Human cytomegalovirus has a complex double-stranded DNA genome of Ϸ240,000 bp that contains Ϸ150 ORFs likely to encode proteins, most of whose functions are not well understood. We have used an infectious bacterial artificial chromosome to introduce 413 defined insertion and substitution mutations into the human cytomegalovirus AD169 genome by random and sitedirected transposon mutagenesis. Mutations were produced in all unique ORFs with a high probability of encoding proteins for which mutants have not been previously documented and in many previously characterized ORFs. The growth of selected mutants was assayed in cultured human fibroblasts, and we now recognize 41 essential, 88 nonessential, and 27 augmenting ORFs. Most essential and augmenting genes are located in the central region, and nonessential genes generally cluster near the ends of the viral genome. Human cytomegalovirus (HCMV) is the prototypic ␤-herpes virus and a ubiquitous human pathogen. Although infections in healthy children and adults are generally asymptomatic, HCMV is a leading viral cause of birth defects and a major cause of morbidity and mortality in immunocompromised individuals (1).HCMV contains a complex double-stranded DNA genome of Ϸ240,000 bp, the largest genome for a virus known to infect humans. The laboratory strain of HCMV, AD169, contains Ϸ150 ORFs likely to encode proteins (2-5). Most ORFs have not been well studied due to the limited host range and slow growth of HCMV in cultured cells and the lack of efficient tools to generate mutant viruses. Recently, the HCMV genome has been cloned as an infectious bacterial artificial chromosome (BAC) (6-9), greatly facilitating its genetic manipulation (8, 10).We previously described an infectious BAC clone of HCMV AD169, termed pAD͞Cre (6). The BAC vector is flanked by LoxP sites and contains a Cre-recombinase gene that is modified by the insertion of an intron into its coding sequence. Consequently, Cre is not expressed in bacterial cells, but it is expressed when its transcript is spliced in human cells and the BAC vector is excised from the virus. Now we report the use of both random and site-directed transposon mutagenesis to introduce 413 defined insertion and substitution mutations into the HCMV AD169 genome residing in pAD͞Cre. Mutations were produced in all ORFs with a high probability of encoding proteins for which mutants have not been previously documented and in many previously characterized ORFs. We have begun to systematically delineate the functions of viral ORFs in HCMVinfected cells by analyzing the growth of HCMV mutants in cultured human fibroblasts. We now recognize 41 essential, 88 nonessential, and 27 augmenting ORFs. This work describes a functional map of the complete HCMV genome and provides a foundation for future genetic studies. MethodsCells, Viruses, and Plasmids. Primary human foreskin fibroblasts at passage 8-15 were propagated in medium supplemented with 10% FCS. The HCMV strain AD169 BAC, pAD͞Cre (6), was the wild-type parent of all mutant viruses. A...

Six strains of human cytomegalovirus have been sequenced, including two laboratory strains (AD169 and Towne) that have been extensively passaged in fibroblasts and four clinical isolates that have been passaged to a limited extent in the laboratory (Toledo, FIX, PH, and TR). All of the sequenced viral genomes have been cloned as infectious bacterial artificial chromosomes. A total of 252 ORFs with the potential to encode proteins have been identified that are conserved in all four clinical isolates of the virus. Multiple sequence alignments revealed substantial variation in the amino acid sequences encoded by many of the conserved ORFs

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