Rev. Med. Virol. 2003; 13: 111–121. Published online in Wiley InterScience (www.interscience.wiley.com). Reviews in Medical Virology DOI: 10.1002/rmv.380 R E V I E W Cloning of herpesviral genomes as bacterial artificial chromosomes Heiko Adler1*, Martin Messerle2 and Ulrich H. Koszinowski3 1GSF—Research Center for Environment and Health, Institute of Molecular Immunology, Clinical Cooperation Group Hematopoietic Cell Transplantation, Munich, Germany 2Virus Cell Interaction Group, Medical Faculty, University of Halle-Wittenberg, Halle, Germany 3Max von Pettenkofer-Institute, Department of Virology, Genecenter, Ludwig-Maximilians-University, Munich, Germany SUMMARY Herpesviruses, which are important pathogens for both animals and humans, have large and complex genomes with a coding capacity for up to 225 open reading frames (ORFs). Due to the large genome size and the slow replication kinetics in vitro of some herpesviruses, mutagenesis of viral genes in the context of the viral genome by conventional recombination methods in cell culture has been difficult. Given that mutagenesis of viral genes is the basic strategy to investigate function, many of the herpesvirus ORFs could not be defined functionally. Recently, a completely new approach for the construction of herpesvirus mutants has been developed, based on cloning of the virus genome as a bacterial artificial chromosome (BAC) in E. coli. This technique allows the maintenance of viral genomes as a plasmid in E. coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. Any genetic modification of the viral genome in E. coli using prokaryotic recombination proteins is possible, thereby allowing the generation of mutant viruses and facilitating the analysis of herpesvirus genomes cloned as infectious BACs. In this review, we describe the principle of cloning a viral genome as a BAC using murine gammaherpesvirus 68 (MHV-68), a mouse model for gammaherpesvirus infections, as an example. Copyright # 2003 John Wiley & Sons, Ltd. Accepted: 10 October 2002 INTRODUCTION ing up to 225 open reading frames (ORFs) [1]. Herpesviruses are important pathogens for both These large and complex genomes can code for animals and humans [1]. After primary infection, an extensive repertoire of gene functions required they persist lifelong in their host in a latent state, to interfere efficiently with the host immune interrupted by occasional episodes of reactivation. response. Although the complete sequence of the A prerequisite for establishing a persistent infec- genome of many herpesviruses has been deter- tion despite the host’s immune system is the abil- mined, the precise function for many of the viral ity of herpesviruses to modulate the virus–host genes, both in vitro, and in particular in vivo, has interaction [2–6]. Herpesviruses possess double- not been elucidated. Knowing the function of indi- stranded DNA genomes of 100–250 kbp, contain- vidual or families of viral genes is important: (a) to understand their role in pathogenesis; (b) to exam- ine their potential as a therapeutic target; (c) for *Corresponding author: Dr H. Adler, GSF—Research Center for the rational design of vaccines; and (d) for utilisa- Environment and Health, Institute of Molecular Immunology, Clini- cal Cooperation Group Hematopoietic Cell Transplantation, Marchio- tion as a tool in gene therapy. ninistrasse 25, D-81377 Munich, Germany. E-mail: [email protected] In principle, the strategy to assign a function to a viral gene in the context of the whole viral genome Abbreviations used BAC, bacterial artificial chromosome; gfp, green fluorescent protein; is mutagenesis of the gene of interest, followed by gpt, guanosine phosphoribosyl transferase; HCMV, human cytomega- the analysis of the phenotype of the mutant virus lovirus; HHV-8, human herpesvirus 8; HVS, herpesvirus saimiri; both in vitro and in vivo. Conventional methods KSHV, Kaposi’s sarcoma herpesvirus; MCMV, mouse cytomegalo- virus; MHV-68, murine gammaherpesvirus 68; PRV, pseudorabies- for mutagenesis of herpesviruses included chemi- virus; Tn, transposon. cal mutagenesis, site-directed mutagenesis by Copyright # 2003 John Wiley & Sons, Ltd. 112 H. Adler et al. homologous recombination in eukaryotic cells, and MHV-68 is a mouse model of -herpesvirus manipulating virus genomes using overlapping infection [25–32]. In humans, the prototypic 1- cosmid clones (reviewed, for example, in [7–9]). herpesvirus, EBV, is associated with lymphomas These methods proved very useful for the genera- and nasopharyngeal carcinoma [33], and HHV-8, tion of a variety of mutants but their construction is a 2-herpesvirus, is associated with Kaposi’s sar- often inefficient, laborious and time consuming, coma, primary effusion lymphomas and multi- mainly due to the large genome size and, in case centric Castleman’s disease [34]. In vivo studies of the - and -herpesviruses, the slow replication of -herpesvirus pathogenesis have been limited kinetics or the lack of replication in vitro. Conven- to clinical investigation of the infection