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deletions and the formation of illegitimate recombinant viruses have frequently been observed (refs. 7 and 9; I.C., unpublished data).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...

SummaryVirus shedding from the epithelial cells of the serous acini of salivary glands is a major source for the horizontal transmission of cytomegalovirus. These cells are, different to other tissues, exempt from CD8 T lymphocyte control. CD4 T lymphocytes are essential to terminate the productive infection. Here, we prove that T-B cooperation and the production of antibodies are not required for this process. For the infection with murine cytomegalovirus, mutant mice were used which do not produce antibodies because of a disrupted membrane exon of the immunoglobulin # chain gene. Also, in these mice the virus clearance from salivary glands is a function of CD4 T lymphocytes. However, these mice clear the virus and establish viral latency with a kinetics that is distinguishable from normal mice. Reactivation from virus latency is the only stage at which the absence of antibodies alters the phenotype of infection. In immunoglobulindeficient mice, virus recurrence results in higher virus titers. The adoptive serum transfer proved that antibody is the limited factor that prevents virus dissemination in the immunodeficient host.

Interferon gamma (IFNT) represents an essential cytokine involved in murine cytomegalovirus (MCMV) clearance from the salivary gland and the control of horizontal transmission. Because IFN 7 cannot be responsible for all cytokine effects during recovery from MCMV infection we have now tested the potential participation of tumour necrosis factor alpha (TNFct) in the antiviral defence. Neutralization of endogenous TNF~ abolished the antiviral activity of CD4 T cells in immunocompetent as well as in CD8 subset-deficient mice. These data suggest that the antiviral effect of the CD4 subset requires the presence of at least two cytokines, namely IFN7 and TNF~. Depletion of endogenous TNF~ in adoptive cell transfer recipients diminished the antiviral function of CD8 T lymphocytes suggesting that TNFct also participates in CD8 T cell effector functions. Furthermore, endogenous cytokines were found to be required for survival after infection with lethal doses of MCMV, whereas immunotherapy with recombinant TNF~ and IFN~ could not limit virus replication in vivo. The results suggest that, similar to IFNy, TNFct is an integral part of the protective mechanisms involved in cytomegalovirus clearance.

It has been claimed that MHC class I proteins serve as receptors for murine cytomegalovirus (MCMV) and that this interaction is the most important mechanism for virus entry in most cells. This claim is based on the observation that the MHC haplotype contributes to the susceptibility to cytomegalovirus (CMV) infection in vivo. Results from in vitro studies support the concept that stable expression of correctly folded MHC class I molecules contributes to infection, since the individual properties of MHC class I alleles, the availability offl2-microglobulin (fl2m) and also the degree of peptide charging of the MHC class I heavy chain fl2m heterodimers determined the infection phenotype of cell lines. To assess the biological relevance of proper MHC class I expression we investigated CMV infection in fl2m-deficient mice which fail to express ternary MHC class I complexes and lack peripheral CD8 + T lymphocytes. We found that organ virus titres and virus clearance kinetics were not altered in fl2m mutant mice. In addition, there was no indication of diminished virus propagation in fl2m / embryonic fibroblasts, fl2m -lmice suffered from the lack ofCD8 + T lymphocytes that was partially compensated for by the function of CD4 ÷ T lymphocytes. An organ-specific anti-virus function of natural killer (NK) cells was observed, independent from the p2 m deletion. The immune control unique for salivary gland infection was maintained. From the data presented here, we confirm the role of MHC class I molecules in the immune surveillance of CMV infection but question the biological impact of correct MHC class I complexes for productive infection.