Metabolomics in drug target discovery.

Most diseases result in metabolic changes. In many cases, these changes play a causative role in disease progression. By identifying pathological metabolic changes, metabolomics can point to potential new sites for therapeutic intervention. Particularly promising enzymatic targets are those that carry increased flux in the disease state. Definitive assessment of flux requires the use of isotope tracers. Here we present techniques for finding new drug targets using metabolomics and isotope tracers. The utility of these methods is exemplified in the study of three different viral pathogens. For influenza A and herpes simplex virus, metabolomic analysis of infected versus mock-infected cells revealed dramatic concentration changes around the current antiviral target enzymes. Similar analysis of human-cytomegalovirus-infected cells, however, found the greatest changes in a region of metabolism unrelated to the current antiviral target. Instead, it pointed to the tricarboxylic acid (TCA) cycle and its efflux to feed fatty acid biosynthesis as a potential preferred target. Isotope tracer studies revealed that cytomegalovirus greatly increases flux through the key fatty acid metabolic enzyme acetyl-coenzyme A carboxylase. Inhibition of this enzyme blocks human cytomegalovirus replication. Examples where metabolomics has contributed to identification of anticancer drug targets are also discussed. Eventual proof of the value of metabolomics as a drug target discovery strategy will be successful clinical development of therapeutics hitting these new targets.

Human cytomegalovirus genome.

Human cytomegalovirus (HCMV) contains a large and complex E-type genome. There are both clinical isolates of the virus that have been passaged minimally in fibroblasts and so-called laboratory strains that have been extensively passaged and adapted to growth in fibroblasts. The genomes of laboratory strains have undergone rearrangements. To date, the genomes of five clinical isolates have been sequenced. We have re-evaluated the coding content of clinical isolates by identifying the set of open reading frames (ORFs) that are conserved in all five sequenced clinical isolates. We have further determined which of these ORFs are present in the chimpanzee cytomegalovirus (CCMV) genome. A total of 173 ORFs are present in all HCMV genomes and the CCMV genome, and we conclude that these ORFs are very likely to be functional. An additional 59 ORFs are present in the genomes of all five HCMV isolates, but not in CCMV. We have discounted 26 of this latter set of ORFs, because they reside in regions of the genome unlikely to encode functional ORFs. The remaining 33 ORFs are potentially functional ORFs that are specific to HCMV.

Altered cellular mRNA levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mRNAs.

The effect of human cytomegalovirus (HCMV) infection on cellular mRNA accumulation was analyzed by gene chip technology. During a 48-h time course after infection of human diploid fibroblasts, 1,425 cellular mRNAs were found to be up-regulated or down-regulated by threefold or greater in at least two consecutive time points. Several classes of genes were prominently affected, including interferon response genes, cell cycle regulators, apoptosis regulators, inflammatory pathway genes, and immune regulators. The number of mRNAs that were up-regulated or down-regulated were roughly equal over the complete time course. However, for the first 8 h after infection, the number of up-regulated mRNAs was significantly less than the number of down-regulated mRNAs. By analyzing the mRNA expression profile of cells infected in the presence of cycloheximide, it was found that a minimum of 25 mRNAs were modulated by HCMV in the absence of protein synthesis. These included mRNAs encoded by a small number of interferon-responsive genes, as well as beta interferon itself. Cellular mRNA levels in cytomegalovirus-infected cells were compared to the levels in cells infected with UV-inactivated virus. The inactivated virus caused the up-regulation of a much greater number of mRNAs, many of which encoded proteins with antiviral roles, such as interferon-responsive genes and proinflammatory cytokines. These data argue that one or more newly synthesized viral gene products block the induction of antiviral pathways that are triggered by HCMV binding and entry.

In silico structural and functional analysis of the human cytomegalovirus (HHV5) genome.

