We assessed several replicase-encoded nsps of MHV (strain A59), SARS-CoV, and human coronavirus 229E (HCoV-229E) for their ability to interfere with host cell gene expression. Using transient gene expression studies, we found that MHV-A59, SARS-CoV, and HCoV-229E nsp1 significantly reduced luciferase reporter gene expression under the control of IFN-β, IFN-stimulated response element (ISRE), and SV40 promoters (Figure 1). This is consistent with a recent report suggesting that SARS-CoV nsp1 induces general host cell mRNA degradation [18]. Nevertheless, it should be noted that the nsp1-mediated reduction in reporter protein expression appeared more robust for ISRE and SV40 than for IFN-β promoter-driven expression. Our data also support the hypothesis that MHV encodes a SARS-CoV nsp1 homolog that displays the same function [23]. Although comparative sequence analyses suggest that nsp1 of group 2a/2b coronaviruses (e.g., MHV and SARS-CoV, respectively) and the nsp1 of group 1 coronaviruses (e.g., HCoV-229E) may belong to different protein families [22,23], we also observed reduced reporter gene expression in HCoV-229E nsp1-transfected cells (Figure 1). Whether functional similarities may exist between nsp1 molecules encoded by coronaviruses of different phylogenetic lineages remains to be established in future studies. Importantly, our data also revealed that reporter gene expression from all tested promoters was not affected when C-terminally truncated MHV nsp1 molecules were tested (Figure 1).

To assess the role of nsp1 in the context of virus replication, we constructed a recombinant MHV encoding a truncated nsp1 protein using our reverse genetic system [24]. Based on the results shown in Figure 1, we decided to delete MHV nucleotides (nts) 829–927 (99 nts). In the resulting mutant virus, MHV-nsp1Δ99, the replicase gene start codon, the translational reading frame, and the residues required for proteolytic release of nsp1 from the replicase polyprotein were maintained (Figure 2A). As reported for a set of similar MHV mutants by Brockway et al. [25], viral growth and peak titers of MHV-nsp1Δ99 in murine 17Clone1 cells were indistinguishable from that of wild-type virus (Figure 2B). To assess the stability of the recombinant MHV-nsp1Δ99, we analyzed the nsp1-coding region by RT-PCR sequencing after seven passages in tissue culture and no nucleotide changes were detected (unpublished data).

Infection of conventional DCs (cDCs) is an early and crucial event for the generation of protective antiviral immunity [26]. MHV productively infects cDCs and activates plasmacytoid DCs (pDCs) to generate a first wave of protective type I IFN [27]. To assess whether the mutant MHV-nsp1Δ99 has retained the ability to infect pAPCs, peritoneal macrophages (Figure 2C), bone marrow–derived CD11c⁺ cDCs (Figure 2D), and splenic, FACS-sorted CD11c⁺ cDCs (Figure 2E) were exposed to MHV-nsp1Δ99 and wild-type control virus. Similar to replication kinetics in cell lines (Figure 2B), MHV-nsp1Δ99 showed no significant growth defect in primary pAPCs (Figure 2C–2E), indicating that the deletion of nsp1 did not alter the pronounced tropism of MHV for cDCs and macrophages.

