Use of Recombinant Approaches to Construct Human Cytomegalovirus Mutants
- 1.9k Downloads
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.
Key wordsBacterial artificial chromosome (BAC) Targeted mutagenesis Homologous recombination Antibiotic selection
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).
- 5.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 91:1535–1546PubMedCentralPubMedCrossRefGoogle Scholar
- 10.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 76:9551–9555PubMedCentralPubMedCrossRefGoogle Scholar
- 13.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 120:3191–3208PubMedCentralPubMedCrossRefGoogle Scholar
- 15.Sambrook JF, Russel DW (2000) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.YGoogle Scholar