The open reading frames of human cytomegalovirus (human herpesvirus-5, HHV5) encode some 213 unique proteins with mostly unknown functions. Using the threading program, ProCeryon, we calculated possible matches between the amino acid sequences of these proteins and the Protein Data Bank library of three-dimensional structures. Thirty-six proteins were fully identified in terms of their structure and, often, function; 65 proteins were recognized as members of narrow structural/functional families (e.g. DNA-binding factors, cytokines, enzymes, signaling particles, cell surface receptors etc.); and 87 proteins were assigned to broad structural classes (e.g. all-beta, 3-layer-alphabetaalpha, multidomain, etc.). Genes encoding proteins with similar folds, or containing identical structural traits (extreme sequence length, runs of unstructured (Pro and/or Gly-rich) residues, transmembrane segments, etc.) often formed tandem clusters throughout the genome. In the course of this work, benchmarks on about 20 known folds were used to optimize adjustable parameters of threading calculations, i.e. gap penalty weights used in sequence/structure alignments; new scores obtained as simple combinations of existing scoring functions; and number of threading runs conducive to meaningful results. An introduction of summed, per-residue-normalized scores has been essential for discovery of subdomains (EGF-like, SH2, SH3) in longer protein sequences, such as the eight "open sandwich" cytokine domains, 60-70 amino acids long and having the 3beta1alpha fold with one or two disulfide bridges, present in otherwise unrelated proteins.

Murine cytomegalovirus M78 protein, a G protein-coupled receptor homologue, is a constituent of the virion and facilitates accumulation of immediate-early viral mRNA.

The M78 protein of murine cytomegalovirus exhibits sequence features of a G protein-coupled receptor. It is synthesized with early kinetics, it becomes partially colocalized with Golgi markers, and it is incorporated into viral particles. We have constructed a viral substitution mutant, SMsubM78, which lacks most of the M78 ORF. The mutant produces a reduced yield in cultured 10.1 fibroblast and IC21 macrophage cell lines. The defect is multiplicity dependent and greater in the macrophage cell line. Consistent with its growth defect in cultured cells, the mutant exhibits reduced pathogenicity in mice, generating less infectious progeny than wild-type virus in all organs assayed. SMsubM78 fails to efficiently activate accumulation of the viral m123 immediate-early mRNA in infected macrophages. M78 facilitates the accumulation of the immediate-early mRNA in cycloheximide-treated cells, arguing that it acts in the absence of de novo protein synthesis. We conclude that the M78 G protein-coupled receptor homologue is delivered to cells as a constituent of the virion, and it acts to facilitate the accumulation of immediate-early mRNA.

Interaction between YY1 and the retinoblastoma protein. Regulation of cell cycle progression in differentiated cells.

Overexpression of the transcription factor YY1 activates DNA synthesis in differentiated primary human coronary artery smooth muscle cells. Overexpression of the retinoblastoma protein together with YY1 blocked this effect. In growth-arrested cells, YY1 resides in a complex with the retinoblastoma protein, but the complex is not detected in serum-stimulated S phase cultures, indicating that the interaction of the retinoblastoma protein and YY1 is cell cycle-regulated. Recombinant retinoblastoma protein directly interacts with YY1, destabilizing the interaction of YY1 with DNA and inhibiting its transcription initiator function in vitro. We conclude that in differentiated cells elevation of the nuclear level of YY1 protein favors progression into the S phase, and we propose that this activity is regulated by its interaction with the retinoblastoma protein.

UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirus-infected cells.

The human cytomegalovirus UL82 gene encodes a protein (pp71) that is localized in the tegument domain of the virus particle. The UL82 gene product is delivered to the nucleus at the time of infection, and it is believed to function in gene activation. We have constructed a human cytomegalovirus mutant, ADsubUL82, that lacks a substantial portion of the UL82 coding region. It was propagated on human diploid fibroblasts expressing the UL82 gene product, and it was possible to produce a mutant virus lacking the UL82 protein by passaging virus stocks for one cycle of growth on normal, noncomplementing fibroblasts. The UL82-deficient mutant displays a multiplicity-dependent growth defect in normal human fibroblasts. The growth of ADsubUL82 is severely restricted at low input multiplicities (0.01-0.1 plaque-forming units per cell), producing a yield that is reduced by a factor of about 10(5) in comparison to wild-type virus. At higher input multiplicities (10 plaque-forming units per cell), ADsubUL82 grew nearly as well as the wild-type virus. By using a human cytomegalovirus gene array, we demonstrated that UL82 functions to facilitate virus mRNA accumulation very early during the human cytomegalovirus replication cycle. The growth phenotype associated with the UL82 mutant seems to result from its inability to efficiently activate human cytomegalovirus immediate early genes.

