Abstract
To fully understand the function of cytomegalovirus (CMV) genes, it is imperative that they be studied in the context of infection. Therefore, the targeted deletion of individual viral genes and the comparison of loss of function viral mutants to the wild-type virus allow the identification of the relevance and role for a particular gene in the viral replication cycle. Targeted CMV mutagenesis has made huge advances over the past 15 years. The cloning of CMV genomes into (E. coli) as bacterial artificial chromosomes (BAC) allows not only quick and efficient deletion of viral genomic regions, individual genes, or single nucleotide exchanges in the viral genome but also the insertion of heterologous genetic sequences for gain of function approaches. The conceptual advantage of this strategy is that it overcomes the restrictions of recombinant technologies in cell culture systems. Namely, recombination in infected cells occurs only in a few clones, and their selection is not possible if the targeted genes are relevant for virus replication and are not able to compete for growth against the unrecombined viruses. On the other hand, BAC mutagenesis enables the selection for antibiotic resistance in E. coli, allowing a selective growth advantage to the recombined genomes. Here we describe the methods used for the generation of a CMV BAC, targeted mutagenesis of BAC clones, and transfection of human cells with CMV BAC DNA in order to reconstitute the viral infection process.
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References
Spaete RR, Mocarski ES (1987) Insertion and deletion mutagenesis of the human cytomegalovirus genome. Proc Natl Acad Sci U S A 84:7213–7217
Chee MS, Bankier AT, Beck S, Bohni R, Brown CM, Cerny R, Horsnell T, Hutchison CA 3rd, Kouzarides T, Martignetti JA et al (1990) Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154:125–169
Murphy E, Rigoutsos I, Shibuya T, Shenk TE (2003) Reevaluation of human cytomegalovirus coding potential. Proc Natl Acad Sci U S A 100:13585–13590
Davison AJ, Dolan A, Akter P, Addison C, Dargan DJ, Alcendor DJ, McGeoch DJ, Hayward GS (2003) The human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome. J Gen Virol 84:17–28
Dargan DJ, Douglas E, Cunningham C, Jamieson F, Stanton RJ, Baluchova K, McSharry BP, Tomasec P, Emery VC, Percivalle E, Sarasini A, Gerna G, Wilkinson GW, Davison AJ (2010) Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture. J Gen Virol 91:1535–1546
Sinzger C, Schmidt K, Knapp J, Kahl M, Beck R, Waldman J, Hebart H, Einsele H, Jahn G (1999) Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome. J Gen Virol 80(Pt 11):2867–2877
Borst EM, Hahn G, Koszinowski UH, Messerle M (1999) Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants. J Virol 73:8320–8329
Hahn G, Rose D, Wagner M, Rhiel S, McVoy MA (2003) Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Towne long as BACs and site-directed mutagenesis using a PCR-based technique. Virology 307:164–177
Marchini A, Liu H, Zhu H (2001) Human cytomegalovirus with IE-2 (UL122) deleted fails to express early lytic genes. J Virol 75: 1870–1878
Hahn G, Khan H, Baldanti F, Koszinowski UH, Revello MG, Gerna G (2002) The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells, as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-type characteristics. J Virol 76:9551–9555
Sinzger C, Hahn G, Digel M, Katona R, Sampaio KL, Messerle M, Hengel H, Koszinowski U, Brune W, Adler B (2008) Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E. J Gen Virol 89:359–368
Murphy E, Yu D, Grimwood J, Schmutz J, Dickson M, Jarvis MA, Hahn G, Nelson JA, Myers RM, Shenk TE (2003) Coding potential of laboratory and clinical strains of human cytomegalovirus. Proc Natl Acad Sci U S A 100:14976–14981
Stanton RJ, Baluchova K, Dargan DJ, Cunningham C, Sheehy O, Seirafian S, McSharry BP, Neale ML, Davies JA, Tomasec P, Davison AJ, Wilkinson GW (2010) Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication. J Clin Invest 120:3191–3208
Tischer BK, Smith GA, Osterrieder N (2010) En passant mutagenesis: a two step markerless red recombination system. Methods Mol Biol 634:421–430
Sambrook JF, Russel DW (2000) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y
Borst EM, Irena C-M, Messerle M (2004) Cloning of β-herpesvirus genomes as bacterial artificial chromosomes. In: Zhao SS, Stodolsky M (eds) Bacterial artificial chromosomes: volume 2: functional studies. Humana., Totowa, NJ, pp 221–240
Tischer BK, von Einem J, Kaufer B, Osterrieder N (2006) Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40:191–197
Muyrers JP, Zhang Y, Testa G, Stewart AF (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27:1555–1557
Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97:5978–5983
Wagner M, Gutermann A, Podlech J, Reddehase MJ, Koszinowski UH (2002) Major histocompatibility complex class I allele-specific cooperative and competitive interactions between immune evasion proteins of cytomegalovirus. J Exp Med 196:805–816
Yu D, Smith GA, Enquist LW, Shenk T (2002) Construction of a self-excisable bacterial artificial chromosome containing the human cytomegalovirus genome and mutagenesis of the diploid TRL/IRL13 gene. J Virol 76:2316–2328
Acknowledgments
I.D. is supported by a stipend from the Helmholtz International Graduate School for Infection Research; L.C.S. and M.M. are supported by the Helmholtz Virtual Institute “Viral Strategies of Immune Evasion” (VH-VI-424).
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Dekhtiarenko, I., Čičin-Šain, L., Messerle, M. (2014). Use of Recombinant Approaches to Construct Human Cytomegalovirus Mutants. In: Yurochko, A., Miller, W. (eds) Human Cytomegaloviruses. Methods in Molecular Biology, vol 1119. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-788-4_5
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DOI: https://doi.org/10.1007/978-1-62703-788-4_5
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