Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA1
Advanced Biomedical Computing Center, Science Application International Corporation, National Cancer Institute, Frederick, MD 21702-1201, USA2
Author for correspondence: Konstantin Kousoulas. Fax +1 225 578 9701. e-mail vtgusk{at}lsu.edu
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Abstract |
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Main text |
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Previously, we reported nucleotide sequences of approximately 9·5 kbp from the 3' region downstream of the replicase genes specified by respiratory and enteric BCoV isolates from different animals (Chouljenko et al., 1998 ). We report here a comparative analysis of the newly determined complete genomes of the LUN and ENT strains of BCoV. Besides being the first report of complete genome sequences of BCoV, this analysis also provides insight into replicase strain-specific differences, which are associated with the evolution of BCoV in the natural host.
BCoV strains were propagated in HRT-18G cells (Storz et al., 1996 ). After the second passage, viruses were further plaque-purified three times and genomic RNA was extracted from gradient-purified virus to produce cDNAs, as described previously (Chouljenko et al., 1998
). In order to ensure that sequences were derived from the predominant virus species, only direct RTPCR products were used as templates for rTth DNA polymerase-XL enzyme (PE Applied Biosystems). To minimize the potential damage to nucleotides caused by UV irradiation and ethidium bromide, all templates were purified by the crystal violet method (Invitrogen) and DNA was recovered from the gel by using the Zymoclean gel DNA recovery kit (Zymo Research). PCRs were performed by using the GeneAmp XL PCR kit, AmpliWax PCR Gem 100 (providing hot-start PCR) and the GeneAmp PCR System 9600 instrument (all from PE Applied Biosystems). Sequencing was performed on an ABI Prism 377 DNA sequencer (PE Applied Biosystems) according to the strategy outlined in Fig. 1
. Oligonucleotide primers were based on either the replicase sequence of the mouse hepatitis virus (MHV) A59 c12 mutant (GenBank accession no. AF029248) or an available BCoV nucleotide sequence (AF058944). After the respective BCoV sequences were elucidated, BCoV-based primers (Fig. 1
) were used and overlapping cDNA fragments of 26 kbp were re-amplified and re-sequenced. Sequences of the cDNA fragments were aligned to assemble the entire BCoV genome of LUN and ENT strains using the Sequencher 3.1.1 sequence analysis software (Gene Code Corporation).
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A multiple sequence alignment of replicase polyproteins pp1ab of BCoV and other coronaviruses (not shown) was produced using the programs CLUSTAL X (Thompson et al., 1997 ), Dialign2 (Morgenstern, 1999
) and Macaw (Schuler et al., 1991
) and used for phylogenetic analysis of pp1b. By conducting a parsimonious exhaustive tree search with a UNIX version of the program PAUP* 4.0.0d55 (Swofford, 2000
), which is part of the GCG Wisconsin package, we found that the most closely related replicases were those of BCoV and MHV (Fig. 2A
). A similar observation was reported recently for the most conserved fragment of pp1b using a larger set of coronaviruses (Stephensen et al., 1999
). This replicase clustering parallels that of the structural proteins of MHV and BCoV, which previously led to the placement of these viruses in coronavirus group 2 (Siddell, 1995
). The pp1ab polyprotein amino acid sequences of BCoV and MHV share approximately 75% identity, distributed unevenly (Fig. 2B
). Identities of 4793% were found between BCoV and MHV for 16 replicase protein sequences (Table 1
). The domain organization and end-products of proteolytic processing of pp1a/pp1ab were predicted from comparisons of BCoV and other, better-characterized coronaviruses (reviewed in Ziebuhr et al., 2000
; see also Kanjanahaluethai & Baker, 2000
; Ziebuhr et al., 2001
). The viral proteinases and the X, Cys-rich (GFL) and predicted (Rost et al., 1995
) membrane-spanning domains (TM1, TM2 and TM3) are derived from the pp1a part of the pp1ab polyprotein. The major replication enzymes, including the putative RNA-dependent RNA polymerase (RdRp) and RNA helicase (HEL), as well as Cys/His-rich putative Zn-binding domain (ZD) and nidovirus-specific conserved domain (ND), are derived from pp1b (Table 1
; Fig. 1A
).