Efficient replication of adeno-associated virus type 2 vectors: a cis-acting element outside of the terminal repeats and a minimal size.

Recombinant adeno-associated virus type 2 (AAV2) can be produced in adenovirus-infected cells by cotransfecting a plasmid containing the recombinant AAV2 genome, which is generally comprised of the viral terminal repeats flanking a transgene, together with a second plasmid expressing the AAV2 rep and cap genes. However, recombinant viruses generally replicate inefficiently, often producing 100-fold fewer virus particles per cell than can be obtained after transfection with a plasmid containing a wild-type AAV2 genome. We demonstrate that this defect is due, at least in part, to the presence of a positive-acting cis element between nucleotides 194 and 1882 of AAV2. Recombinant AAV2 genomes lacking this region accumulated 14-fold less double-stranded, monomer-length replicative-form DNA than did wild-type AAV2. In addition, we demonstrate that a minimum genome size of 3.5 kb is required for efficient production of single-stranded viral DNA. Relatively small recombinant genomes (2,992 and 3,445 bp) accumulated three- to eightfold less single-stranded DNA per monomer-length replicative-form DNA molecule than wild-type AAV2. In contrast, recombinant AAV2 with larger genomes (3,555 to 4,712 bp) accumulated similar amounts of single-stranded DNA per monomer-length replicative-form DNA compared to wild-type AAV2. Analysis of two recombinant AAV2 genomes less than 3.5 kb in size indicated that they were deficient in the production of the extended form of monomer-length replicative-form DNA, which is thought to be the immediate precursor to single-stranded AAV2 DNA.

Replication of wild-type and mutant human cytomegalovirus in life-extended human diploid fibroblasts.

A cDNA encoding the catalytic subunit of human telomerase was used to generate life-extended derivatives of primary human diploid fibroblasts. The life-extended cells supported efficient human cytomegalovirus (HCMV) replication. A subclone of the life-extended cells was generated containing the HCMV UL82 gene and used to isolate and propagate a virus that exhibited a profound growth defect after infection at a low input multiplicity.

A subset of viral transcripts packaged within human cytomegalovirus particles.

A human cytomegalovirus gene array was used to identify a previously unidentified class of viral transcripts. These transcripts, termed virion RNAs, were packaged within infectious virions and were delivered to the host cell on infection. This mechanism of herpesvirus gene expression allows for viral genes to be expressed within an infected cell immediately after virus entry and in the absence of transcription from the viral genome.

Human cytomegalovirus UL69 protein is required for efficient accumulation of infected cells in the G1 phase of the cell cycle.

Human cytomegalovirus blocks cell-cycle progression in the G(1) compartment upon infection of primary human fibroblasts. The virus-coded UL69 protein can institute a G(1) block when expressed in cells in the absence of virus infection. We have constructed a cytomegalovirus mutant, TNsubUL69, that lacks the UL69 coding region. This virus grows slowly in fibroblasts, but produces a wild-type yield after an extended delay. It grows with normal kinetics in cells coinfected with a recombinant retrovirus, retroUL69, which expresses UL69 protein, demonstrating that its growth defect results from the mutation in the UL69 gene. UL69 protein is packaged within virus particles, and it was possible for us to produce two types of virus stocks. TNsubUL69(+pUL69) lacks the UL69 gene but contains UL69 protein in virus particles. It is produced by growth in fibroblasts that are coinfected with retroUL69. TNsubUL69(-pUL69) lacks the UL69 gene and protein. It is produced by growth in fibroblasts that do not contain UL69 protein. The mutant virions lacking both the UL69 gene and protein fail to induce a cell-cycle block with normal efficiency, whereas the mutant particles lacking the gene but containing the protein can institute the block. These results are consistent with the view that the UL69 protein contributes to the cytomegalovirus-induced cell-cycle block, and they suggest that UL69 protein delivered to cells within virions can induce the block without the synthesis of additional UL69 protein encoded by the infecting viral genome.

