1 Institute of Virology and Immunology, University of Würzburg, Würzburg, Germany
2 Research Department, Cantonal Hospital St Gallen, CH-9007 St Gallen, Switzerland
3 Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, UK
Correspondence
Volker Thiel
volker.thiel{at}kssg.ch
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ABSTRACT |
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T. H. and E. S. contributed equally to this work.
Published online ahead of print on 19 March 2004 as DOI 10.1099/vir.0.80044-0.
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INTRODUCTION |
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Although the knowledge of coronavirus replication at the molecular level is still rudimentary, the general principles of coronavirus replication have been elucidated (Lai & Holmes, 2001; Sawicki & Sawicki, 1998
; Thiel et al., 2003a
; Ziebuhr et al., 2000
). Coronavirus gene expression starts with the translation of the replicase gene from the infectious genomic RNA. The replicase gene comprises two large open reading frames (ORFs), designated ORF1a and ORF1b, that are located at the 5' end, and extend over more than two-thirds of the genome. The upstream ORF1a encodes a polyprotein of 450500 kDa, termed polyprotein (pp) 1a, whereas ORF1a and ORF1b together encode pp1ab (750800 kDa), which is synthesized by a (1) ribosomal frameshift during translation. Virally encoded proteinases, namely papain-like and 3C-like proteinases, process the coronavirus polyproteins pp1a and pp1ab to generate at least 15 or 16 end-products and an unknown number of intermediate products. These non-structural proteins (nsp) assemble to form the functional replication/transcription complex in the cytoplasm of the infected cell. The replication/transcription complex mediates the replication of the coronavirus genomic RNA and the transcription of multiple subgenomic mRNAs. Although it has been shown that the replicase gene products suffice for coronavirus transcription (Thiel et al., 2001b
), it is still a matter of debate whether the replicase gene products may also suffice for coronavirus genome replication.
A recent analysis of coronavirus replicase genes by comparative genomics revealed a new view of the (putative) enzymic functions encoded by coronaviruses and identified a number of conserved replicase domains that remain uncharacterized (Snijder et al., 2003). Thus, the list of potential targets for antiviral intervention, such as the viral RNA-dependent RNA polymerase, helicase and proteases, has been extended to newly predicted enzymes that might be associated with RNA processing and metabolism. However, since the function of most coronavirus replicase gene products is still unknown, the identification of antiviral drugs that interfere with coronavirus replication will, most probably, be dependent on random screening of compound libraries. In contrast, the rational design of antivirals may be limited to a few well-characterized target enzymes (Anand et al., 2002
, 2003
; Davidson & Siddell, 2003
; Tanner et al., 2003
; Thiel et al., 2003a
; Yang et al., 2003
).
The recent development of reverse genetic systems for coronaviruses (Almazan et al., 2000; Casais et al., 2001
; Thiel et al., 2001a
; Yount et al., 2000
, 2002
, 2003
) provides an opportunity to carry out an extensive biological characterization of the viral replicative proteins and functions. Hopefully, these analyses will elucidate the unique features of coronavirus replication at the molecular level. In the long term, these studies may lead also to the development of new strategies to prevent and control coronavirus infection. In the short term, however, the reverse genetic systems can be used to facilitate the identification of coronavirus replicase inhibitors. For example, the concept of using autonomously replicating RNAs (replicon RNAs) has been explored in a number of positive-strand RNA virus systems and has led to the establishment of novel antiviral screening assays (Bartenschlager, 2002
; Frolov et al., 1996
; Khromykh, 2000
; Lo et al., 2003
; Randall & Rice, 2001
). Stable cell lines containing non-cytopathic, selectable replicon RNAs are currently used to assess the efficacy of candidate inhibitors of viruses that cannot be propagated efficiently in tissue culture, such as hepatitis C virus (Bartenschlager, 2002
; Randall & Rice, 2001
). Moreover, since no structural genes and, therefore, no infectious viruses are formed, replicon-based assays represent an attractive tool for the identification of antivirals if the pathogenicity of the virus is a concern.
