SARS-CoV-1 ORF3 ja ORF8 Katsahdus taaksepäin entiseen SARS-koronavirukseen vuodelta 2003

Hong Kong Med J 2012;18(Suppl 3):S16-9

Impact of SARS-coronavirus-encoded proteins on cellular signalling pathways and cytokine/chemokine gene expression

JKL Chan, P Cheung, M He, Z Wu

Department of Biochemistry, Hong Kong University of Science and Technology, Hong Kong

 

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1. Although GFP-tagged ORF8 and ORF3 potently activate the JNK and p38 MAPK pathways, expression of non-tagged SARS-CoV-encoded ORF8 or ORF3 has no obvious effect.
2. Although both GFP-tagged ORF8 and ORF3 induce cell death, expression of non-tagged ORF8 or ORF3 has no obvious effect on cell survival.
3. Addition of an epitope tag to a protein of interest, a common way to study novel proteins in the absence of suitable antibodies, may generate unexpected artefacts. Caution should be taken with any results derived from epitope-tagged proteins.
4. When studying a novel protein, it is essential to prepare suitable antibodies to facilitate detection and purification (eg by immunoprecipitation) of the native or endogenous proteins.

Introduction 

Severe acute respiratory syndrome coronavirus (SARS-CoV) was responsible for the global SARS pandemic in 2003.1,2 

 Although all CoVs have similar microscopic appearance, gene products, and genomic organisation, SARS-CoV is unique in that it is associated with high mortality rates in humans. Spike, membrane, envelope, and nucleocapsid proteins as well as replicase are commonly conserved among all CoVs. The genome of SARS-CoV also encodes nine other novel open-reading frames (ORFs) with unknown functions (Fig 1).1,2  

Various viruses exert their pathogenic effects through interaction of their viral proteins with distinct cellular targets. We hypothesised that the severe inflammation and high mortality caused by SARS-CoV are contributed in part by these novel ORFs. Therefore, we aimed to evaluate the functions of these novel ORFs by overexpressing them in human cell lines.

 

  Mitogen-activated protein kinases (MAPK) are important cellular signalling molecules involved in cell growth, differentiation, and apoptosis under both normal and pathological conditions. Three major classes of MAPKs, namely extracellular signal-regulated kinases, cJun N-terminal kinases (JNKs), and p38 MAPKs, have been extensively characterised in the past 15 years. Many viral proteins are known to activate these MAPKs to exert their cytotoxic effects and trigger host inflammatory responses. 

For example, the Tax protein of human T-cell leukaemia virus type 1 and the latent membrane protein 1 (LMP1) of the Epstein-Barr virus potently activate the JNK pathway. We hypothesised that the novel ORFs of SARS-CoV may trigger inflammation and promote apoptosis of host cells through activation of MAPKs, especially the JNK and p38 MAPKs, which are known to be activated by pro-inflammatory and apoptotic stimuli

 

Impact of SARS-coronavirus-encoded proteinsHong Kong Med J Vol 18 No 4 Supplement 3 August 2012 19 
Owing to the high mortality rate caused by SARS-CoV, it is essential to understand the molecular mechanisms underlying the pathogenesis. We undertook the project at a time when many key reagents (including the antibodies againt ORF8) were not available. GFP-tagged ORF8 was found to potently induce cell death, which was consistent with other reports.3,4 In addition, GFP-tagged ORF8 strongly activated the JNK and p38 MAPK pathways in host cells. Non-tagged ORF failed to induce these changes; this suggested that artefacts were generated in the GFP fusion proteins. Before obtaining the ORF8 antibody, a segment of ORF8 gene encoding aa 17-94 was used as the bait in the yeast two-hybrid screening. Several interesting clones were found. Owing to the lack of a suitable biological assay for the native ORF8, these clones were not further characterised. 

 In addition to ORF8, ORF3 (also known as ORF3a), another novel ORF that was found to be expressed in SARS-CoV-infected cells was also extensively studied. Similarly, GFP-ORF3 induced cell death and activated both JNK and p38 in host cells. In contrast, the non-tagged ORF3 failed to induce cell death and activate JNK and p38 as well as IkB kinase

.Conclusions 
The native (non-tagged) ORF8 and ORF3 did not significantly induce cell death, nor did they activate JNK and p38 MAPK pathways. 
 We believe that results in several reports on ORF8 and ORF3 were most likely due to artefacts generated by inappropriate fusion of an epitope tag at either end of the viral proteins.3,4 Therefore, caution should be exercised in interpreting results derived from epitope-tagged proteins. Suitable antibodies to the protein of interest should be prepared to facilitate the study of the native proteins. 

AcknowledgementThis study was supported by the Research Fund for the Control of Infectious Diseases, Food and Health Bureau, Hong Kong SAR Government (#01030802).References 1. Rota P, Oberste MS, Monroe SS, et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 2003;300:1394-9. 2. Marra MA, Jones SJ, Astell CR, et al. The genome sequence of the SARS-associated coronavirus. Science 2003;300:1399-404.3. Tan YJ, Fielding BC, Goh PY, et al. Overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway. J Virol 2004;78:14043-7.4. Yuan X, Wu J, Shan Y, et al. SARS coronavirus 7a protein blocks cell cycle progression at G0/G1 phase via the cyclin D3/pRb pathway. Virology 2006;346:74-85.5. Fielding BC, Tan YJ, Shuo S, et al. Characterization of a unique group-specific protein (U122) of the severe acute respiratory syndrome coronavirus. J Virol 2004;78:7311-8.