Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo
© Pfefferle et al; licensee BioMed Central Ltd. 2009
Received: 30 July 2009
Accepted: 24 August 2009
Published: 24 August 2009
During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells).
In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection in-vivo, ORF 7b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the SARS epidemic.
The severe acute respiratory syndrome (SARS) emerged in the end of 2002 in China and caused an international epidemic . Its causative agent, a hitherto unknown Coronavirus (CoV) is thought to have been circulating in an animal reservoir before it crossed species barriers into humans [2–7]. Bats have been implicated as the original reservoir of all CoV, and the large range of relevant human and animal CoV has been suggested to be resulting from host switching events [8–16].
In the context of viral host switching, it is interesting that several SARS-CoV proteins encoded on subgenomic (sg) RNAs seem to be dispensable for virus replication in cultured cells of primate or rodent origin, as well as in rodent models [17–19]. Because these ORFs are not shared between different CoV groups, they are referred to as group-specific ORFs . Proteins encoded by group-specific ORFs have been shown to influence pathogenesis, virus replication, or host immune response [17, 20–24]. During the human SARS epidemic, SARS-CoV has rapidly acquired deletions in several of its group-specific ORFs [7, 25–27]. The original functions of associated proteins might exemplify mechanisms through which highly pathogenic zoonotic viruses such as the SARS-CoV can persist in their reservoirs without causing disease.
The characterization of virus proteins can be unreliable if only the protein of interest is studied on its own. The study of proteins in the whole virus context reflects virus-host interactions more realistically, and takes into account intraviral protein interactions. Such experiments can be done using reverse genetics techniques which for most plus-strand viruses rely on cloned cDNA copies of the whole RNA genome that can be mutagenized in-vitro [28–30]. Different approaches have been followed to implement CoV reverse genetics. A great challenge in this regard is the huge size of the CoV genome, making cloning procedures difficult because plasmid-based cDNA constructs are instable in E. coli. In-vitro ligation of subgenomic cDNA fragments without the assembly of full-length plasmids has been successfully used to establish CoV reverse genetics [31–33]. As an alternative, full-length cDNA copies have been reconstructed and kept in vaccinia virus [34, 35]. A third approach is based on bacterial artificial chromosomes (BAC) for keeping full-length CoV cDNA stable, owing to a low copy number of BAC DNA per E. coli cell [36–39]. The first two systems use T7 RNA polymerase promoter-driven in-vitro transcription of capped, infectious RNA that is transfected into cells. The latter uses a CMV promoter and relies on the transfection of full-length cDNA into cells, which is then transcribed in the nucleus into infectious RNA. In this study we have implemented a modified approach to CoV reverse genetics by cloning the entire SARS-CoV genome downstream of a T7 RNA polymerase promotor in a BAC. Using the linearized BAC construct as a template for in vitro transcription, this system combines plasmid-based handling of cDNA constructs with direct delivery of genome-like RNA into the cytoplasm.
The novel system was used to characterize a 45 nucleotide in-frame deletion in ORF 7b that is present in the primary isolate of SARS-CoV prototype strain Frankfurt-1 . This specific deletion is not present in any other of > 150 SARS-CoV ORF 7b sequences in GenBank, and in none of the SARS-like bat CoV. However, deletions of the whole ORF 7b and beyond have been acquired by SARS-CoV during the SARS epidemic in humans [25–27].
The ORF 7b protein is a 44 amino acid protein that is transcribed by a noncanonical leaky scanning mechanism from the second ORF encoded on subgenomic RNA 7 [20, 40]. The protein is a type III integral transmembrane protein located in the Golgi compartment . It has been shown previously that the protein is a structural virion component, that it is dispensable for replication in various cell cultures, and that it induces apoptosis in cultured cells if overexpressed [18, 40]. The pro-apoptotic effect seems to be limited to late stages of the apoptotic cascade . Qualitatively the same effect was confirmed in studies on a recombinant virus, containing a combined deletion of ORF 7a and ORF 7b . However, it is unclear to what extent either the ORF 7a or ORF 7b proteins, respectively, contribute to the effect. It is also unclear to what degree the ORF 7b protein alone influences virus replication in-vivo. This is relevant for the Frankfurt-1 virus because it has been used as a model virus in several studies on pathogenesis and antiviral drug research (e.g [42–45]). Finally it is unclear whether the Frankfurt-1 ORF 7b deletion has been acquired during cell culture, or whether it may have been present already in the patient and may have undergone transmission.
In this study, primary clinical samples from the Frankfurt index patient and a secondary case who acquired her infection from him were re-analyzed. Frankfurt-1 viruses with and without the deletion were then reconstructed by reverse genetics. Effects of the deletion on interferon induction and response, on induction of apoptosis, and on in-vivo replication in Syrian Golden hamsters were determined.
