The development of the thymus depends initially on epithelial-mesenchymal and subsequently on reciprocal lympho-stromal interactions. The genetic steps governing development and differentiation of the thymic microenvironment are unknown. With the use of a targeted disruption of the whn gene, which recapitulates the phenotype of the athymic nude mouse, the WHN transcription factor was shown to be the product of the nude locus. Formation of the thymic epithelial primordium before the entry of lymphocyte progenitors did not require the activity of WHN. However, subsequent differentiation of primitive precursor cells into subcapsular, cortical, and medullary epithelial cells of the postnatal thymus did depend on activity of the whn gene. These results define the first genetically separable steps during thymic epithelial differentiation.

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...

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Human cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not been available in a phenotypically homogeneous form compatible with genetic analysis. To overcome this problem, we cloned the TB40/E strain into a bacterial artificial chromosome (BAC) vector. Both highly endotheliotropic and poorly endotheliotropic virus clones, representing three distinct restriction fragment patterns, were reconstituted after transfection of BAC clones derived from previously plaque-purified strain TB40/E. For one of the highly endotheliotropic clones, TB40-BAC4, we provide the genome sequence. Two BACs with identical restriction fragment patterns but different cell tropism were further analysed in the UL128-UL131A gene region. Sequence analysis revealed one coding-relevant adenine insertion at position 332 of UL128 in the BAC of the poorly endotheliotropic virus, which caused a frameshift in the C-terminal part of the coding sequence. Removal of this insertion by markerless mutagenesis restored the highly endotheliotropic phenotype, indicating that the loss of endothelial cell tropism was caused by this insertion. In conclusion, HCMV strain TB40/E, which combines the high endothelial cell tropism of a clinical isolate with the high titre growth of a cell culture adapted strain, is now available as a BAC clone suitable for genetic engineering. The results also suggest BAC cloning as a suitable method for selection of genetically defined virus clones.

Natural killer (NK) cells are innate lymphocytes that lack antigen-specific rearranged receptors, a hallmark of adaptive lymphocytes. In some people infected with human cytomegalovirus (HCMV), an NK cell subset expressing the activating receptor NKG2C undergoes clonal-like expansion that partially resembles anti-viral adaptive responses. However, the viral ligand that drives the activation and differentiation of adaptive NKG2C NK cells has remained unclear. Here we found that adaptive NKG2C NK cells differentially recognized distinct HCMV strains encoding variable UL40 peptides that, in combination with pro-inflammatory signals, controlled the population expansion and differentiation of adaptive NKG2C NK cells. Thus, we propose that polymorphic HCMV peptides contribute to shaping of the heterogeneity of adaptive NKG2C NK cell populations among HCMV-seropositive people.

Cytomegalovirus (CMV) infection is characterized by host immunosuppression and multiorganic involvement. CMV-infected dendritic cells (DC) were recently shown to display reduced immune functions, but their role in virus dissemination is not clear. In this report, we demonstrated that CMV could be captured by DC through binding on DC-SIGN and subsequently transmitted to permissive cells. Moreover, blocking DC-SIGN by specific antibodies inhibited DC infection by primary CMV isolates and expression of DC-SIGN or its homolog DC-SIGNR rendered susceptible cells permissive to CMV infection. We demonstrated that CMV envelope glycoprotein B is a viral ligand for DC-SIGN and DC-SIGNR. These results provide new insights into the molecular interactions contributing to cell infection by CMV and extend DC-SIGN implication in virus propagation.

SummaryAccording to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity.

We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Here, we have subjected the HCMV BAC to random transposon (Tn) mutagenesis using a Tn1721-derived insertion sequence and have provided the conditions for excision of the BAC cassette. We report on a fast and efficient screening procedure for a Tn insertion library. Bacterial clones containing randomly mutated full-length HCMV genomes were transferred into 96-well microtiter plates. A PCR screening method based on two Tn primers and one primer specific for the desired genomic position of the Tn insertion was established. Within three consecutive rounds of PCR a Tn insertion of interest can be assigned to a specific bacterial clone. We applied this method to retrieve mutants of HCMV envelope glycoprotein genes. To determine the infectivities of the mutant HCMV genomes, the DNA of the identified BACs was transfected into permissive fibroblasts. In contrast to BACs with mutations in the genes coding for gB, gH, gL, and gM, which did not yield infectious virus, BACs with disruptions of open reading frame UL4 (gp48) or UL74 (gO) were viable, although gO-deficient viruses showed a severe growth deficit. Thus, gO (UL74), a component of the glycoprotein complex III, is dispensable for viral growth. We conclude that our approach of PCR screening for Tn insertions will greatly facilitate the functional analysis of herpesvirus genomes.