Abstract
Abstract The non‐structural proteins (nsp or replicase proteins) of coronaviruses are relatively conserved and can be effective targets for drugs. Few studies have been conducted into the function of the severe acute respiratory syndrome coronavirus (SARS‐CoV) nsp5. In this study, bioinformatics methods were employed to predict the secondary structure and construct 3‐D models of the SARS‐CoV GD strain nsp5. Sequencing and sequential comparison was performed to analyze the mutation trend of the polymerase nsp5 gene during the epidemic process using a nucleotide‐nucleotide basic local alignment search tool (BLASTN) and a protein‐protein basic local alignment search tool (BLASTP). The results indicated that the nsp5 gene was steady during the epidemic process and the protein was homologous with other coronavirus nsp5 proteins. The protein encoded by the nsp5 gene was expressed in COS‐7 cells and analyzed by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). This study provided the foundation for further exploration of the protein's biological function, and contributed to the search for anti‐SARS‐CoV drugs.
Edited by Bing SUN
Keywords: severe acute respiratory syndrome (SARS), severe acute respiratory syndrome coronavirus(SARS‐CoV), non‐structural protein (nsp), replicase, secondary structure, 3‐D structure
This research was supported by a grant from the Science Foundation for SARS of Guangdong Province (No. 2003Z3‐E0461)
References
- 1. Enserink M, Infectious diseases. Second suspect in the global mystery outbreak. Science 2003, 299: 1963. [DOI] [PubMed] [Google Scholar]
- 2. Gao F, Ou HY, Chen LL, Zheng WX, Zhang CT, Prediction of proteinase cleavage sites in polyproteins of coronaviruses and its application in analyzing SARS‐CoV genomes. FEBS Lett 2003, 553: 451–456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP, Peñaranda S et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 2003, 300: 1394–1399. [DOI] [PubMed] [Google Scholar]
- 4. Marra MA, Jones SJ, Astell CR, Holt RA, Brooks‐Wilson A, Butterfield YSN, Khattra J et al. The genome sequence of the SARS‐associated coronavirus. Science 2003, 300: 1399–1404. [DOI] [PubMed] [Google Scholar]
- 5. Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LLM, Guan Y et al. Unique and conserved features of genome and proteome of SARS‐coronavirus, an early split‐off from the coronavirus group 2 lineage. J Mol Biol 2003, 331: 991–1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Sutton G, Fry E, Carter L, Sainsbury S, Walter T, Nettleship J, Berrow N et al. The nsp9 replicase protein of SARS‐coronavirus, structure and functional insights. Structure 2004, 12: 341–353. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Thiel V, Ivanov KA, Putics A, Hertzig T, Schelle B, Bayer S, Weißbrich B et al. Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol 2003, 84: 2305–2315. [DOI] [PubMed] [Google Scholar]
- 8. Tijms MA, Dinten LC, Gorbalenya AE, Snijder EJ, A zinc finger‐containing papain‐like protease couples subgenomic mRNA synthesis to genome translation in a positive‐stranded RNA virus. Proc Natl Acad Sci USA 2001, 98: 1889–1894. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Bermejo Martin JF, Jimenez JL, Munoz‐Fernandez A, Pentoxifylline and severe acute respiratory syndrome (SARS): A drug to be considered. Med Sci Monit 2003, 9: 29–34. [PubMed] [Google Scholar]
- 10. Anand K, Ziebuhr J, Wadhwani P, Mesters J R, Hilgenfeld R, Coronavirus main proteinase (3CL‐Pro) structure: Basis for design of anti‐SARS Drugs. Science 2003, 300: 1763–1767. [DOI] [PubMed] [Google Scholar]
- 11. Lu JH, Yan XG, Guo ZM, Zheng HY, Zhang X, Wan ZY, Zhang RL et al. Establishment of SARS virus vaccine line. Guangdong Med J (Chin) 2003, 24: 194–195. [Google Scholar]
- 12. Zhang CH, Guo ZM, Zheng HY, Lu JH, Wang YF, Yan XG, Zhao Y et al. Humoral immune responses in rabbits induced by an experimental inactivated severe acute respiratory syndrome coronavirus vaccine prepared from F69 strain. Chin Med J 2004, 117: 1625–1629. [PubMed] [Google Scholar]
- 13. McGuffin LJ, Bryson K, Jones DT, The PSIPRED protein structure prediction server. Bioinformatics 2000, 16: 404–405. [DOI] [PubMed] [Google Scholar]
- 14. Kelley LA, MacCallum RM, Sternberg MJE, Enhanced genome annotation using structural profiles in the program 3D‐PSSM. J Mol Biol 2000, 299: 499–520. [DOI] [PubMed] [Google Scholar]
- 15. Lu Y, Denison MR, Determinants of mouse hepatitis virus 3C‐like proteinase activity. Virology 1997, 230: 335–342. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Ahmad K, New human coronavirus isolated. Lancet Infect Dis 2004, 4: 255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Davidkova G, Zhang SP, Nichols RA, Weiss B, Reduced level of calmodulin in PC12 cells induced by stable expression of calmodulin antisense RNA inhibits cell proliferation and induces neurite outgrowth. Neuroscience 1996, 75: 1003–1019. [DOI] [PubMed] [Google Scholar]