MHV-A59 is a hepatotropic and neurotropic virus that can cause acute hepatitis and encephalitis. Following intraperitoneal infection, virus replication is first detectable in spleen and liver, followed by virus spread to other organs, including the central nervous system. Hepatitis is the first clinical sign of disease, accompanied by elevated liver enzyme values in serum. Associated with the appearance of cytotoxic T cell responses approximately at day 5 post infection (p.i.), virus titers usually decline and are no longer detectable after day 7 p.i. Infections with a high dose (≥ 5 × 10⁶ pfu, intraperitoneal) may, however, occasionally result in fatality. To evaluate the importance of nsp1 for virus replication and viral pathogenicity in vivo, C57BL/6 mice were infected intraperitoneally with different doses of wild-type MHV or MHV-nsp1Δ99. Both viruses replicated in the spleen, whereby MHV-nsp1Δ99 titers were consistently lower than wild-type virus titers (Figure 3A). Furthermore, MHV-nsp1Δ99 was rapidly cleared and not detectable after day 2 p.i. (Figure 3A). Wild-type, but not mutant virus, was detectable in the liver at low and intermediate dose (50 pfu and 5,000 pfu, respectively) (Figure 3B). When high virus doses (5 × 10⁶ pfu) were applied, MHV-nsp1Δ99 eventually reached the liver at day 2 p.i., but was not detectable at later time points (Figure 3B). MHV-nsp1Δ99 was not detectable in other non-hematopoietic organs, such as lung and central nervous system (unpublished data). Mice infected with wild-type virus showed acute liver disease with elevated liver enzyme values in serum. Furthermore, after high dose infection with wild-type virus (5 × 10⁶ pfu), a significant weight loss that peaked at approximately 10%–15% at day 4 was observed (Figure 3E). In contrast, mice infected with the nsp1 mutant virus remained healthy after low, intermediate, or high dose infections. Even at the highest dose applied (5 × 10⁶ pfu), MHV-nsp1Δ99–infected mice did not lose weight (Figure 3E), and no elevated liver enzyme values were detected in the serum (Figure 3C). This observation correlated well with the absence of hepatocyte necrosis and parenchymal inflammation following MHV-nsp1Δ99 infection (Figure 3D). To further assess the attenuation of the MHV nsp1 mutant, mice were infected intracranially with 200 pfu and 20,000 pfu of MHV-nsp1Δ99 or MHV-A59. All mice infected with 200 pfu survived for at least 30 d (unpublished data). Mice infected with 20,000 pfu of MHV-A59 succumbed to the infection, whereas mice infected with 20,000 pfu of MHV-nsp1Δ99 survived and showed no signs of clinical disease (Figure 3F). Collectively, these data demonstrate that MHV-nsp1Δ99 is strongly attenuated in vivo, but has retained the ability to replicate in secondary lymphoid organs, such as the spleen.

We have previously shown that pDCs are the major source of IFN-α in the early stages of MHV infection and that type I IFN responses in CD11c⁺ cDCs are only weakly triggered by MHV [27]. To test whether nsp1 has an influence on the induction of IFN-α, we infected both cDCs and pDCs with MHV-nsp1Δ99 or wild-type MHV. Both viruses elicited rapid and high IFN-α production in Flt3-L–differentiated bone marrow–derived pDCs (Figure 4A) and FACS-sorted primary pDCs (Figure 4B). Furthermore, both wild-type and mutant MHV elicited only a late and weak IFN-α production in cDCs (Figure 4A and 4B). These results suggest that nsp1 does not affect the induction of type I IFN. To assess a potential impact of nsp1 on type I IFN signaling and antiviral effector mechanisms in target cells that efficiently support MHV replication, cDCs and macrophages were pretreated with different dosages of IFN-α and infected with MHV-nsp1Δ99 or wild-type MHV. In cDCs, IFN-α treatment had a comparable effect on the replication of both MHV-nsp1Δ99 and the wild-type control virus (Figure 4C). However, replication of MHV-nsp1Δ99 was, in a dose-dependent manner, more vulnerable to IFN-α treatment in macrophages (Figure 4D), suggesting that nsp1 might counteract IFN signaling and/or the antiviral activities of IFN-induced effector proteins.

Indeed, in vivo experiments in type I IFN receptor-deficient (IFNAR^−/−) mice [28] strongly support this interpretation. Infection of IFNAR^−/− mice with wild-type MHV led to high titers in all tested organs (Figure 5A–5D), indicating that signals transmitted via the IFNAR are crucial for preventing uncontrolled spread of the virus [27]. Surprisingly, the severe attenuation of MHV-nsp1Δ99 in wild-type 129Sv mice was not present in IFNAR^−/− mice (Figure 5A–5D). Replication of MHV-nsp1Δ99 in IFNAR^−/− mice was largely restored and virus titers reached about 10⁴–10⁵ pfu/g tissue in several organs after only 36 h (figures 5A–5D). These data strongly suggest that nsp1 has a pivotal role in counteracting type I IFN host responses and provide an explanation for the rapid clearance of MHV-nsp1Δ99 in wild-type mice. Interestingly, liver damage, measured as alanine 2-oxoglutarate-aminotransferase (ALT) levels in serum, was not yet detectable in MHV-nsp1Δ99–infected IFNAR^−/− mice at 36 h p.i. (Figure 5E). At 72 h p.i., MHV-nsp1Δ99 reached titers and ALT levels in IFNAR^−/− mice comparable to those observed in MHV-A59–infected IFNAR^−/− mice at 36 h p.i., demonstrating that MHV-nsp1Δ99 replication in IFNAR^−/− is, although with slower kinetics, restored.