We report here the generation and analysis of the first selectable, coronavirus-based replicon RNA. We have introduced the gene for a selectable marker into the HCoV 229E replicase gene and we have replaced three structural genes, namely the S (spike), E (envelope) and M (membrane) genes, and two genes of unknown function (genes 4a and 4b), by a reporter gene encoding green fluorescent protein (GFP). Our data indicate that this approach facilitates the rapid and biosafe evaluation of candidate coronavirus replicase inhibitors.
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METHODS |
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Cloning of plasmid DNAs and recombinant viruses.
Recombinant VV vHCoV-inf-1 containing the full-length cDNA of HCoV 229E (Thiel et al., 2001a) was used as a basis to construct the recombinant VV vHCoV-Rep-1 cDNA encoding the HCoV 229E-based replicon RNA. The construction of vHCoV-Rep-1 involved several steps of VV-mediated recombination using the E. coli guanine phosphoribosyltransferase (gpt) gene as a marker for positive or negative selection (Isaacs et al., 1990
; Kerr & Smith, 1991
).
To perform VV-mediated homologous recombination, the plasmid DNAs described below were constructed using standard procedures. The precise details, plasmid maps and sequences are available from the authors upon request.
To construct the plasmid pGPT-1 a 2·1 kb HpaISphI fragment, derived from pTM-3 (Elroy-Stein et al., 1989) and encoding the gpt gene downstream of a VV promoter, was inserted into the SmaI restriction site of pBluescriptII KS+ (Stratagene). Plasmid pRec-1 is based on pBR322 (New England Biolabs) and a DNA insert has been cloned between the EcoRIEagI sites of pBR322 comprising the HCoV 229E-derived nucleotides 1960121145, a 2·1 kb EcoRIXbaI fragment derived from pGPT-1 (containing the gpt gene) and HCoV 229E-derived nucleotides 2420125874. pVec-GN is based on pBR322 and a DNA insert has been cloned at the BamHI site of pBR322 comprised of the HCoV 229E-derived nucleotides 1948520568, one C nucleotide, the GFP gene and the HCoV 229E-derived nucleotides 2565427273. pRec-2 is based on pGPT-1 and contains a 500 bp HindIIINotI fragment derived from VV vNotI/tk (Merchlinsky & Moss, 1992
), the 2·1 kb fragment derived from pTM-3 and HCoV 229E-derived nucleotides 21773323. pTNI is based on pRec-2 and contains the 500 bp HindIIINotI fragment derived from VV vNotI/tk, the bacteriophage T7 RNA polymerase promoter, one G nucleotide, the HCoV 229E-derived nucleotides 1615, the sequence encoding the TaV 2A-like element (Donnelly et al., 2001
), the neo gene derived from pTET/ON (Clontech), the EMCV-IRES element derived from pTM-3 and the HCoV 229E-derived nucleotides 6263323.
VV-mediated homologous recombination was done as follows. CV-1 cells (5x105) were infected (m.o.i.=1) with the respective recombinant VV indicated below followed by transfection of 5 µg plasmid DNA 1 h post-infection using Lipofectin transfection reagent (Invitrogen). Two days later, the cells were harvested and recombinant VV were isolated by three rounds of plaque purification under gpt-positive or negative selection as appropriate. In order to isolate gpt-containing VV, gpt-positive selection was done on CV-1 cells in the presence of mycophenolic acid (25 µg ml1; Sigma), xanthine (250 µg ml1; Calbiochem) and hypoxanthine (15 µg ml1; Calbiochem); gpt-negative selection was done on D980R cells in the presence of 6-thioguanine (1 µg ml1; Sigma). The identity of all recombinant VVs were confirmed by Southern blot and sequencing analysis.
The insertion of the GFP gene into the HCoV 229E-derived cDNA of vHCoV-inf-1 was done in two steps as follows. First, recombinant VV vRec-1 was isolated after homologous recombination of vHCoV-inf-1 with plasmid DNA pRec-1 using gpt-positive selection. Second, recombinant VV vVec-GN was isolated after homologous recombination of vRec-1 with plasmid DNA pVec-GN using gpt-negative selection. As a result of this cloning procedure the HCoV 229E genes S, 4a, 4b, E and M were replaced by the GFP gene.