Origin of the ORF 7b deletion
Expression of ORF 7b but not ORF 7b with the 45 nt deletion induces apoptosis and the type I interferon response
To examine the effect of the ORF 7b deletion on the type I IFN response, reporter gene assays were performed. Cells were transfected with plasmids encoding ORF 7a, ORF 7b or ORF 7b del, respectively. All cells were co-transfected with the pHISG-54 reporter plasmid containing the firefly luciferase gene under the control of the ISRE region of the human IFN-stimulated gene 54. Expression plasmid pRL-SV40 encoding Renilla luciferase was co-transfected to normalize for interferon-independent stimulation of transcription. Twenty-four hours later, the cells were infected with SeV to induce IFN-mediated reporter gene expression. Cells were lyzed 24 h post infection and subjected to reporter gene assays. As shown in Figure 2B, expression of both ORF 7a and ORF 7b but not ORF 7b del induced IFN-dependent reporter gene expression. In those cultures superinfected with SeV, none of the plasmids reduced the SeV-associated, IFN-dependent reporter gene expression. The Ebolavirus VP35, a known antagonist of interferon induction, clearly showed reduction of reporter gene expression if used in the same system (Figure 2B) [48, 49].
These distinct findings prompted us to elucidate 7b protein functions in the natural virus context. To be able to measure even marginal phenotypical differences we decided to reconstruct both genotypes while establishing a novel reverse genetic system.
Construction of a full-length infectious cDNA clone
The linearized BAC cDNA and a PCR product containing the nucleocapsid gene were in-vitro transcribed and co-transfected in BHK cells. Because BHK cells did not support SARS-CoV replication, supernatants were transferred to Vero cells susceptible for SARS-CoV infection. Virus progeny was identified by immunofluorescence analysis with anti-SARS-CoV patient serum after 24 h (Figure 4B), as well as by plaque assays after 48 h (Figure 4B). Electron microscopy showed intracellular structures compatible with sites of virion assembly as well as mature virus particles (Figure 4C).
The 7b protein is expressed in cells during SARS-CoV infection
It was concluded that the ORF 7b protein of the recombinant viruses was expressed in infected cells, and that its principal properties are not affected by a C-terminal flag-tag epitope. These findings, including the pattern of fluorescence when expressing ORF 7b, were consistent with earlier reports by Pekosz et al [18, 40].
The deletion in ORF 7b enhances growth of virus in cell culture
Growth properties of rSCV and r7bΔTMD on different cell lines were compared. Plaque morphology was determined for both viruses, with no discernible differences (Figure 5B). Because plaque assay could only show cells that die from virus infection, the same experiment was repeated and read out by immunofocus assay, using serum of a human SARS survivor. There was no difference in immunofocus morphology (Figure 5B).
Growth curves in three different cell cultures were determined next. Virus RNA was measured in supernatant by real-time RT-PCR. A multiplicity of infection (MOI) of 0.001 was used for both recombinant viruses in Vero and CaCo-2 cells. For HuH7 cell, an MOI of 0.01 was used, due to their lower susceptibility to SARS-CoV infection. In Vero cells, very similar increases in RNA concentration were observed with both viruses during 48 hours (Figure 5C). In CaCo-2 and HuH7 cells, respectively, r7bΔTMD accumulated about 4- and 6-fold more RNA than rSCV. It was concluded that the deleted virus had a slight but reproducible growth advantage in the latter cell lines. In the absence of mechanisms of adaptive immunity, replication of RNA viruses is controlled by production of and response to type-I interferons, as well as apoptosis of infected cells. Taking into account our findings in overexpression experiments, central elements of these systems were therefore examined in cells infected with both virus variants.
ORF 7b is not involved in the ablation of interferon induction observed during SARS-CoV infection
ORF 7b does not interfere with interferon alpha production
HEK 293-lp cells were used to study release of interferon alpha in the supernatants of infected cells. It has been reported by Spiegel et al. that interferon alpha expression is induced in SARS-CoV-infected 293-lp cells to a certain level . Exactly the same cells were obtained from F. Weber, University of Freiburg, and interferon alpha transcription after infection with SARS-CoV was qualitatively confirmed by RT-PCR in our laboratory (not shown). The level of interferon alpha was then determined by EIA in supernatant of 293-lp cells, 48 h after infection of both viruses at an MOI of 5. As shown in Figure 7A, infection with the control virus NDV elevated the interferon alpha level in supernatant by a factor of 3, while neither rSCV nor r7bΔTMD caused detectable secretion.
Virus with the deletion in ORF7b has a slight replicative advantage in cells pretreated with interferon beta
To study the effects of interferon on replication of both viruses, Vero cells were pre-treated with increasing concentrations of interferon beta in order to induce an antiviral state. Cells were infected with either rSCV or r7bΔTMD at an MOI of 0.001. As shown in Figure 7B, r7bΔTMD replicated to marginally higher virus concentrations than rSCV in presence of interferon (up to 3.4 fold increase). Since in our hands CaCo-2 cells were more resistant to interferon beta pre-treatment than Vero cells, experiments were repeated with higher concentrations of interferon using CaCo-2 cells. More efficient replication (up to 7.9-fold increase) was again observed for r7bΔTMD (Figure 7B).