The phenotypic analysis of MHV-nsp1Δ99 revealed a number of features that are advantageous for live attenuated vaccines. MHV-nsp1Δ99 grows to high titers in cell culture, infects pAPCs, replicates almost exclusively in secondary lymphoid organs, and is strongly attenuated in vivo. To assess the potential of MHV-nsp1Δ99 as an attenuated live vaccine, we replaced accessory gene 4 of MHV-nsp1Δ99 and wild-type MHV-A59 by a gene encoding a fusion protein of the immunodominant cytotoxic T lymphocyte (CTL) epitope (KAVYNFATC) of the lymphocytic choriomeningitis virus (LCMV) and the enhanced green fluorescent protein (GP33-GFP) [29]. The resulting recombinant viruses, MHV-nsp1Δ99-GP33-GFP and MHV-GP33-GFP, were used to infect C57BL/6 mice with different doses (50 and 5,000 pfu, intraperitoneal), and CD8⁺ T cell responses were assessed using flow cytometry–based detection of intracellular IFN-γ following antigen-specific short-term in vitro restimulation. As shown in Figure 6A and 6B, infection with as few as 50 pfu of MHV-nsp1Δ99-GP33-GFP elicited strong CD8⁺ T cell responses against both the H2-D^b–restricted GP33 and the H2-K^b–restricted MHV S598 epitope.

To assess the level of protection against homologous MHV-A59 challenge, groups of C57BL/6 mice were immunized (5,000 pfu) with MHV-nsp1Δ99-GP33-GFP, MHV-GP33-GFP, or treated with PBS. Sixteen days p.i., mice were challenged with wild-type MHV (5,000 pfu) and viral titers were determined 5 d post challenge infection. Viral titers were below the limit of detection in MHV-nsp1Δ99-GP33-GFP– and MHV-GP33-GFP–immunized mice (Figure 6C). Together with the absence of elevated liver enzyme values in immunized mice (Figure 6D), these data indicate that vaccination with the attenuated MHV nsp1 mutant provides complete protection against homologous virus challenge.

The reverse genetic system facilitates incorporation of antigens derived from other infectious organisms. In order to determine whether the attenuated nsp1 mutant virus could confer protection against heterologous virus infection, MHV-nsp1Δ99-GP33-GFP–immunized C57BL/6 mice were challenged after 4 wk with LCMV (200 pfu, intravenous). LCMV titers in spleens were significantly reduced both in mice vaccinated with MHV-GP33-GFP and the attenuated MHV-nsp1Δ99-GP33-GFP virus (Figure 6E). Remarkably, only 50 pfu of nsp1 mutant virus expressing the GP33 epitope were sufficient to achieve a reduction of LCMV titers by more than 4 orders of magnitude, indicating that nsp1 mutant viruses are well-suited to serve as attenuated recombinant virus vectors against heterologous viral infections.

C57BL/6 mice were obtained from Charles River Laboratories (http://www.criver.com/). 129Sv and type I IFN receptor-deficient mice (IFNAR^−/−) [28] were obtained from the Institut für Labortierkunde (University of Zürich) and bred in our facilities. All mice were maintained in individually ventilated cages and were used between 6 and 9 wk of age. All animal experiments were performed in accordance with the Swiss Federal legislation on animal protection.

MC57, BHK-21, L929, 293, and CV-1 cells were purchased from the European Collection of Cell Cultures (http://www.ecacc.org.uk/). D980R cells were a kind gift from G. L. Smith, Imperial College, London, United Kingdom. 17Clone1 cells were a kind gift from S. G. Sawicki, Medical University of Ohio, Toledo, Ohio, United States. BHK-MHV-N cells, expressing the MHV-A59 nucleocapsid protein under the control of the TET/ON system (Clontech, http://www.clontech.com/), have been described previously [24]. All cells were maintained in minimal essential medium supplemented with fetal bovine serum (5%–10%) and antibiotics.

Murine cDCs and pDCs were obtained from spleens of C57BL/6, 129Sv, or IFNAR^−/− mice following digestion with collagenase type II (Gibco-BRL, http://www.invitrogen.com/) for 20 min at 37 °C. Cells were resuspended in PBS supplemented with 2% FCS and 2 mM EDTA and overlaid on 20% Optiprep density gradient medium (Sigma-Aldrich, http://www.sigmaaldrich.com/). After centrifugation at 700g for 15 min, low density cells were depleted of CD3- and CD19-positive cells using DYNAL magnetic beads according to the instructions of the manufacturer (Invitrogen, http://www.invitrogen.com/). The DC-enriched preparations were stained with α-PDCA-1, α-CD11b, and α-CD11c, and the distinct pDC and cDC populations were sorted using a FACS ARIA (BD Biosciences, http://www.bdbiosciences.com/) sorter. Purity of both cell preparations was always >98%.