The insertion of the sequence encoding the TaV 2A-like element, the neo gene and the EMCV-IRES element at the sequence encoding the HCoV 229E nsp1/nsp2 polyprotein cleavage site was done in two steps as follows. First, recombinant VV vRec-2-GN was isolated after homologous recombination of vVec-GN with plasmid DNA pRec-2 using gpt-positive selection. Second, recombinant VV vRep-1 was isolated after homologous recombination of vRec-2-GN with plasmid DNA pTNI using gpt-negative selection. The recombinant VV vRep-1 represents the source of template DNA used to generate the replicon RNA Rep-1 (Fig. 1).
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Analysis of replicon RNA-containing BHK cells.
Individual cell clones obtained after G418 selection of Rep-1 RNA-transfected BHK-21 cells were analysed by fluorescence microscopy using a Leica DM R fluorescence microscope and Leica IM 1000 software. Flow cytometry analysis was done using a FACSCalibur and CellQuest software (BD Pharmingen).
Poly(A)-containing RNA from BHK-Rep-1 cells or HCoV 229E-infected MRC-5 cells was isolated using oligo(dT)25 Dynabeads (Dynal) (Thiel et al., 1997). Northern blot analysis of poly(A)-containing RNA involved electrophoresis and transfer to nylon membranes as described (Thiel et al., 2003a
). To detect RNAs derived from HCoV 229E and Rep-1, a 32P-labelled probe corresponding to the HCoV 229E nucleotides 2629727273 was produced using the Multiprime DNA-labelling system (Amersham). A complete sequencing analysis of re-isolated Rep-1 RNA from BHK-Rep-1 cells was done by standard RT-PCR sequencing methods (Thiel et al., 2003a
).
Inhibition of coronavirus replication.
BHK-Rep-1 cells were seeded out in 96-well plates at a concentration of 5x103 per well in MEM medium containing 10 % FCS and 400 µg G418 ml1; 24 h later, the medium was replaced by fresh medium without G418 and graded doses of IFN-, ribavirin or glycyrrhizin (all purchased from Sigma) were added. As the parental BHK-21 cells are derived from hamster kidneys, an IFN-
hybrid constructed from recombinant human interferons
A and
D that crosses the species barrier was used for the assay (Sigma, product no. I 4401). An untreated well served as control. Tissue culture media and inhibitors were replenished after 24 h and 48 h, and the cells were harvested and analysed by flow cytometry on day three. Inhibition of reporter gene expression was calculated as reduction of the mean fluorescence intensity (MFI) of GFP-positive cells by setting the MFI of untreated BHK-Rep-1 cells as 0 % inhibition and the complete down regulation of GFP to background levels as 100 % inhibition. Cells that were subjected to fluorescence microscopy were harvested on day two, transferred to glass slides (Falcon) and cultivated for another 24 h.
Cytotoxicity of candidate inhibitors was assayed using BHK-21 cells that were grown in MEM medium containing 10 % FCS. Cell proliferation was analysed 2 and 3 days after treatment with inhibitors using the CellTitre 96 AQueous One kit (Promega).
