The large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequence motifs suggested a common ancestry and related functions for these gene products. Two members of this family, m140 and m141, were recently shown to affect MCMV replication on macrophages. To test the role of all US22 members in cell tropism, we analyzed the growth properties in different cell types of MCMV mutants carrying transposon insertions in all 12 US22 gene family members. When necessary, additional targeted mutants with gene deletions, ATG deletions, and ectopic gene revertants were constructed. Mutants with disruption of genes M23, M24, m25.1, m25.2, and m128 (ie2) showed no obvious growth phenotype, whereas growth of M43 mutants was reduced in a number of cell lines. Genes m142 and m143 were shown to be essential for virus replication. Growth of mutants with insertions into genes M36, m139, m140, and m141 in macrophages was severely affected. The common phenotype of the m139, m140, and m141 mutants was explained by an interaction at the protein level. The M36-dependent macrophage growth phenotype could be explained by the antiapoptotic function of the gene that was required for growth on macrophages but not for growth on other cell types. Together, the comprehensive set of mutants of the US22 gene family suggests that individual family members have diverged through evolution to serve a variety of functions for the virus.Herpesviruses are large and complex DNA viruses, widely found in nature. Human cytomegalovirus (HCMV), an important human pathogen, defines the betaherpesvirus family. Mouse CMV (MCMV) and rat CMV serve as biological model systems for HCMV. HCMV, MCMV, and rat CMV display the largest genomes among the herpesviruses (13,34,43). These genomes are essentially colinear over the central 180 kb of the 230-kb genomes. Betaherpesviruses, which include the CMVs as well as human herpesviruses 6 and 7, differ from alpha-and gammaherpesviruses by the presence of additional gene families such as the US22 gene family, which are mainly clustered at the ends of the genome (29, 30).The US22 family was first described in HCMV (13). This gene family comprises 12 members in both HCMV and MCMV and 11 in rat CMV. Members of the US22 gene family are characterized by stretches of hydrophobic and charged residues as well as up to four conserved sequence motifs which are specific for betaherpesviruses. Motif I differs between the HCMV US and UL family members (30). In MCMV, m128 and m139 to m143 share the HCMV US-like motif I, while M23, M24, m25.1, m25.2, M36, and M43 share UL-like motif I. Motifs I and II have consensus sequences, while motifs III and IV are less well defined but have stretches of nonpolar residues (18, 24). The m139 to m141 genes contain all four of these motifs, whereas m142 and m143 (and IRS1/TRS1 of HCMV) lack motif II. In addition, m139, m140, m142, and m143 each have an acidic domain, common to he...

Human cytomegalovirus infects vascular tissues and has been associated with atherogenesis and coronary restenosis. Although established laboratory strains of human cytomegalovirus have lost the ability to grow on vascular endothelial cells, laboratory strains of murine cytomegalovirus retain this ability. With the use of a forward-genetic procedure involving random transposon mutagenesis and rapid phenotypic screening, we identified a murine cytomegalovirus gene governing endothelial cell tropism. This gene, M45, shares sequence homology to ribonucleotide reductase genes. Endothelial cells infected with M45-mutant viruses rapidly undergo apoptosis, suggesting that a viral strategy to evade destruction by cellular apoptosis is indispensable for viral growth in endothelial cells.

Genes that inhibit apoptosis have been described for many DNA viruses. Herpesviruses often contain even more than one gene to control cell death. Apoptosis inhibition by viral genes is postulated to contribute to viral fitness, although a formal proof is pending. To address this question, we studied the mouse cytomegalovirus

Herpesviruses are important pathogens in animals and humans. The large DNA genomes of several herpesviruses have been sequenced, but the function of the majority of putative genes is elusive. Determining which genes are essential for their replication is important for identifying potential chemotherapy targets, designing herpesvirus vectors, and generating attenuated vaccines. For this purpose, we recently reported that herpesvirus genomes can be maintained as infectious bacterial artificial chromosomes (BAC) in Escherichia coli. Here we describe a one-step procedure for random-insertion mutagenesis of a herpesvirus BAC using a Tn1721-based transposon system. Transposon insertion sites were determined by direct sequencing, and infectious virus was recovered by transfecting cultured cells with the mutant genomes. Lethal mutations were rescued by cotransfecting cells containing noninfectious genomes with the corresponding wild-type subgenomic fragments. We also constructed revertant genomes by allelic exchange in bacteria. These methods, which are generally applicable to any cloned herpesvirus genome, will facilitate analysis of gene function for this virus family.

Mice lacking g 2-microglobulin (g 2 m − mice) express greatly reduced levels of MHC class I molecules, and cells from g 2 m − mice are therefore highly sensitive to NK cells. However, NK cells from g 2 m − mice fail to kill g 2 m − normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of g 2 m − NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to g 2 m − NK cells, we made the following observations: First, g 2 m − NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to g 2 m − concanavalin A (Con A)-activated T cell blasts. Secondly, g 2 m − NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than g 2 m + NK cells. Thirdly g 2 m − NK cells were not only tolerant to g 2 m − Con A blasts but also to Con A blasts from H-2K b− /D b− double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of g 2 m − NK cells to distinguish between MHC class I-negative and-positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in g 2 m − mice, which may instead operate through mechanisms involving triggering pathways. Abbreviations: I 2 m: g 2-Microglobulin TAP: Transporter associated with antigen processing