The deletion in ORF 7b does not alter the capability of virus to induce apoptosis in cell culture
The deletion in ORF 7b confers a significant replicative advantage in Syrian golden hamsters
Virus replication levels in hamster lungs
Virus replication (mean* of N animals)
(Virus titer, RNA concentration, N animals)
(Virus titer, RNA concentration, N animals)
Day 1 p.i.
1.04 × 10 7 (9.00 × 106 - 1.2 × 107) PFU/g
9.86 × 10 8 (2.4 × 107 - 1.8 × 108) PFU/g
6.65 × 10 7 ( 1.84 × 107 - 2.4 × 108) copies/g
1.53 × 10 9 (9.98 × 108 - 2.2 × 109) copies/g
n = 2
n = 3
Day 3 p.i.
9.86 × 10 6 (8.00 × 105 - 1.20 × 108) PFU/g
1.63 × 10 8 (1.00 × 108 - 3.6 × 108) PFU/g
4.27 × 10 8 (1.68 × 108 - 2.1 × 109) copies/g
7.70E × 10 8 (7.50 × 108 - 8.10 × 108) copies/g
n = 3
n = 3
The replication advantage for r7bΔTMD was in concordance with findings in CaCo-2 and HuH-7 cell cultures (Figure 5).
In the present study we have characterized a naturally-acquired deletion in the ORF 7b of the primary SARS-CoV Frankfurt-1 isolate by reverse genetics. In contrast to other plus strand RNA viruses it has taken rather long to complete the first coronavirus reverse genetics systems [28, 30, 31, 34, 37, 54]. It has been difficult to clone complete CoV genomes due to their large sizes and toxicity or lability of constructs in E. coli [31, 34, 37]. This has been circumvented by Baric et al. by the use of subgenomic plasmids that are ligated in-vitro to full genomic cDNA, prior to transcription and electroporation . We tried this approach initially, but we failed to generate sufficient amounts of full-length cDNA for in-vitro transcription. Thiel et al. have described an approach to generating full-length cDNA by stepwise assembly of an entire coronavirus genome in a pox virus backbone . As we had not worked with pox viruses before, this technique appeared rather difficult to establish. As a third alternative, Enjuanes and coworkers have presented an approach based on cloning of the entire genome in BAC and transfecting the BAC-contained viral cDNA under the control of a CMV promoter . The use of BAC DNA provides the remarkable benefit of being able to handle full length genomic DNA in one plasmid backbone, using standard DNA cloning techniques. As demonstrated in several studies of that group [24, 36, 37, 55–57], BAC manipulations are rather fast and straightforward, while providing little opportunity for de-novo mutations resulting from DNA manipulation steps. In our strategy we used a bacteriophage T7-derived RNA polymerase promoter instead of the CMV promoter because we wanted to provide a genome that most closely resembled that of the virus, using cytoplasmic sites for replication and circumventing transcription and possible splicing in the nucleus [37, 38, 56]. A T7 promoter has not been used before with a plasmid-contained CoV cDNA genome; it was conceivable that leaky transcription might enhance underlying toxicity of CoV genomes in E. coli. Our study shows that the SARS-CoV genome is stable in BAC despite the T7 promoter. Interestingly, Enjuanes and colleagues have made BAC-based full length clones for different CoV and reported that their SARS-CoV BAC clone was more stable than, e.g., the one they developed for TGEV . The SARS-CoV genome may thus be more stable in E. coli than that of other CoVs. It remains to be seen whether combined T7/BAC infectious cDNA clones can also be constructed for other CoVs.
The 45 nucleotide in-frame deletion in the transmembrane domain of ORF 7b is a paramount feature of the Frankfurt-1 strain. This strain has been employed as a prototypic SARS-CoV in several studies on pathogenesis and antiviral therapy (e.g., [42–45]). By analysis of primary clinical samples from the patients treated in 2003 for SARS in Frankfurt, we could show that the mutation has been selected during initial isolation in cell culture, and that it did not stem from the Frankfurt index patient . Initial characterizations of the protein by overexpression experiments suggested reduced induction of interferon and apoptosis in association with the deletion, which led us to reconstruct the corresponding viruses with and without the deletion by reverse genetics. In concordance with earlier findings, type I interferon was neither induced nor produced by either SARS-CoV variant in our study [50, 58–61]. It is assumed that CoV either encode a range of proteins interacting with interferon sensing, or shield their RNA from immune recognition through the formation of double membrane vesicle-based replication compartments [60, 62–64]. Our experiments suggest that ORF 7b is not necessary for SARS-CoV counteraction against the induction of the interferon beta promoter. It also seems unlikely that ORF 7b contributes to the interference of SARS-CoV with secretion of interferon alpha . However, the deleted virus showed slightly decreased sensitivity to pretreatment of cells with interferon. This effect was remarkable since earlier studies only determined opposite (= evasive) effects on the interferon response for CoV accessory proteins. These include interference with the interferon signalling cascade in the case of SARS-CoV protein 6, or prevention of activation of interferon-sensitive genes for mouse hepatitis virus nucleocapsid protein [61, 65]. Here we observed an ORF 7b-dependent extension of the replication-attenuating effect of interferon. However, the additional extent of attenuation on top of the effect of interferon beta was of the same size as that observed in untreated cell cultures (compare Figure 5 and Figure 7) and did hardly increase with increasing interferon concentrations. This suggests an additive rather than a synergistic effect of ORF 7b and interferon on the attenuation of virus replication. In spite of the high relevance of the interferon response for controlling SARS-CoV replication, we should therefore assume that ORF 7b plays no role in the context of the type I interferon system [62, 66].