because tional methods are performed in eukaryotic cells of the restricted host range of these viruses. and therefore are difficult to control. One problem MHV-68 is a natural pathogen of wild murid is that the analysis of the mutant viral genomes is rodents [35], and is capable of infecting laboratory only possible at the very end of the lengthy experi- mice. Clinically, MHV-68 infection of mice induces mental procedure and thus additional changes in a syndrome very similar to EBV in humans [25]. the viral genome such as deletions, rearrangements Genetically, MHV-68 is similar to EBV, but more or illegitimate recombinations are frequently only closely related to herpesvirus saimiri (HVS) and observed then. Furthermore, the generation of a HHV-8 [36]. A major advantage of this model is viral mutant requires selection against nonrecom- the availability of genetically defined mouse binant wt virus and finally separation of the strains rendered deficient for specific parameters, mutant virus from the wt virus, for example by pla- for example by gene-knockout technology. The que-purification. Additionally, complementing cell genome of MHV-68 consists of 118 237 bp of lines are required when mutations are introduced unique sequence flanked by multiple copies of a into essential viral genes. The construction of a 1213 bp terminal repeat, and contains genes com- mutant is therefore often a limiting step for the mon to other members of the gammaherpes- analysis of a viral gene function in the genomic viruses, homologues of cellular genes, but also context. genes specific for MHV-68 only [36]. The analysis Recently, a completely new approach for the of MHV-68 mutants will significantly contribute construction of herpesvirus mutants has been to the understanding of viral gene functions developed. This approach is based on cloning of and to the evaluation of their role in the patho- the virus genome as a bacterial artificial chromo- genesis of gammaherpesvirus infections. some (BAC) in E. coli [10]. Soon after the pioneering work of Messerle et al. [10], who cloned the entire CONSTRUCTION OF A RECOMBINANT genome of mouse cytomegalovirus (MCMV) as VIRUS CONTAINING THE BAC VECTOR BAC, the cloning of several herpesviruses includ- SEQUENCES ing human cytomegalovirus (HCMV), HSV, pseu- For the generation of a virus BAC, the viral genome dorabiesvirus (PRV), EBV, Kaposi’s sarcoma and the BAC vector have to be joined. Usually, the herpesvirus (KSHV, also called human herpes- BAC vector is inserted into the virus genome by virus 8 [HHV-8]) and murine gammaherpesvirus homologous recombination in eukaryotic cells 68 (MHV-68) as BACs has been described [11– which is comparable to the construction of a 24]. This technique allows the maintenance of viral recombinant virus by the conventional mutagen- genomes as a BAC in E. coli and the reconstitution esis technique (Figure 1). This could in theory of viral progeny by transfection of the BAC plas- also be achieved by ligation of the linear viral gen- mid into eukaryotic cells. Mutagenesis of the virus ome to the BAC vector in vitro. In the case of MHV- genome in E. coli using prokaryotic recombination 68, the left end of the genome was chosen for the functions is possible, thereby allowing the genera- integration of the BAC vector sequences since this tion of mutant viruses. Using this method, any region has been shown to be dispensable for lytic genetic modification should be possible, thereby replication in vitro and for latent infection in vivo facilitating the analysis of herpesvirus genomes [37]. Additionally, that study and previous work cloned as infectious BACs. Here, we describe the describing the generation of HVS recombinants principle of cloning a viral genome as BAC using showed that in the case of gammaherpesviruses, MHV-68 as an example [18]. insertions between the unique region of the DNA Copyright # 2003 John Wiley & Sons, Ltd. Rev. Med. Virol. 2003; 13: 111–121. Cloning of herpesviral genomes 113 TRANSFER OF THE RECOMBINANT GENOME FROM INFECTED CELLS TO E. COLI Since the linear, double-stranded DNA genome of herpesviruses circularises during replication, cir- cular viral DNA can be isolated from infected cells. In the case of MHV-68, this was done after three rounds of selection. The circular replication inter- mediate was then transferred to E. coli by DNA transformation. It is important to note that this transfer into E. coli is needed only once. Isolation of plasmids from single E. coli colonies and restric- tion enzyme analysis led to the identification of a Figure 1. Construction of the MHV-68 BAC genome. The BAC- bacterial clone containing a BAC plasmid with the cloned genome was generated in eukaryotic cells by homologous recombination of MHV-68 DNA with a recombination plasmid full-length MHV-68 genome. containing 1.5 kbp of flanking homologous sequence (white box) as well as the BAC vector (BAC), the guanosine phosphori- PROPAGATION AND MUTAGENESIS bosyl transferase gene (gpt) and the gene for the green fluorescent IN E.