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RESULTS |
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Generation and analysis of a stable cell line containing HCoV replicon RNA
To clone a cDNA encoding the HCoV replicon RNA, we modified the full-length HCoV 229E cDNA, which is cloned and propagated in a VV vector (Thiel et al., 2001a). VV-mediated homologous recombination was used to replace the structural genes S, E and M and non-structural genes 4a and 4b by the GFP gene as well as to insert the TaV-2A/neo/EMCV-IRES sequence. The genomic DNA of the resulting recombinant VV was used as template for in vitro transcription to synthesize the replicon RNA, designated Rep-1. Two days after transfection of Rep-1 RNA into BHK-21 cells, green fluorescent cells could be detected, which is indicative of replicon RNA-mediated GFP expression. To establish a stable cell line containing autonomously replicating Rep-1 RNA (Fig. 1b
), transfected cells were selected with G418. Several clones displaying green fluorescence have been obtained and analysed for the presence of replicon RNAs. Northern blot analysis of poly(A)-containing RNA derived from several individual clones revealed the presence of full-length Rep-1 RNA and Rep-1-derived transcripts encoding GFP and N (Fig. 2a
). GFP expression of one clone, designated BHK-Rep-1, has been analysed in detail by fluorescence microscopy and flow cytometry for a period of 4 months (over 50 passages under G418 selection). Throughout this period the percentage of green fluorescent cells remained at a constant level of 4060 % (Fig. 2b, c
). Furthermore, BHK-Rep-1 cells that have been stored in liquid nitrogen and re-cultured still displayed green fluorescence indistinguishable from cells that have been passaged continuously. A complete sequence analysis of the replicon RNA, re-isolated from BHK-Rep-1 cells that had been passaged for 2 months in tissue culture, showed five nucleotide changes that were not encoded in the Rep-1 cDNA (Table 1
). The nucleotide changes are located in the replicase gene (in the coding sequences of nsp1 and nsp2), in the EMCV-IRES element (two changes) and, interestingly, in the N gene (one change). Since these nucleotide changes were not encoded by the cloned cDNA, they, most likely, have been acquired during replication in tissue culture and thus indicate the capability of autonomous replication of Rep-1 RNA. Whether they contribute to the establishment of autonomously replicating Rep-1 RNA in BHK cells remains to be elucidated.
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DISCUSSION |
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In order to provide a convenient and safe assay for the identification of coronavirus replicase inhibitors, we have established a protocol that is based on the use of replicon RNAs generated by reverse genetic techniques. The replicon RNAs have been modified by the introduction of the neomycin-resistance gene into the replicase gene and the introduction of a downstream reporter gene (GFP) that is expressed via the replicase-mediated synthesis of a subgenomic mRNA. This strategy enabled us to select for stable cell lines containing coronavirus-derived, autonomously replicating RNAs that mediate the expression of GFP as a marker for coronavirus replication.
Our results clearly demonstrate that the coronavirus replicon system facilitates the identification of replicase inhibitors in tissue culture. We were able to show that IFN- greatly reduces the replicon-mediated GFP expression in our system. This finding confirms a study by Cinatl et al. (2003b)
, who showed that IFN-
effectively inhibited SARS-CoV replication in tissue culture. The observed inhibition of coronavirus replication by IFN-
in our assay reached a maximum of about 80 %, indicating that a small number of cells still displayed green fluorescence. Whether these cells do not respond to IFN-
-treatment or may contain resistant replicon RNAs remains to be determined.
Our finding that ribavirin only moderately affected coronavirus replication is also in agreement with reports showing that SARS patients may not benefit from ribavirin treatment and that ribavirin does not inhibit SARS-CoV replication in tissue culture (Booth et al., 2003; Cinatl et al., 2003a
; So et al., 2003
). Although it has been claimed that ribavirin forces some RNA viruses into error catastrophe (Crotty et al., 2000
, 2001
), our data further support the notion that coronaviruses may be resistant to this drug. The mechanisms of this resistance are unclear but may be linked to a recently identified 3'5' exonuclease domain (ExoN) encoded by coronaviruses (Snijder et al., 2003
). It has been proposed that the ExoN activity may be linked to mechanisms such as RNA proofreading, repair and/or recombination which ensure the genetic stability of the extraordinarily large coronavirus RNA genome. Although this idea has still to be proven, our data that re-isolated replicon RNAs after 2 months in tissue culture showed only five nucleotide changes already indicate a low error frequency of coronavirus replication.