Apoptosis of target cells can limit virus infection in-vitro and in-vivo. Our initial overexpression experiments pointed to a strong pro-apoptotic effect of intact ORF 7b, which was in concordance with a study by Schaecher et al. who found that sgRNA 7-derived proteins activated caspase 3 if overexpressed . Complementary to their study, however, our experiments did not confirm any similar effect specifically for the ORF 7b protein in the full virus context. Schaecher et al. studied a recombinant virus with a double deletion of both ORF 7a and 7b, and this virus induced apoptosis clearly less efficiently than the parent full-length virus [18, 19]. The most likely explanation for the difference between both viruses is that the pro-apoptotic effect of gene 7 proteins observed by Schaecher et al. was contributed by ORF 7a rather than ORF 7b.
Even though the ORF 7b deletion in Frankfurt-1 was not affecting interferon and apoptosis systems, the virus with a deletion seems to have been selected during isolation in cell culture and shows a replicative advantage in two of three cell lines. This is remarkable because SARS-CoV variants with deletions in the ORF 7 (and also ORF 8) gene region have been transmitted and maintained in humans in the late phases of the 2003 epidemic [25–27]. It has never been formally addressed whether these viruses might have undergone particularly efficient transmission. We therefore determined whether the ORF 7b deletion in Frankfurt-1 conferred a replicative advantage in-vivo, using Syrian Golden Hamsters as a model of human SARS-CoV infection [52, 53]. Interestingly, the enhancing effect of the ORF 7b deletion was even more pronounced in hamsters than in cell culture. Hamsters infected with the deleted variant had significantly more virus RNA and a 95-fold increase of infectious virus titers in their lungs after 24 h. The rate of successful infections was 6/6 with the deleted virus and 5/6 with the full virus. In concordance with these observations, Roberts et al. have described ca. 10-fold more efficient replication of Frankfurt-1 in hamsters as compared to Urbani and HKU-39849 [52, 53]. Mortality in Hamsters was only observed with Frankfurt-1 (3 of 20 animals) but not Urbani and HKU-39849 [52, 53]. It was suggested that an amino acid exchange (L1148F) in the S2-domain of the spike protein of Frankfurt-1 against both Urbani and HKU-39849 might explain the difference. However, a replicative difference in extent similar to that reported by Roberts et al. was observed in our study between two variants of Frankfurt-1 that differed only by the ORF 7b deletion. As the deletion is not present in Urbani or any other prototype strain, this identifies the 7b protein as a potential attenuating factor within the genome of SARS-CoV.
We have seen in this study that the attenuating effect of ORF 7b was focused on the early phase of infection in-vivo. Because it has been suggested that delayed accumulation of high virus concentrations in infected patients has limited the spread of SARS-CoV in the population, it is tempting to speculate that the occurrence of viruses with deletions in the ORF7/8 region in the late phase of the 2003 epidemic might have added to the efficiency of virus transmission in humans [67–69]. It will be interesting in the future to investigate the exact mechanism of ORF 7b-dependent attenuation, and to determine whether this might contribute to the maintenance of virus in its natural reservoir.
Materials and methods
Cells and viruses
The original Vero cells on which Frankfurt-1 was primarily isolated (hereafter termed Vero FM, obtained from Jindrich Cinatl, Universtiy of Frankfurt), human hepatoma cells HuH7 (ATCC CCL-185), human colonic cancer cells CaCo-2 (ATCC HTB-37) and human embryonic kidney cells HEK 293-low passage (hereafter termed 293-lp, obtained from Friedemann Weber, University of Freiburg ) were maintained and grown in Dulbecco's modified Eagle medium (DMEM) containing 10% foetal calf serum (FCS, PAA, Pasching, Austria), 1 mM glutamine (PAA), 1 mM sodium pyruvate (PAA), 1% non-essential amino acids (PAA), 100 U/ml penicillin (PAA), and 100 μg/ml streptomycin (PAA). All experiments with 293-lp cells were performed between cell passage 42 and 48.
