With current techniques, genetic alterations of herpesviruses are difficult to perform, mostly because of the large size of their genomes. To solve this problem, we have designed a system that allows the cloning of any ␥-herpesvirus in Escherichia coli onto an F factor-derived plasmid. Immortalized B cell lines were readily established with recombinant Epstein-Barr virus (EBV), demonstrating that the F factorcloned EBV genome has all the characteristics of wild-type EBV. Because any genetic modification is possible in E. coli, this experimental approach opens the way to the genetic analysis of all EBV functions. Moreover, it is now feasible to generate attenuated EBV strains in vitro such that vaccine strains can be designed. Because we incorporated the genes for hygromycin resistance and green f luorescent protein onto the E. coli cloned EBV genome, the still open question of the EBV target cells other than B lymphocytes will be addressed.

Epstein-Barr virus (EBV), a herpes virus, infects human B lymphocytes in vitro and efficiently immortalizes them. About 10 of the approximately 100 genes of EBV are expressed in recently immortalized B cells and although there is circumstantial evidence that at least three of these may contribute to the process of immortalization, there is no direct evidence that any particular gene is required. We have developed a genetic analysis of EBV that uses a transformation-defective strain of the virus as a helper virus in conjunction with DNA that contains all of the viral cis-acting elements required for replication, cleavage and packaging during the lytic phase of the viral life cycle. This DNA can include viral genes required for immortalization that complement the transformation-defective virus strain. The DNA can be amplified and packaged by the products of the helper virus and the packaged DNA is infectious. We have analysed two viral genes expressed in immortalized cells and find that the gene encoding EBV nuclear antigen-2 is required for immortalization, whereas the gene for the EBV nuclear antigen leader protein is not.

The propagation of herpesviruses has long been viewed as a temporally regulated sequential process that results from the consecutive expression of speci®c viral transactivators. As a key step in this process, lytic viral DNA replication is considered as a checkpoint that controls the expression of the late structural viral genes. In a novel genetic approach, we show that both hypotheses do not hold true for the Epstein±Barr virus (EBV). The study of viral mutants of EBV in which the early genes BZLF1 and BRLF1 are deleted allowed a precise assignment of the function of these proteins. Both transactivators were absolutely essential for viral DNA replication. Both BZLF1 and BRLF1 were required for full expression of the EBV proteins expressed during the lytic program, although the respective in¯uence of these molecules on the expression of various viral target genes varied greatly. In replication-defective viral mutants, neither early gene expression nor DNA replication was a prerequisite for late gene expression. This work shows that BRLF1 and BZLF1 harbor distinct but complementary functions that in¯uence all stages of viral production.

A strategy for cloning and mutagenesis of an infectious herpesvirus genome is described. The mouse cytomegalovirus genome was cloned and maintained as a 230 kb bacterial artificial chromosome (BAC) in E. coli. Transfection of the BAC plasmid into eukaryotic cells led to a productive virus infection. The feasibility to introduce targeted mutations into the BAC cloned virus genome was shown by mutation of the immediate-early 1 gene and generation of a mutant virus. Thus, the complete construction of a mutant herpesvirus genome can now be carried out in a controlled manner prior to the reconstitution of infectious progeny. The described approach should be generally applicable to the mutagenesis of genomes of other large DNA viruses.Cytomegalovirus (CMV) is an important human pathogen with a high prevalence in the human population that causes severe and even fatal disease in immunologically immature or immunocompromised patients (1). Because human and mouse CMV (MCMV) show a series of similarities in biology and pathogenesis (2) infection of the mouse with MCMV has become an extensively used in vivo model to study the pathogenesis of CMV infection. The 235-kb genomes of both human and mouse CMV are the largest genomes of mammalian DNA viruses. Sequence analysis of the human and mouse CMV genomes revealed a similar genetic organization and a coding capacity for presumably more than 220 polypeptides (3-5). However, information on the function of the majority of CMV gene products is still rather limited. This is in sharp contrast to the alphaherpesviruses, where the study of a wealth of viral mutants contributed significantly to the understanding of viral gene functions (reviewed in ref. 6). There is a lack of CMV mutants because due to the large genome size and slow replication kinetics construction of CMV recombinants turned out to be difficult.The technique of insertional mutagenesis has been developed for disruption and deletion of CMV genes (7,8). Because the frequency of homologous recombination in eukaryotic cells is low the technique is quite ineffective. In addition adventitious deletions and the formation of illegitimate recombinant viruses have frequently been observed (refs. 7 and 9; I.C., unpublished data). Although selection procedures have improved the original technique (9-11) generation of CMV mutants remains a laborious, time-consuming, and often unsuccessful task. Recently, the technique for construction of recombinant herpesviruses from cloned overlapping fragments (12) has been extended to CMV (13). This is a major improvement in that the technique generates only recombinant virus and obviates selection against nonrecombinant wild type (wt) virus. Still, the resultant mutant is the product of several recombination events in eukaryotic cells that are difficult to control. Correct reconstitution of the viral genome can only be verified after growth and isolation of the mutant virus.Here we describe an approach for production of CMV mutants. Construction of the mutant genome is completely inde...