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Amino acid differences between the two BCoV isolates are listed in Fig. 1. Within ORF1a, the isolates differed in 32 positions, six of which were non-synonymous and were located in the third (five mutations) and fourth (one) putative proteins (Fig. 1
; Table 1
). The third protein is the largest (1899 aa) replicase subunit in BCoV and is also known as p195 or p210 (p240) in other coronaviruses (Denison & Perlman, 1987
; Lim et al., 2000
; Schiller et al., 1998
). The replicase mutations were found within or proximal to the interdomain junctions involving the PL1pro, PL2pro, TM1 and TM2 domains (Fig. 1A
). The BCoV and MHV p195/p210 proteins are known to tolerate substantial changes including large deletions and insertions (compare the sizes of proteins in Table 1
; see also Fig. 2B
). Therefore, the predominant clustering of mutations within p195/p210 may be due to the rapid evolution of this protein. However, it is evident that only two of the five mutations, those at positions 910 and 2091, are located in the least conserved regions (hot spots) in p195/p210. Furthermore, the first and second proteins, which have not accepted mutations after the divergence of the LUN and ENT strains, have been shown to be the fastest evolving proteins in coronaviruses and specifically in the BCoV/MHV lineage (compare the percentages of identical residues in the first three proteins in Table 1
; see also Fig. 2B
; Lee et al., 1991
; A. E. Gorbalenya, unpublished). These observations suggest that certain amino acid differences observed between LUN and ENT p195/p210 may be meaningful because they have evolved under selective pressure. Mutation-induced changes in PL1pro/PL2pro functions might affect the expression pattern of the poorly characterized p195/p210 and flanking proteins, which are under the control of these proteases. Also, these mutations could affect expression of subgenomic mRNAs, which may be stimulated by a non-proteolytic activity associated with these proteases (Herold et al., 1999
; Tijms et al., 2001
). Mutations in the vicinity of transmembrane domains, which are thought to form a scaffold for the RNA-synthesizing machinery (Gorbalenya et al., 1989
; Shi et al., 1999
; van der Meer et al., 1999
), might affect the biogenesis and/or functions of this multisubunit structure.
Downstream of the replicase genes, the LUN and ENT isolates differed in 59 positions, 18 of which were non-synonymous and were scattered over all proteins (Fig. 1B). Comparison of all available BCoV nucleotide sequences revealed that only the amino acid change at position 179 (Arg for LUN and Gln for ENT) of the S protein may be specific for respiratory versus enteric isolates, as has been demonstrated for transmissible gastroenteritis virus (Almazán et al., 2000
; Ballesteros et al., 1995
, 1997
; Krempl et al., 1997
; Rasschaert et al., 1990
; Sanchez et al., 1999
).
Previous work with BCoV and other coronaviruses has suggested that S also plays an important role in virus-induced membrane fusion and plaque size (Chambers et al., 1990 ; Gallagher et al., 1991
; Kubo et al., 1993
; Routledge et al., 1991
; Sturman et al., 1990
; Yoo et al., 1991
). Interestingly, when the LUN and ENT strains were assayed on HRT-18G cells at 48 h after infection, the LUN strain caused extensive cell-to-cell fusion and formed two- to threefold larger plaques than did the ENT strain (data not shown). Single amino acid changes within S are prime candidates for causing changes in virus-induced cell fusion and plaque size. However, other strain-specific mutations may also contribute to these phenotypes.
The genome structures of BCoV reported here extend the list of known coronavirus genomes (Boursnell et al., 1987 ; Eleouet et al., 1995
; Bonilla et al., 1994
; Bredenbeek et al., 1990
; Herold et al., 1993
; Lee et al., 1991
). A theoretical analysis of amino acid differences between the LUN and ENT isolates indicated that the replicative protein p195/p210 and structural protein S might control strain-specific properties. Current work is focusing on the evaluation of the relative importance of the isolate-specific mutations of BCoV in cell culture and in the natural host through the construction and use of specific LUN/ENT recombinant viruses.
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Acknowledgments |
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Footnotes |
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References |
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Received 11 May 2001;
accepted 30 August 2001.