The SARS-CoV Frankfurt-1 isolate [2, 20, 70] was titrated on Vero FM cells. Sindbis virus (SV) derived from infectious cDNA clone pTOTO  was obtained from Beate Kümmerer, BNI, Hamburg and titrated on Vero FM cells. Sendai virus (SeV) strain Cantell was obtained from Christopher Basler, Mount Sinai School of Medicine, New York, propagated in 11-day-old embryonated chicken eggs, and titrated by standard hemagglutination test. Newcastle disease virus (NDV) strain PPMV-1/pigeon/Germany/R151 was obtained from the virus collection of the Friedrich Löffler Institute, Riems, Germany, and titrated on Vero FM cells.
Virus quantification by cell culture and RT-PCR
Plaque assays were done with Avicel overlays (RC581, FMC BioPolymer, Belgium) as described elsewhere . Immunofocus assay used the same overlay and was otherwise performed as described previously . Viral RNA quantification using in-vitro transcribed RNA standards was done as described previously .
General cloning and mutagenesis techniques
Standard cloning techniques were used. All gel purifications were done with the QIAEX II kit (Qiagen, Hilden, Germany). DNA constructs were electroporated into E. cloni (Lucigen, Middleton, USA) or Stbl 3 E. coli cells (Invitrogen, Karlsruhe, Germany). Prior to digestion with methylation-sensitive endonucleases plasmids were transformed in Sure cells (Stratagene, La Jolla, USA). BAC preparations were done with the Nucleobond®AX-kit (Macherey Nagel, Germany) as instructed. Plasmid-based inverse PCR was performed with QuikChange XXL kit (Stratagene, USA). PCR mutagenesis by overlap-extension PCR used Phusion® DNA polymerase and around 50 ng of input plasmid DNA. SARS-CoV coding sequence within constructs was fully sequenced after every mutagenic step.
Cloning of subgenomic plasmids
Total RNA was extracted from infected Vero cells with the Qiagen RNeasy kit. Using primers described by Yount et al. , cDNA fragments spanning the SARS-CoV genome were generated by RT-PCR using Superscript III reverse transcriptase and Expand High Fidelity DNA polymerase mixture. These primers inserted Bgl I restriction sites at fragment borders and a T7 promoter in front of the 5'end of the genome . In addition to the strategy described by Yount et al., a Not I restriction site was introduced downstream of the genomic poly-A tail. Figure 3 gives an overview of cloned fragments. Fragment A was cloned in two parts (A1 and A2, Figure 3), using primer Afwd 5'-TACTAATACGACTCACTATAGATATTAGGTTTTTACCTACCCAGG-3' and A1rev 5'-aatgccagtatgacctgagccaatatc-3' and A2fwd 5'-GATATTGGCTCAGGTCATACTGGCATT-3' and Arev 5'-ACACCATAGTCAACGATGCC-3'. After correction of errors both inserts were amplified from plasmids and used as templates in an overlap-extension PCR. A naturally existing Bgl I restriction site at genome position 1572 was thereby deleted. The extension product was subcloned in pSMART, resulting in clone pA. PCR products B, D, and E were cloned in pCR2.1 (Invitrogen). Fragments C and F were cloned in pSMART Low Copy Kanamycin vectors (Lucigen) after instability was observed in pCR2.1. A 45 nt deletion present in the Frankfurt-1 virus isolate (nt 27654 to 27699 in Genbank Accession No AY310120), was filled in by overlap-extension PCR. A region including restriction sites BamH I (genome position 26045) and Not I (following the 3'end of genome) was amplified from subclone pF in two halves using appropriate outer primers and overlap-extension primers 5'-TTTCTGCTATTCCTTGTTTTAATAATGCTTATTATATTTTGGTTTTCACTCGAAATCCAGGATCTAGAAG-3' and 5'-ATTATTAAAACAAGGAATAGCAGAAAGGCTAAAAAGCACAAATAGAAGTCAATTAAAGTGAGCTCATTC-3'.
The fragments were overlap-extended, digested with BamH I and Not I, and cloned back into the corresponding restriction sites in clone pF. All clones were verified by sequencing. Using the same technique, a DDDDK (flag-) tag sequence was introduced at the C-terminus of ORF 7b, with overlap-extension primers 5'-GATTACAAGGATGACGACGATAAGTAAACGAACATGAAACTTCTC-3' and 5'-CTTATCGTCGTCATCCTTGTAATCGACTTTGGTACAAGGTTCT-3'.