Murine bone marrow–derived cDCs or pDCs were generated by 6 to 7 d of culture with either granulocyte-monocyte colony stimulating factor (GM-CSF)-containing supernatant from the cell line X63-GM-CSF (kindly provided by Antonius Rolink, University of Basel, Basel, Switzerland) or Flt3-L (R&D Systems, http://www.rndsystems.com/) at 20 ng/ml, respectively. Bone marrow–derived cDCs were further purified using Optiprep density gradient centrifugation. Bone marrow–derived pDCs were purified using the mouse pDC isolation kit (Miltenyi Biotec, http://www.miltenyibiotec.com/) adapted for the isolation of in vitro–derived pDCs by adding CD11b-biotin to the negative selection cocktail. Antibodies used in this study were purchased from BioLegend (http://www.biolegend.com/): CD11c-PE, B220-APC, CD11b-FITC; or from Miltenyi Biotec: mPDCA-1-FITC and CD11c-APC. Thioglycolate-elicited macrophages were collected from the peritoneal cavity of mice and cultured at 4 × 10⁵ cells per well in DMEM (with 10% FCS, L-glutamine, and penicillin/streptomycin) for 2 h at 37 °C. Non-adherent cells were removed by washing with cold PBS.

LCMV-WE strain, originally obtained from F. Lehmann-Grube (Hamburg, Germany), was propagated on L929 cells. MHV A59 was generated from a molecularly cloned cDNA [24] based on the Albany strain of MHV A59. Coronaviruses and recombinant vaccinia viruses were propagated, titrated, and purified as described [24,50,51].

Mutant vaccinia viruses are based on the recombinant vaccina virus vMHV-inf-1 (containing the full-length MHV-A59 cDNA) and were generated using our reverse genetic system as described previously [24]. Briefly, the gene to be mutated was replaced by the Escherichia coli guanine-phosphoribosyltransferase (GPT) gene through vaccinia virus–mediated homologous recombination, and GPT-positive clones were selected by three rounds of plaque purification on CV-1 cells in the presence of xanthine, hypoxanthine, and mycophenolic acid (GPT-positive selection) [50]. In a second round, the GPT gene was replaced by the mutated gene, and GPT-negative clones, containing the mutated gene, were selected by three rounds of plaque purification on D980R cells in the presence of 6-thioguanine (GPT-negative seletion) [50]. To construct the recombinant vaccinia virus encoding the MHV-nsp1Δ99 cDNA, the 5′ end of the MHV-A59 cDNA in vMHV-inf-1 was replaced by GPT using the plasmid pRec4. This plasmid is based on pGPT-1 [50], and the GPT gene is flanked to its left by 500 bp of vaccinia DNA and to its right by an internal ribosomal entry sequence (IRES) followed by MHV-A59 nts 952-1315. The GPT-negative selection was carried out using the plasmid pRec15. This plasmid contains 250 bp of vaccinia DNA followed by the bacteriophage T7 RNA polymerase promoter, one G nucleotide, and MHV nts 1–828 linked to MHV nts 928-1315. To replace the MHV-A59 accessory gene 4 in vMHV-inf-1 and vMHV-nsp1Δ99 by a gene encoding a fusion protein of EGFP and the LCMV-derived CTL epitope KAVYNFATC (GP33-GFP) [29], the plasmid pRec8 was used for recombination with vaccinia viruses vMHV-inf-1 and vMHV-nsp1Δ99 . This plasmid contains the GPT gene flanked to its left by MHV nts 27500–27967 and to its right by MHV nts 28265–28700. GPT-negative selection was carried out using the plasmid pRec9. This plasmid contains MHV bp 27500–27967, the GP33-GFP gene, and MHV nts 28265–28700. Further cloning details, plasmid maps, and sequences are available from the authors upon request. Recombinant coronaviruses were rescued from cloned cDNA using purified vaccinia virus DNA as template for the in vitro transcription of recombinant MHV genomes as described [51].

