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Viruses rely on the metabolic network of their cellular hosts to provide energy and building blocks for viral replication. We developed a flux measurement approach based on liquid chromatography-tandem mass spectrometry to quantify changes in metabolic activity induced by human cytomegalovirus (HCMV). This approach reliably elucidated fluxes in cultured mammalian cells by monitoring metabolome labeling kinetics after feeding cells 13 C-labeled forms of glucose and glutamine. Infection with HCMV markedly upregulated flux through much of the central carbon metabolism, including glycolysis. Particularly notable increases occurred in flux through the tricarboxylic acid cycle and its efflux to the fatty acid biosynthesis pathway. Pharmacological inhibition of fatty acid biosynthesis suppressed the replication of both HCMV and influenza A, another enveloped virus. These results show that fatty acid synthesis is essential for the replication of two divergent enveloped viruses and that systems-level metabolic flux profiling can identify metabolic targets for antiviral therapy.The capability of mass spectrometry and nuclear magnetic resonance spectroscopy to quantify numerous metabolites simultaneously has given rise to the systems-level examination of metabolites (metabolomics) and their fluxes (fluxomics) [1][2][3] . Initial efforts to apply metabolomics to investigate human disease have focused largely on analysis of biofluids in normal versus affected individuals 4-7 . Although promising, such analysis is complicated by variation between individuals 8 . Moreover, the complexity of metabolic

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...

Human cytomegalovirus replicates in many different cell types, including epithelial cells, endothelial cells, and fibroblasts. However, laboratory strains of the virus, many of which were developed as attenuated vaccine candidates by serial passage in fibroblasts, have lost the ability to infect epithelial and endothelial cells. Their growth is restricted primarily to fibroblasts, due to mutations in the UL131-UL128 locus. We now demonstrate that two products of this locus, pUL130 and pUL128, form a complex with gH and gL, but not gO. The AD169 laboratory strain, which lacks a functional UL131 protein, produces virions containing only the gH-gL-gO complex. An epithelial and endothelial cell tropic AD169 variant in which the UL131 ORF has been repaired, termed BADrUL131, produces virions that carry both gH-gL-gO and gH-gL-pUL128-pUL130 complexes. Antibodies against pUL130 and pUL128 block infection of epithelial and endothelial cells by BADrUL131 and the fusion-inducing factor X clinical human cytomegalovirus isolate but do not affect the efficiency with which fibroblasts are infected.glycoprotein complex ͉ host range