Isolation of differentially expressed cDNAs from p53-dependent apoptotic cells: activation of the human homologue of the Drosophila peroxidasin gene.

Inactivation of the p53 tumor suppressor protein has been observed in a large number of human cancers. Overexpression of p53 induces either growth arrest or programmed cell death (apoptosis). The growth arrest function of p53 is mediated by induction of p21 (WAF1/CIP1), but the mechanisms underlying p53-dependent apoptosis are still largely unknown. To investigate these mechanisms, we have identified six differentially expressed transcripts in a human colon cancer cell line undergoing p53-dependent apoptosis. One of the p53-responsive genes showed significant homology to Drosophila peroxidasin, an extracellular matrix-associated peroxidase, and is likely to be its human homologue. Our results suggest a possible connection between p53-dependent apoptosis and the production of reactive oxygen species.

Human cytomegalovirus UL36 protein is dispensable for viral replication in cultured cells.

Consistent with earlier analyses of human cytomegalovirus UL36 mRNA, we find that the UL36 protein is present throughout infection. In fact, it is delivered to the infected cell as a constituent of the virion. Curiously, much less UL36 protein accumulated in cells infected with the AD169 strain of human cytomegalovirus than in cells infected with the Towne or Toledo strain, and localization of the protein in cells infected with AD169 is strikingly different from that in cell infected with the Towne or Toledo strain. The variation in steady-state level of the proteins results from different stabilities of the proteins. The UL36 proteins from the three viral strains differ by several amino acid substitutions. However, this variability is not responsible for the different half-lives because the AD169 and Towne proteins, which exhibit very different half-lives within infected cells, exhibit the same half-life when introduced into uninfected cells by transfection with expression plasmids. We demonstrate that the UL36 protein is nonessential for growth in cultured cells, and we propose that the ability of the virus to replicate in the absence of UL36 function likely explains the striking strain-specific variation in the half-life and intracellular localization of the protein.

Early region 1 transforming functions are dispensable for mammary tumorigenesis by human adenovirus type 9.

Some human adenoviruses are tumorigenic in rodents. Subgroup A and B human adenoviruses generally induce sarcomas in both male and female animals, and the gene products encoded within viral early region 1 (E1 region) are both necessary and sufficient for this tumorigenicity. In contrast, subgroup D human adenovirus type 9 (Ad9) induces estrogen-dependent mammary tumors in female rats and requires the E4 region-encoded ORF1 oncoprotein for its tumorigenicity. Considering the established importance of the viral E1 region for tumorigenesis by adenoviruses, we investigated whether this viral transcription unit is also necessary for Ad9 to generate mammary tumors. The nucleotide sequence of the Ad9 E1 region indicated that the gene organization and predicted E1A and E1B polypeptides of Ad9 are closely related to those of other human adenovirus E1 regions. In addition, an Ad9 E1 region plasmid demonstrated focus-forming activity in both low-passage-number and established rat embryo fibroblasts, whereas a large deletion within either the E1A or E1B gene of this plasmid diminished transforming activity. Surprisingly, we found that introducing the same transformation-inactivating E1A and E1B deletions into Ad9 results in mutant viruses that retain the ability to elicit mammary tumors in rats. These results are novel in showing that Ad9 represents a unique oncogenic adenovirus in which the E4 region, rather than the E1 region, encodes the major oncogenic determinant in the rat.

Human cytomegalovirus inhibits transcription of the CC chemokine MCP-1 gene.

