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1、BIOCHEMICAL ASPECTS OF CORONAVIRUS REPLICATION Luis Enjuanes, Fernando Almaz n, Isabel Sola, Sonia Ziga, Enrique Alvarez, Juan Reguera, and Carmen Capiscol* 1. INTRODUCTION infected cells. Nevertheless, there is a limited knowledge of the biochemical events occurring in host cells and in the biochem
2、istry of the infection. Infections by CoVs cause alterations in host cells in transcription and translation patterns, in the cell cycle, in the cytoskeleton, and in apoptosis pathways. In addition, in the host, CoV infection may cause inflammation, alterations of the immune response, of cytokine and
3、 chemokine levels, of interferon (INF)-induced gene expression and of stress responses, and modification of coagulation pathways. This chapter will focus on selected biochemical aspects of CoV replication and transcription with special attention to the interaction between cell and viral factors. 2.
4、INFLUENCE OF VIRAL AND CELLULAR PROTEINS IN CoV REPLICATION 2.1. Nuclear Localization of CoV Proteins There are at least three CoV proteins that have been localized within the nucleus of infected cells: nucleoprotein (N), 3b, and nsp1 (Table 1). The nucleolus has been implicated in many aspects of c
5、ell biology that include functions such as ribosomal rRNA synthesis and ribosome biogenesis, gene silencing, senescence, and cell cycle regulation.1-5 The nucleolus contains different factors including nucleolin, fibrillarin, spectrin, B23, rRNA, and ribosomal proteins S5 and L9. Viruses interact wi
6、th the nucleolus and its antigens; viral proteins co-localize with factors such as nucleolin, B23, and fibrillarin and cause their redistribution during infection.2 N proteins from CoV genus *Centro Nacional de Biotecnologa, CSIC, Darwin, 3, Cantoblanco, 28049 Madrid, Spain. 13 Extensive morphologic
7、al and biochemical changes occur in coronavirus (CoV)- L. ENJUANES ET AL. Table 1. NiV proteins in the nucleus of infected cells. known protein. Protein 3b is predominantly localized in the nucleolus. A functional nuclear localization signal is located in amino acids 134 to 154. Ectopic over-express
8、ion of protein 3b in Vero, 293, and COS-7 cells induced cell cycle arrest at Go/G1 phase.13 EAV nsp1 also has been localized in the nucleus.10,14 Therefore, in total, at least three nidovirales proteins (N, 3b and nsp1) have been detected in the nucleus of infected cells, suggesting that nidovirales
9、 may modify cell behavior through the nucleus. 2.2. CoV Genome Replication In CoV replication, recognition of RNA genome 5 and 3 ends by viral and cellular proteins is most likely essential. Furthermore, the interaction of these ends probably is a requirement for replication and transcription, as th
10、ese are processes that must be initiated at the 3 end of the genome, and it has been shown that these processes are influenced by sequences mapping at the 5 end of the genome.15,16 There may be a direct interaction between the CoV 5 end 3 ends, as predicted for MHV and TGEV RNA genomes in the absenc
11、e of protein using computer programs.17,18 Nevertheless, this direct interaction seems unlikely inside cells in the presence of cell and viral proteins. In fact, during genome synthesis, the first end synthesized (3) will be most likely immediately folded and nonspecifically covered by proteins, suc
12、h as the N protein or nsp9, or by proteins binding specific RNA motifs with characteristic secondary structures. In fact, the PROTEIN VIRUS REFERENCE N IBV Chen, et al., 2003 MHV Wurm, et al., 2001 SARS-CoV Timani, et al., 2004 TGEV Wurm, et al., 2001 PRRSV Rowland, et al.,1999 EAV Tijms, et al., 20
13、02 3b SARS-CoV Yuan, et al., 2005 nsp1 EAV van de Meer, et al., 1999 14 (transmissible gastroenteritis virus, TGEV),6? (mouse hepatitis virus, MHV, and severe and acute respiratory syndrome coronavirus, SARS-CoV),6-8 and ? (infectious bronchitis virus, IBV),9 and also from two arteriviruses porcine
14、respiratory and reproductive syndrome virus (PRRSV) and equine arteritis virus (EAV)10,11 localize in the nucleolus, and this may be a common feature among nidovirus N proteins that influences host cell proliferation.6 The association of N protein with the nucleus may be cell dependent. In 6 12 fact
15、, until now, N protein has been located in the nucleus of LLC-PK1 and Vero cells plasmids expressing N protein or infected with TGEV . Overall, these data indicate that significance. transfected with plasmids expressing N protein , but not in ST cells transfected with the presence of N protein in th
16、e nucleus of the infected cells might be of functional SARS-CoV ORF 3b encodes a protein of 154 amino acids, lacking similarities to any BIOCHEMICAL ASPECTS OF CoV REPLICATION postulated cross-talk between 5 and 3 ends and CoV replication has been shown in our laboratory.19 Precipitation of digoxigeninated 3-ends by biotinylated 5-ends using streptavidin sepharos
