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Review
. 2008 Apr;133(1):101-12.
doi: 10.1016/j.virusres.2007.03.015. Epub 2007 Apr 23.

SARS coronavirus and innate immunity

Matthew Frieman  1 , Mark Heise, Ralph Baric
Affiliations
Review

SARS coronavirus and innate immunity

Matthew Frieman et al. Virus Res. 2008 Apr.

Abstract

The emergence of the highly pathogenic SARS coronavirus (SARS-CoV) has reignited interest in coronavirus biology and pathogenesis. An emerging theme in coronavirus pathogenesis is that the interaction between specific viral genes and the host immune system, specifically the innate immune system, functions as a key determinant in regulating virulence and disease outcomes. Using SARS-CoV as a model, we will review the current knowledge of the interplay between coronavirus infection and the host innate immune system in vivo, and then discuss the mechanisms by which specific gene products antagonize the host innate immune response in cell culture models. Our data suggests that the SARS-CoV uses specific strategies to evade and antagonize the sensing and signaling arms of the interferon pathway. We summarize by identifying future points of consideration that will contribute greatly to our understanding of the molecular mechanisms governing coronavirus pathogenesis and virulence, and the development of severe disease in humans and animals.

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Figures

Fig. 1
Fig. 1
IFN sensing and signaling pathway. RNA viruses are internalized through several mechanisms, either fusion with the plasma membrane or binding to a surface receptor (ACE2 for SARS-CoV, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) for MHV). That internalization exposes the genomic RNA to the dsRNA sensing machinery in the cell; TLR3, RIGI and MDA5. These proteins signal the IRF-3 cascade leading to induction of IFNb and production of secreted IFNβ protein. That IFNβ protein can then bind IFNα/β receptors (INFAR1) on the surface of the same cell or surrounding cells. This activates the Stat1 signaling pathway to activate the many anti-viral genes found with ISRE promoter elements.
Fig. 2
Fig. 2
Lack of an IFN beta induction by SARS-CoV. SARS-CoV was tested for its ability to activate an IFN beta promoter by transfection of 293T cells with a plasmid containing the IFNβ promoter driving expression of firefly luciferase. After 24 h post transfection either PBS, SARS-CoV or Sendai Virus was added at an MOI of 5. Samples were taken at 8, 24, 36 and 48 h post infection and analyzed for luciferase production. We find no induction of IFNβ resulting from SARS-CoV infection however a robust expression level is seen in the Sendai Virus infected cells. The depletion of luciferase seen in the Sendai infections after 24 h is due to cell death.
Fig. 3
Fig. 3
Lack of type I IFN secreted from infected cells. A type I IFN bioassay was performed on media from infected MA104 cells (IFN competent for signaling and production). Cells were infected with an MOI of 5 for either SARS-CoV strains TOR2 and Urbani. Parainfluenza Virus was used as a positive control. Over a timecourse of 2, 24 and 48 h of infection no type I IFN was produced from SARS-CoV infected cells while PIV produced large amounts of IFN. LOD means level of detection for the assay.
Fig. 4
Fig. 4
Real-time PCR for innate immune response genes. Caco2 cells were infected with the infectious clone for SARS-CoV (icSARS), the Urbani strain of SARS or Sendai Virus at an MOI of 5 for each. After 18 h cells were harvested for RNA extraction and used for real-time analysis of the denoted genes. Low levels expression was seen in SARS-CoV infected samples for IFNα, IFNβ, IL-6 and IL-8 although large inductions were seen in the Sendai Virus infected samples. Mx was found to be induced by all three viruses to a very large extent while IP-10 was increased up to 100-fold for SARS infected cells and minimal for Sendai infection. All expression levels are shown relative to an 18S standard.
Fig. 5
Fig. 5
(A) SARS-CoV genome organization. The SARS-CoV genome is shown divided into two main regions, the replicase consisting of ORFs 1a and 1b and the subgenomic ORFs comprising the structural and accessory ORFs. (B) Generation of SARS-CoV infectious clone. The SARS-CoV genome is broken into 6 fragments noted A through F. Each fragment is cloned so as to encode a type II restriction site at either end allowing for directed ligation of all fragments while not changing the amino acids of the encoded proteins. The plasmids are digested and ligated all together to form a single 30 kb fragment. This is used as the template for transcription by T7 polymerase which the 5′ most piece has a start site encoded in it. The resulting RNA is electroporated into Vero cells and virus is collected in the media 24 h after electroporation.
Fig. 6
Fig. 6
The coronavirus life cycle. Coronavirus entry is mediated by binding of S glycoprotein to the ACE2 receptor, cleavage by cathepsin L and activation of a fusion peptide in S2 that mediates entry via fusion through endocytic compartments [1]. Following fusion with the endosomal compartment the viral genome release into the cytosol where it is translated into the viral replicase proteins ORF1a and 1b [2]. These polyproteins are then cleaved by 2 proteases, Main Protease (Mpro) and Papain like protease, PLP, into the individual proteins necessary for replication [3]. Subgenomic RNA synthesis occurs from discontinuous transcription which joins leader RNA sequences encoded at the 5′ end of the genome to the body sequences of each subgenomic RNA. The eight different subgenomic negative strands serve as template for the synthesis of like sized subgenomic mRNA [4]. Subgenomic RNAs are then translated into viral proteins which localize to their relevant compartments [5]. Assembly of virions occurs in an ERGIC like compartment in the cell. Here S, E, M and N bound to genomic viral RNA are assembled into virions in vesicles [6]. The vesicles are then exported to the cell surface where fusion occurs with release of virions into the exterior environment [7,8].
Fig. 7
Fig. 7
SARS does not block NFκB and IFNβ promoter induction. Vero cells were transfected with either plasmids containing the NFκB (A) or the IFNβ (B) promoter expressing luciferase. 24 h post transfection cells were infected with the designated viruses. The 12 h post infection cells were then infected with Sendai Virus and luciferase was assayed 6 h later using Steady-Glo Luciferase Reagent (Promega). Triplicate wells were averaged and then compared to mock infected wells to graph the fold induction values.
Fig. 8
Fig. 8
SARS-CoV ORF6 blocks nuclear import of Stat1. (a) Two hundred and ninety three cells were transfected with a plasmid containing an ISRE promoter driving luciferase and either CAGGS/GFP or CAGGS/ORF6. The 24 h post transfection half of the wells were treated with IFNβ for 4 h and then assayed by Steady-Glo Luciferase Reagent (Promega) for ISRE promoter induction. (b) Two hundred and ninety three cells were transfected with HA tagged ORF6. The 24 h post transfection cells were treated with 100 IU/ml of IFNβ for 1 h. Cells were then lysed and assayed by western blot for the presence of total STAT1 (top panel) or phosphorylated STAT1 (bottom panel). (c) Vero cells were transfected with either STAT1/GFP alone or co-transfected with the designated plasmids for 24 h. After 24 h, cells were either untreated or treated with IFNβ or IFNγ as designated. Notice STAT1 is cytoplasmic when untreated but is transported to the nucleus when treated with either IFNβ or IFNγ. Co-expression of SARS ORF6 retains STAT1 in the cytoplasm while SARS 3a expression does not. Also notice that SARS ORF6 blocks IFNγ induced STAT1 nuclear translocation as well.
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