In primary human diploid fibroblasts, infection with an unpurified stock of human cytomegalovirus induced accumulation of the CC chemokine MCP-1 in the cell culture medium. By 24 h postinfection, the level of MCP-1 returned to that in uninfected cultures. When cells were infected with UV-inactivated human cytomegalovirus, the induction of MCP-1 was still observed, but no reduction was seen by 24 h postinfection or later. This effect was the result of a decrease in the level of MCP-1 mRNA present within the infected cell. Infection with purified virus revealed that the induction of MCP-1 was due to an activity found in the medium of infected cells; purified virions did not induce the expression of MCP-1. However, infection with purified virions repressed the level of MCP-1 mRNA below that found in uninfected cells. Additionally, infection with human cytomegalovirus prevented the induction of MCP-1 expression by tumor necrosis factor alpha and interleukin-1beta. The CC chemokine receptor encoded by the human cytomegalovirus US28 open reading frame (ORF) did not appear to play a role in this process, since a mutant virus in which the US28 ORF had been deleted downregulated MCP-1 in the same manner.

Human cytomegalovirus UL69 protein induces cells to accumulate in G1 phase of the cell cycle.

Earlier studies have revealed that human cytomegalovirus rapidly inhibits the growth of fibroblasts, blocking cell cycle progression at multiple points, including the G1-to-S-phase transition. The present study demonstrates that the UL69 protein, a virus-encoded constituent of the virion, is able to arrest cell cycle progression when introduced into uninfected cells. Expression of the UL69 protein causes U2 OS cells and primary human fibroblasts to accumulate within the G1 compartment of the cell cycle, and serum fails to induce the progression of quiescent human fibroblasts into the S phase when the protein is present. Therefore, the UL69 protein is at least partially responsible for the cell cycle block that is instituted after infection of permissive cells with human cytomegalovirus.

Adenovirus E4orf6 oncoprotein modulates the function of the p53-related protein, p73.

Recently, several proteins have been identified that are related in their sequence to the p53 tumor-suppressor protein. One of these proteins, which is termed p73, exhibits sequence homology to the p53 transcriptional activation, DNA binding, and oligomerization domains. The adenovirus E1B 55-kDa protein, the adenovirus E4orf6 protein, and SV40 T antigen each can bind to p53 and inhibit p53 function. Here we demonstrate that the adenovirus E4orf6 protein, but not the E1B 55-kDa protein or T antigen, interacts with p73. The E4orf6 protein inhibits p73-mediated transcriptional activation and cell killing in a manner similar to its effect on p53. Thus, only a subset of viral oncoproteins that antagonize p53 function also interacts with the related p73 protein.

Cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays.

Mechanistic insights to viral replication and pathogenesis generally have come from the analysis of viral gene products, either by studying their biochemical activities and interactions individually or by creating mutant viruses and analyzing their phenotype. Now it is possible to identify and catalog the host cell genes whose mRNA levels change in response to a pathogen. We have used DNA array technology to monitor the level of approximately 6,600 human mRNAs in uninfected as compared with human cytomegalovirus-infected cells. The level of 258 mRNAs changed by a factor of 4 or more before the onset of viral DNA replication. Several of these mRNAs encode gene products that might play key roles in virus-induced pathogenesis, identifying them as intriguing targets for further study.

Increased anxiety of mice lacking the serotonin1A receptor.

Brain serotonin (5-HT) has been implicated in a number of physiological processes and pathological conditions. These effects are mediated by at least 14 different 5-HT receptors. We have inactivated the gene encoding the 5-HT1A receptor in mice and found that receptor-deficient animals have an increased tendency to avoid a novel and fearful environment and to escape a stressful situation, behaviors consistent with an increased anxiety and stress response. Based on the role of the 5-HT1A receptor in the feedback regulation of the 5-HT system, we hypothesize that an increased serotonergic neurotransmission is responsible for the anxiety-like behavior of receptor-deficient animals. This view is consistent with earlier studies showing that pharmacological activation of the 5-HT system is anxiogenic in animal models and also in humans.

E1B 55-kilodalton-associated protein: a cellular protein with RNA-binding activity implicated in nucleocytoplasmic transport of adenovirus and cellular mRNAs.

The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.