Assembly of full length BAC cDNA clone
BAC vector pBeloBAC11 was obtained from NEB, Boston, USA. The Nco I site at position 890 was oblated by primer extension mutagenesis, resulting in pBelodNco. The Not I-Not I multiple cloning site fragment was removed from pBelodNco and replaced by an oligonucleotide adapter containing Nsi I, BsaH I, Sph I and Not I restriction sites in sequence, resulting in pBeloAd4. Fragment A was amplified from plasmid pA with primers 5'-AGTAAT GGGCCC TAAGTACTAATACGACTCACTATAGATATTAGG-3' and 5'-ACACCATAGTCAACGATGCC-3', thereby introducing a PspOM I site upstream of the T7-promotor (Figure 3). The fragment was digested with PspOM I and Bgl I and ligated to the long EcoR I - Not I fragment of pBeloAd4 (pBeloAd4A, Figure 1). The 5'-most 3,062 nt were amplified from plasmid pB using primers 5'-GCCTATATGCATGGATGTTAGAG-3' and 5'-ATGAAT GCGGCCGC TACACTCAACACGTGTGGCACGC-3', thereby introducing a Not I site immediately downstream of the Mlu I site at position 7453. The PCR product was digested with Bgl I and Not I, gel purified, and ligated to the dephosphorylated short Not I - EcoR I fragment of pBeloAd4 (pBeloAd4B1, Figure 3). pBeloAd4A and pBeloAd4B were gel purified and ligated, resulting in quarter clone pAB, (Figure 3).
The 3'-most 5,536 nt were amplified from plasmid B3 in two parts, using primers 5'-TAGACTA CGCCGGCG TAGCCTTAGGTTTAAAAACAATTGCCACTC-3' and 5'-TACACTCAACACGTGTGGCACGATTGCGCT-3' (5'-part); and primers 5'-AGCGCAATCGTGCCACACGTGTTGAGTGTA-3'and 5'-TGAACCGCCACGCTGGCTAAACC-3' (3'-part), respectively. Both products were overlap-extended, resulting in a PCR product with a depleted Mlu I site at position 7453. The product also contained a Not I site upstream of the Bsu36 I site at position 6544, introduced by a primer 5'-overhang. The product was Not I and Bgl I digested and ligated to the long EcoR I - Not I fragment of pBeloAd4 (pBeloAd4B2, Figure 3).
Plasmid pD was digested with Bgl I and Bcl I (compatible to BamH I). The fragment was ligated to the dephosphorylated short BamH I - EcoR I fragment of pBelodNco (pBeloNcoD1, Figure 3). Fragment C was cut out of its pSMART vector with Bgl I and dephosphorylated, followed by ligation to pBeloNcoD1 and gel purification. This product was ligated to pBeloAd4B2, generating quarter clone pBCD.
The Acl I - Bgl I fragment of vector D-24-5 was ligated to the long EcoR I - BsaH I fragment of pBelodNco (Bsa HI is compatible with Acl I) (pBeloNcoD2, Figure 3). The dephosphorylated Pst I - Bgl I fragment of vector pE was ligated to the short Nsi I - EcoR I fragment of pBeloAd4 (Nsi I is compatible with Pst I) (pBeloAd4E1, Figure 3). This product was ligated with pBeloNcoD2 to yield quarter clone pDE.
The 2,793 bp SpH I - Bgl I fragment of subclone pE was ligated to the long EcoR I - SpH I fragment of pBeloAd4 (pBeloAd4E2, Figure 3).
The Bgl I - Not I fragment of plasmid pF was ligated to the short Not I - EcoR I fragment of pBeloAd4 (pBeloAd4F, Figure 1). This fragment was ligated with pBeloAd4E2, yielding quarter clone pEF.
Quarter clones pAB and pBCD were digested with Bsu36 I and PspOM I, the latter cut destroying the replicative element sopC. Fragments of interest were gel-purified and ligated to yield half clone pL. Quarter clones pDE and pEF were digested with Nco I. One Nco I cut was in the virus cDNA insert on each BAC, and the other in the sopC gene. Fragments of interest were purified and ligated to yield half clone pR. Half clones were digested with Mlu I and PspOM I. Fragments of interest were gel purified and ligated into the full length clone prSCV.
Rescue of recombinant virus
Full-length BAC clones were linearized with Not I, extracted with phenol-chloroform, and transcribed with the mMessage-mMachine® T7 (Ambion, USA) at an input of 1 μg of DNA per 20 μl reaction. A PCR product spanning the nucleocapsid reading frame and the genomic 3'-prime end was generated with primers N-fwd (5'-GGCC ATTTAGGTGACACTATAG ATGTCTGATAATGGACCCCAATC), the underlined sequence representing an SP6 promoter) and Frev (5'-TTTTTTTTTTTTTTTTTTTTGTCATTCTCCTAAGAAGC-3'). The product was purified and transcribed with mMessage-mMachine SP6 kit. Transcripts from both in-vitro transcription reactions were quantified photometrically. Genomic transcripts and N transcripts were co-electroporated at a 10:1 ratio into 107 BHK-21 cells, using a GenePulser instrument (Biorad, Germany) with two pulses of 1.5 kV, 25 μF and maximal resistance. Cells were left at room temperature for 10 minutes and seeded in 75 cm2 flasks. In a biosafety-4 laboratory, electroporated BHK-21 cells were incubated at 37°C for 24 hours. Supernatants were serially diluted and transferred to Vero cells. Using 1% SeaPlaque® agarose overlay (Biozym, Germany), three rounds of plaque purification were performed for each recombinant virus.
