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1、Host cell proteins interacting with the 3 end of TGEV coronavirus genome infl uence virus replication Carmen Galn a, Isabel Solaa, Aitor Nogalesa, Benjamin Thomasb, Alexandre Akoulitchevb,1, Luis Enjuanes a, Fernando Almazna a Department of Molecular and Cell Biology, Centro Nacional de Biotecnologa
2、, CSIC, C/Darwin 3, Cantoblanco, 28049 Madrid, Spain b Oxford Central Proteomics Facility, Sir William Dunn School of Pathology, University of Oxford, UK a b s t r a c ta r t i c l ei n f o Article history: Received 27 April 2009 Returned to author for revision 25 May 2009 Accepted 3 June 2009 Avail
3、able online 5 July 2009 Keywords: Coronavirus Proteomics RNA-binding proteins siRNAs RNA synthesis Coronavirus RNA synthesis is performed by a multienzymatic replicase complex together with cellular factors. This process requires the specifi c recognition of RNA cis-acting signals located at the end
4、s of the viral genome. To identify cellular proteins involved in coronavirus RNA synthesis, transmissible gastroenteritis coronavirus (TGEV) genome ends, harboring essential cis-acting signals for replication, were used as baits for RNA affi nity protein purifi cation. Ten proteins were preferential
5、ly pulled down with either the 5 or 3 ends of the genome and identifi ed by proteomic analysis. Nine of them, including members of the heterogeneous ribonucleoprotein family of proteins (hnRNPs), the poly(A)-binding protein (PABP), the p100 transcriptional co-activator protein and two aminoacyl-tRNA
6、 synthetases, showed a preferential binding to the 3 end of the genome, whereas only the polypyrimidine tract-binding protein (PTB) was preferentially pulled down with the 5 end of the genome. The potential function of the 3 end-interacting proteins in virus replication was studied by analyzing the
7、effect of their silencing using a TGEV-derived replicon and the infectious virus. Gene silencing of PABP, hnRNP Q, and glutamyl-prolyl-tRNA synthetase (EPRS) caused a signifi cant 2 to 3-fold reduction of viral RNA synthesis. Interestingly, the silencing of glyceraldehyde 3-phosphate dehydrogenase (
8、GAPDH), initially used as a control gene, caused a 2 to 3-fold increase in viral RNA synthesis in both systems. These data suggest that PABP, hnRNP Q, and EPRS play a positive role in virus infection that could be mediated through their interaction with the viral 3 end, and that GAPDH has a negative
9、 effect on viral infection. 2009 Elsevier Inc. All rights reserved. Introduction Transmissible gastroenteritis virus (TGEV) is a member of the Coronaviridae family, included in the Nidovirales order (Enjuanes et al., 2000, 2008). Coronaviruses (CoVs) have been classifi ed in three different groups b
10、ased on antigenic and genetic criteria. The most extensively studied group members are the TGEV and the human coronavirus (HCoV) 229E from group 1, the mouse hepatitis virus (MHV) and the severe acute respiratory syndrome (SARS) virus (SARS-CoV) from group 2, and the infectious bronchitis virus (IBV
11、) from group 3. CoV infections cause a variety of diseases relevant in animal and human health, being of special relevance the SARS in humans (Drosten et al., 2003; Perlman et al., 2000). After the SARS outbreak in 2002 the interest to understand the molecular basis of CoV replication increased and,
12、 as a result, in the last years more than 30 new CoVs have been identifi ed with a broad host distribution (Vijaykrishna et al., 2007). CoVs contain the largest known single-stranded positive-sense infectious RNA genomes, varying between 27 and 31 kb in length (Enjuanes et al., 2008; Masters, 2006).
13、 The CoV genome resembles the structure of most cellular mRNAs, containing a cap structure at the 5 end, a poly(A) tail at the 3 end, and 5 and 3 untranslated regions (UTRs). The replicase gene, that occupies the 5 two thirds of the genome, includes two overlapping open reading frames (ORF 1a and OR
14、F 1b) that are translated into two large co-amino-terminal polyproteins,pp1aandpp1ab,thesecondoneexpressedbyaribosomal frameshift mechanism (Brierley et al., 1989). Both polyproteins are autoproteolytically cleaved into up to 16 mature products (nsp1 to nsp16), which are believed to be part of the r
15、eplicationtranscription complex (Ziebuhr et al., 2000). CoV replicase gene is extremely complex and, besides the RNA-dependent RNA-polymerase and heli- case activities, it encodes other enzymes less frequent or exclusive amongRNAviruses(Masters,2006;Snijderetal.,2003;Ziebuhr,2005), such as an endori
16、bonuclease, a 35 exoribonuclease, a 2-O-ribose methyltransferase, a ribose ADP 1 phosphatase, and a second RNA- dependent RNA-polymerase (Imbert et al., 2006). In addition to the Virology 391 (2009) 304314 Corresponding author. Fax: +34 91 585 4915. E-mail address: L.Enjuanescnb.csic.es (L. Enjuanes). 1 Present address: Oxford BioDynamics Limited. Oxford University Begbroke Science Park. Sandy Lane, Yarnton OX5 1PF, UK. 0042-6822/$ see front matter 2009 Elsevier Inc. All rights reserved. do
