fig. 2, B–E). Although SARS-CoV showed a higher degree of amino acid sequence homology to Group 2 species, its genomic organization is more closely resembles that of Group 1 species (data not shown). Nevertheless, this controversy exists, and it is clear that SARS-CoV, including the Tor2 strain (isolated from SARS patient number 2 in Toronto [1]), Urbani strain (GenBank accession number AY278741), and CUHK-W1 strain (isolated from a patient suffering from SARS in Hong Kong, GenBank accession number AY278554 [unpublished data]), does not fall into any of the existing phylogenetic groups, and is distant from all known human CoVs such as the human 229E and human CoV OC43, although they share a common host. SARS CoV is a new member of the CoV family, and is very distinct from all CoVs characterized hitherto.
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). Initiation codons were usually found immediately after or a few bases away from the IGS, except for ORF1 and M. An 11-codon "mini-ORF" was predicted 31 bp downstream of the IGS and 128 bp upstream of the initiation codon of AUG at the 5'-UTR, which is similar to that of IBV (26). Eighteen specific primers located at different sites of the genome were used to amplify the 3' ends of the transcripts. Sequencing of the 3'-RACE products showed that only the regions at the 3' end of the genome were amplified. The above results support the unique discontinuous transcription system in CoVs, which generate a nested set of transcripts with common 3' ends and a common leader sequence on the 5' ends. 3'-UTR (sequence downstream the N protein sequence) has been shown to be crucial in the regulation of transcription in a CoV. SimPlot analysis of 3'-UTR showed a remarkably high degree of similarity with IBV, which contradicts that of the other regions of the genome (Fig. 2F). A 32-bp conserved motif (nucleotides 29590-29621) was found in the 3'-UTR of SARS CoV HK-39. Such a motif shares a very high homology with the stem-loop II-like motif (s2m) found in IBV (27). Jonassen et al. (27) pointed out that such a motif was also found in some viruses that are distinct from IBV, like some animal astroviruses and picornavirus, and that it may be due to the consequence of the RNA transferring event that occurred between these viruses. Their findings in IBV, together with the identified putative s2m motif in HK-39, imply that these two CoVs are evolutionarily related.
Putative Functional Domains of ORF 1.
Sequence alignment on the predicted amino acid sequence of ORF1 revealed the uniqueness of this novel SARS CoV. In general, it shows an average of less than 50% similarity with any other groups of CoVs. In ORF1a, one papain-like (PL) domain, a 3C-like (3CL) protease, and a growth factor/receptor-like (GFL) domain were predicted with the reference to that of TGEV (Fig. 3A) (8). The organization is similar to other members of the virus family (12, 13, 28). Two characteristic and remarkably hydrophobic regions located at both sides of the 3CL domain were identified by computer predictions. In total, 10 putative 3CL cleavage sites were predicted in ORF1ab, and their locations are shown in Figure 3A. One of the necessary elements for the ribosomal frame-shifting mechanism (10, 13), a ribosomal slippage site UUAAAC, was also identified at 13392 bp, 15 bp upstream of the stop codon of ORF1a. Alignment of the predicted amino acid sequence of ORF1b to known strains revealed the presence of conserved putative domains, including RNA polymerase domain (POL), metal ion-binding (MIB), and helicase (Hel) domain (Fig. 3A). These findings support the conclusion that the SARS CoV is a typical member of the CoV family.
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, B2). Normally, two to three clusters of heptad repeats are found in the S2 region in other CoVs. The heptad repeats of these coiled-coil regions were identified in other CoVs (Fig. 3, B1). A conserved transmembrane domain was predicted in the C-terminal of the S2 region (Fig. 3, B3). Amino acid sequence alignment of the transmembrane domain has shown that this region is highly conserved. All coronavirus M proteins show to posses a triple membrane-spanning protein with a Nexo-Cendo configuration (17, 29, 30). TMHMM predicts three transmembrane domains on the N-terminal of the M gene of SARS CoV (Fig. 3C). It is observed in the alignment of N protein sequence that there are three stretches of amino acid residues that are highly conserved among the 16 CoV species. Such residues are believed to be involved in its structural maintenance and direct interaction with RNA in the case of IBV (Fig. 3D) (25).
Conclusion.
We have completed the sequencing of a novel CoV HK-39. The distinctive molecular genomic and phylogenetic characteristics of this novel virus seem to warrant its assignment to a new and distinct group IV of the Coronaviridae family.
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Footnotes |
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1 To whom requests for reprints should be addressed at Department of Zoology, Kadoorie Biological Sciences Building, University of Hong Kong, Hong Kong, SAR China. E-mail: fcleung{at}hkucc.hku.hk 
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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