Analysis of mutations in ORF 7b
To distinguish between the two genotypes, two different RT-PCRs were performed. RT-PCR 1 used primers 27500 fwd (5'-CAGCTGCGTGCAAGATCAGT-3') and 27900 rev (5'-CCCTAGTGTTGTACCTTACAAG-3'), thus comprising ORF 7b and yielding a 400 bp fragment for rSCV, while giving a 355 bp fragment for r7bΔTMD. For RT-PCR 2 the identical reverse primer was used but the binding site of the sense primer 27690 fwd (5'-TAGCCTTTCTGCTATTCCTTGT-3') was placed in ORF 7b, recognising the 45 nt only present in rSCV but deleted in r7bΔTMD, hence a PCR product was only obtained for rSCV.
Cloning of ORF 7a, 7b and 7b del
ORF 7a (nts 27258 to 27626 of SARS-CoV genome GenBank accession number AY310120) was amplified using primers 5'-CACCATGAAAATTATTCTCTTCCTGACA-3' (fwd) and 5'-TCATTCTGTCTTTCTCTTAATGGT-3' (rev), and cloned into pCDNA 3.1. Because of low expression rates of the protein (data not shown) the insert was cloned into the high level expression vector pCAGGS , using KpnI and NotI. ORF 7b gene (nts 27623 to 27751) and the deletion mutant ORF 7b del gene (nts 27623 - 27751 deletion of 45 nts 32-76) were amplified using primers 5'-CTAGAATTCCTCGAGACAATGAGAAGTTTCATGTTC-3' and 5'-ATCGTCGACCTCGAGTCACCATTAAGAGAAAGACAG-3', and cloned into pI.18 vector (kind gift of Jim Robertson, National Institute for Biological Standards and Control, Hertfordshire, UK) with a T7 promoter that was inserted by standard cloning procedures. Expression of constructs was verified by coupled in-vitro transcription and translation using the TNT T7 Coupled Reticulocyte Lysate System (Promega, Mannheim, Germany) and immunofluorescence analysis of transfected cells (data not shown).
ISG-54 reporter gene assay
Transfection of 293 cells was performed using the calciumphosphate transfection kit (Invitrogen) according to the manufacturer's instructions. 106 cells were transfected with 0.3 μg of the interferon (IFN)-stimulated response element (ISRE)-driven firefly luciferase reporter plasmid pHISG-54-Luc (kind gift of D. Levy, New York University School of Medicine, New York), 0.3 μg of the constitutive Renilla luciferase expression plasmid pRL-SV40 (Promega) and 4 μg of the plasmid of interest. 16 μg of herring sperm DNA (Promega) were transfected along with the plasmids to optimize DNA uptake. 24 h post transfection, cells were infected with SeV (20 hemagglutinating units) to induce the type I IFN response or were not infected. At 24 h post infection (p.i.), cells were harvested and lysed in 100 μl of passive lysis buffer (Promega, Mannheim, Germany). Subsequent luciferase assays were performed by using the Promega DUAL luciferase assay system according to the manufacturer's instructions. Relative renilla luciferase production was used to normalize for transfection efficiency.
Cells were seeded on chamber slides (μSlide® 8 well, Ibidi, Martinsried, Germany) and infected with SARS-CoV t a multiplicity of infection of 1. After 24 hours cells were washed once with PBS and fixed in ice cold acetone for 15 minutes. Prior to antibody staining, cells were washed three times with PBS. Reconvalescent SARS patient serum from our own diagnostic laboratory was diluted 1:1000 in PBS-T. Rabbit polyclonal antibody against the DDDDK - tag (Abcam, UK) was diluted 1:10000 in PBS-T. Cells were overlaid with 100 μl of antibody solution, incubated at 37°C for 1 hour, and washed four times for 5 minutes with PBS containing 0.1% Tween 20 (PBS-T). Fluorescein-conjugated goat anti-human or anti-rabbit IgG serum (Calbiochem/VWR, Darmstadt, Germany) was diluted 1:10000 in PBS-T and incubated at 37°C for 30 minutes. Cells were washed 4 times with PBS. Chambers were overlaid with 200 μl of PBS and a few drops of mineral oil. Fluorescence was analysed on an inverted fluorescence microscope.