Unlike IFN- and ribavirin, there are few clues to the antiviral mechanism of glycyrrhizin. Our data indicate that, as for ribavirin, glycyrrhizin only moderately affects coronavirus replicase functions. However, in contrast to ribavirin, glycyrrhizin has been shown to inhibit SARS-CoV replication in tissue culture (Cinatl et al., 2003a
). This indicates that glycyrrhizin may not target the coronavirus replication machinery and that antiviral effects may be exerted, for example, during virus adsorption or release.
The replicon system we describe here can be used to screen for coronavirus inhibitors that display antiviral activity against cellular and viral targets involved in viral RNA replication, including proteases, RNA-dependent RNA polymerase, NTPase/helicase and various (putative) functions that have been identified recently [i.e. poly(U)-specific endonuclease, ExoN, S-adenosylmethionine-dependent ribose 2'-O-methyltransferase, adenosine diphosphateribose 1''-phosphatase and cyclic phosphodiesterase]. In addition, the impact of drugs that modify the host cell can be studied in detail. However, because the structural genes S, E and M are not included in the replicon RNA, the assay does not cover cellular and viral targets that are involved in receptor binding, virus entry, genome encapsidation and virus release. On the other hand, since no infectious virus is formed, the assay represents a safe protocol that can be performed in biosafety level 2 laboratories. Another advantage of the coronavirus replicon system is that HCoV 229E vector and replicon RNAs are able to mediate reporter gene expression in a variety of human cells. Thus, the efficacy of candidate inhibitors can be evaluated in human cells from different tissues, irrespectively of their susceptibility to SARS-CoV infection in tissue culture. Furthermore, it should be noted that the replicon system could be further improved, since the expression of other reporter proteins that would improve sensitivity, such as chloramphenicol acetyltransferase or firefly luciferase, is compatible with coronavirus-based transcription (Thiel et al., 2003b).
The replicon-based system will also facilitate the analysis of virus resistance as it may occur under selection pressure. For example, once an efficient inhibitor has been identified, the replicon cell line can be used to select for drug-resistant cells under continuous inhibitor and G418 treatment. Replicon RNA, re-isolated from such cells, can then be analysed for marker mutations related to drug resistance. The detection of such mutations would provide valuable information on the viral target protein(s) and the mechanism of inhibition if the basis of antiviral activity of a particular compound is unknown.
The generation of the first selectable coronavirus replicon RNA represents a novel system and, clearly, there are a number of questions that have to be addressed in future studies. For example, do coronavirus replicon RNAs display any cytotoxicity? Do the observed nucleotide changes in the re-isolated replicon RNA represent adaptive mutations necessary for continuous replication in selected cell lines? Using HCoV-229E vector RNAs we have observed heterologous gene expression for at least 1 week in tissue culture without any indication for vector-related cytotoxicity (unpublished data). On the other hand, the amount of replicon RNA in stable BHK-Rep-1 cell lines is at least 10-fold reduced compared with the amount of viral genomes in coronavirus-infected cells (Fig. 2a), indicating that (co-) selection (replicon/cell) may have taken place. Another important question is whether any host factors are required for coronavirus replication and/or transcription. Are there, for example, eukaryotic cells that are not permissive for coronavirus replication? Studies aimed to identify and analyse such cell lines are currently being performed in our laboratory.
Phylogenetic, biochemical and structural data indicate that the mechanisms and enzymes involved in coronavirus genome expression are, to a high degree, similar amongst different coronaviruses (Anand et al., 2003; Gonzalez et al., 2003
; Snijder et al., 2003
; Thiel et al., 2003a
; Yang et al., 2003
; Ziebuhr et al., 2000
). We therefore predict that inhibitors identified using the HCoV-229E replicon system described here will, most likely, also be effective against SARS-CoV replication. Nevertheless, our data should encourage the development of SARS-CoV replicons that would allow the identification of inhibitors that are specific for SARS-CoV. A reverse genetic system for the construction of SARS-CoV replicons is available (Yount et al., 2003
), and the strategy we have used to generate selectable HCoV 229E replicons should be fully applicable to SARS-CoV.
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ACKNOWLEDGEMENTS |
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Received 18 February 2004;
accepted 5 March 2004.