The firefly luciferase (FF-Luc) plasmid for monitoring IFN-β promoter activation (p125-Luc) was kindly provided by Takashi Fujita, Tokyo Metropolitan Institute of Medical Science, Japan [52]. The FF-Luc reporter construct for monitoring ISRE activation (p(9–27)4tkΔ(−39)lucter) [53] was kindly provided by Stephen Goodbourn, St. George's Hospital Medical School, London, UK. The control plasmid pRL-SV40 (Promega, http://www.promega.com/) encodes the renilla luciferase (REN-Luc) gene under control of the constitutive SV40 promoter. The negative control expression plasmid contained the reading frame of the N-terminus of the human MxA protein. To construct the coronavirus nsp1 expression plasmids, the MHV nts 1–951 (pMHV-nsp1), MHV nts 1–902 (pMHV-Δ49), MHV nts 1–851 (pMHV-Δ100), HCoV-229E nts 293–625 (pHCoV-229E-nsp1), and SARS-CoV nts 265–804 (pSARS-CoV-nsp1) were amplified by standard PCR techniques and cloned downstream of a CMV promoter between the SmaI and XhoI sites of the eukaryotic high-level expression plasmid pI.18 (kindly provided by Jim Robertson, National Institute for Biological Standards and Control, Hertfordshire, UK).

Subconfluent cell monolayers of 293 cells seeded in 12-well plates were transfected with 250 ng p125-Luc reporter plasmid, 50 ng pRL-SV40, and 1 μg of expression plasmid in 200 μl of OPTIMEM (Gibco-BRL) containing 3.9 μl of Fugene HD (Roche, http://www.roche.com). At 8 h post transfection, cells were induced with either 0.2 μg of viral ssRNA containing 5′ triphosphates [54] (p125-Luc), 2.5 μg of poly(I:C) (Sigma), or 500 U/ml IFN-α (p(9–27)4tkΔ(−39)lucter), or left uninduced. After an incubation period of 16 h, cells were harvested and lysed in 100 μl of Reporter Lysis Buffer (Promega). An aliquot of 10 μl lysate was used to measure luciferase activity as decribed by the manufacturer (Promega).

Mice were injected intraperitoneally or intracranially with indicated pfu of MHV A59 or intravenously with the indicated pfu of LCMV and sacrificed at the indicated time points. Organs were stored at −70 °C until further analysis. Blood was incubated at RT to coagulate, centrifuged, and serum was used for ALT measurements using a Hitachi 747 autoanalyzer (http://www.hitachi.com/). Peritoneal exudates cells (PECs) were isolated from the peritoneal cavity by flushing with 4 ml of ice-cold PBS. MHV titers were determined by standard plaque assay using L929 cells. LCMV titers in the spleens were determined 4 d after intravenous challenge in an LCMV infectious focus assay as previously described [55].

Organs were fixed in 4% formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin. Images were acquired using a Leica DMRA microscope (Leica, http://www.leica.com/) with a 25×/0.65 NA objective (total magnification, ×162). Images were processed using Adobe Photoshop (Adobe Systems, http://www.adobe.com). Mouse IFN-α concentration in cell culture supernatants was measured by ELISA (PBL Biomedical Laboratories, http://www.interferonsource.com/) according to the manufacturer's instructions. IFN-α treatment of cells prior to MHV infection was performed using universal type I IFN (IFN-αA/D, Sigma).

Specific ex vivo production of IFN-γ was determined by intracellular cytokine staining. Organs were removed at the indicated time points following infection with recombinant MHV. For intracellular cytokine staining, single cell suspensions of 1 × 10⁶ splenocytes were incubated for 5 h at 37 °C in 96-well round-bottom plates in 200 μl of culture medium containing 25 U/ml IL-2 and 5 μg/ml Brefeldin A (Sigma). Cells were stimulated with phorbolmyristateacetate (PMA, 50 ng/ml) and ionomycin (500 ng/ml) (both purchased from Sigma) as positive control or left untreated as a negative control. For analysis of peptide-specific responses, cells were stimulated with 10⁻⁶ M GP33 peptide or 10⁻⁴ M MHV S598 peptide. The percentage of CD8⁺ T cells producing IFN-γ was determined using a FACSCalibur flow cytometer (BD Biosciences). Both S598 (RCQIFANI) and GP33 (KAVYNFATC) peptides were purchased from Neosystem (http://www.neomps.com/).

All statistical analyses were performed with Prism 4.0 (GraphPad Software, http://www.graphpad.com/). Data were analyzed with the paired Student's t-test assuming that the values followed a Gaussian distribution. A p-value of < 0.05 was considered significant.

The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the viruses and sequences discussed in this paper are HCoV-229E (AF304460), MHV-A59 (AY700211), and SARS-CoV Frankfurt-1 (AY291315).