Western blot analysis
Subconfluent 293-lp cells in six well-plates were infected, harvested at different time points after infection, and pelleted by centrifugation for 5 minutes at 1200 rpm. Pellets were dissolved in 50 μl of 1× Chaps buffer containing 1 mM PMSF and 5 mM DTT followed by three freeze/thaw cycles. Nuclei were pelleted by centrifugation for 10 minutes at 16,000 g and the clarified lysate was dissolved in 1× SDS loading buffer. 10 μl of postnuclear lysate were loaded on precast 4-12% Bis-Tris gradient gels. Separated proteins were electroblotted on nitrocellulose membranes (Whatman, Dassel, Germany) and blocked for 1 h with 1× RotiBlock (Roth, Karlsruhe, Germany). Membranes were washed with PBS and incubated over night with primary antibody at 4°C. Rabbit polyclonal anti-caspase-3 and anti-PARP antibodies (Cell Signaling, Danvers, USA) were diluted 1:1000 in PBS-T. Rabbit anti-flag antibody (Abcam, Cambridge, UK) was diluted 1:5000 in PBS-T. Membranes were washed 4 times for 5 minutes with PBS-T. Horseradish peroxidase-conjugated goat anti-rabbit antibody (Cell Signaling, Danvers, USA) was used at 1:2000 dilution in PBS-T and incubated on membranes for 1 hour. Membranes were washed four times with PBS-T before LumiGLO reagent (Cell Signaling, Danvers, USA) was added. Membranes were exposed to scientific imaging film (Sigma-Aldrich, Munich, Germany) for appropriate times before development.
In overexpression experiments, Vero E6 cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 5 × 104 cells were transfected with 1 μg of empty pI.18 vector, pCAGGS-ORF 7a, pI.18-ORF 7b or pI.18-ORF 7b del, respectively. At 48 h post transfection, cells were lysed in 50 μl 1× Chaps buffer. Proteins were separated on 15% SDS polyacrylamide gels, transferred onto PVDF membranes and blocked for 1 h with 5% skim milk (w/v) in PBS-T. Membranes were washed with PBS-T and incubated over night with primary antibody at 4°C. Western blot detection was done with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody using an enhanced chemiluminescence detection reagent kit (Pierce, Perbio Science, Bonn, Germany) according to the manufacturer's protocol. Immunoreactive bands were visualized using an Optimax 2010 imaging system (PROTEC processor Technology, Oberstenfeld, Germany) with high performance chemiluminescence films (GE Healthcare, Munich, Germany).
Interferon alpha was detected with the Human IFN Alpha ELISA Kit (PBL Interferonsource, Piscataway, USA) according to the manufacturer's instructions. Briefly, 100 μl of supernatant of samples and controls were added to pre-coated microtiter plates and incubated at room temperature for 1 hour, followed by one washing step, addition of antibody solution and another hour of incubation. After three washing steps, 100 μl of HRP conjugate concentrate were added and incubated for 1 hour. The plate was washed four times and TMB substrate was added. After 15 minutes of incubation stop solution was added and absorbance was determined at 450 nm.
Quantification of interferon beta mRNA
Total RNA was prepared from 293-lp cells in 6-well plates with Trizol® reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA was quantified photometrically and 150 ng per reaction were analysed by real-time RT-PCR. Interferon beta mRNA was amplified with primers IFNβFwd (5'-GAACTTTGACATCCCTGAGGAGATT-3') and IFNβRev (5'-GGAGCATCTCATAGATGGTCAATG-3'), and 5'-nuclease probe IFNβ-P (FAM-CAGCAGTTCCAGAAGGAGGACGCC-TAMRA). GAPDH mRNA was detected in parallel with primers GAPDHFwd (5'-AGGTGGTCTCCTCTGACTTCAACA-3'), GAPDHRev (5'-AGTGGTCGTTGAGGGCAATG-3'), and probe GAPDH-P (FAM-CACCCACTCCTCCACCTTTGACGCT-TAMRA). Reactions using the OneStep RT-PCR kit (Qiagen, Hilden, Germany) comprised 50°C for 30 minutes, followed by 95°C for 15 minutes and 40 cycles of 95°C for 10 seconds and 58°C for 30 seconds. For both genes standard curves were generated from limiting dilution series of quantified RNA. The dilution end-points were defined as one PCR unit for each gene. Log PCR units for each experimental sample were calculated from the linear equations of the dilution series. Interferon beta quantity was normalised to GAPDH quantity by subtraction of logarithmic quantities (Interferon - GAPDH).
Infections were performed with rSCV and r7bΔTMD. Heat-inactivated rSCV served as the mock-control. Syrian Golden hamsters (strain LVG, Charles River Laboratories) were infected via the intranasal route with 104 PFU each. 100 μl of virus solution were applied. Hamsters were sacrificed at indicated days post infection. Lungs were prepared in total, weighed, and homogenized. Tissue was suspended to a concentration of 0.5 g/ml in complete DMEM before analysis by RT-PCR or cell culture.
We are grateful to Jindrich Cinatl, Friedemann Weber, Christopher Basler, Beate Kümmerer, and Jim Robertson for donations of viruses or cells. We thank Toni Rieger for his kind help during hamster inoculations.
This study was supported by the German Ministry of Education and Research (Project Code "Ökologie und Pathogenese von SARS"), the European Commission (contract SSPE-CT-2005-022639), the German Research Foundation (Mu1365/1-1 and SFB 535), and the Sino-German Center for Science Promotion.
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