Ganglioside synthase knockout in oncogene-transformed fibroblasts depletes gangliosides and impairs tumor growth.

Biologically active membrane gangliosides, expressed and released by many human tumors, are hypothesized to significantly impact tumor progression. Lack of a model of complete and specific tumor ganglioside depletion in vivo, however, has hampered elucidation of their role. Here, we report the creation of a novel, stable, genetically induced tumor cell system resulting in specific and complete blockade of ganglioside synthesis. Wild-type (WT) and GM3 synthase/GM2 synthase double knockout (DKO) murine embryonic fibroblasts were transformed using amphotropic retrovirus-transduced oncogenes (pBABE-c-Myc(T58A)+H-RasG12V). The transformed cells, WT(t) and DKO(t) respectively, evidenced comparable integrated copy numbers and oncogene expression. Ganglioside synthesis was completely blocked in the DKO(t) cells, importantly without triggering an alternate pathway of ganglioside synthesis. Ganglioside depletion (to <0.5 nmol/10(7) cells from 9 to 11 nmol/10(7) WT(t) or untransfected normal fibroblasts) did not adversely affect cell proliferation kinetics but did reduce cell migration on fibronectin-coated wells, consistent with our previous observations in ganglioside-depleted normal human fibroblasts. Strikingly, despite similar oncogene expression and growth kinetics, DKO(t) cells evidenced significantly impaired tumor growth in syngeneic immunocompetent mice, underscoring the pivotal role of tumor cell gangliosides and providing an ideal system for probing their mechanisms of action in vivo.

The sequence and antiapoptotic functional domains of the human cytomegalovirus UL37 exon 1 immediate early protein are conserved in multiple primary strains.

The human cytomegalovirus UL37 exon 1 gene encodes the immediate early protein pUL37x1 that has antiapoptotic and regulatory activities. Deletion mutagenesis analysis of the open reading frame of UL37x1 identified two domains that are necessary and sufficient for its antiapoptotic activity. These domains are confined within the segments between amino acids 5 to 34, and 118 to 147, respectively. The first domain provides the targeting of the protein to mitochondria. Direct PCR sequencing of UL37 exon 1 amplified from 26 primary strains of human cytomegalovirus demonstrated that the promoter, polyadenylation signal, and the two segments of pUL37x1 required for its antiapoptotic function were invariant in all sequenced strains and identical to those in AD169 pUL37x1. In total, UL37 exon 1 varies between 0.0 and 1.6% at the nucleotide level from strain AD169. Only 11 amino acids were found to vary in one or more viral strains, and these variations occurred only in the domains of pUL37x1 dispensable for its antiapoptotic function. We infer from this remarkable conservation of pUL37x1 in primary strains that this protein and, probably, its antiapoptotic function are required for productive replication of human cytomegalovirus in humans.

Human cytomegalovirus UL37 immediate-early regulatory proteins traffic through the secretory apparatus and to mitochondria.

The human cytomegalovirus (HCMV) UL36-38 immediate-early (IE) locus encodes the UL37 exon 1 (pUL37x1) and UL37 (gpUL37) regulatory proteins, which have anti-apoptotic activities. pUL37x1 shares its entire sequence, including a hydrophobic leader and an acidic domain, with the exception of one residue, with the amino terminus of gpUL37. gpUL37 has, in addition, unique N-linked glycosylation, transmembrane and cytosolic domains. A rabbit polyvalent antiserum was generated against residues 27-40 in the shared amino-terminal domain and a mouse polyvalent antiserum was generated against the full-length protein to study trafficking of individual UL37 proteins in human cells that transiently expressed gpUL37 or pUL37x1. Co-localization studies by confocal laser scanning microscopy detected trafficking of gpUL37 and pUL37x1 from the endoplasmic reticulum to the Golgi apparatus in permissive U373 cells and in human diploid fibroblasts (HFF). Trafficking of gpUL37 to the cellular plasma membrane was detected in unfixed HFF cells. FLAG-tagged gpUL37 trafficked similarly through the secretory apparatus to the plasma membrane. By using confocal microscopy and immunoblotting of fractionated cells, gpUL37 and pUL37x1 were found to co-localize with mitochondria in human cells. This unconventional dual trafficking pattern through the secretory apparatus and to mitochondria is novel for herpesvirus IE regulatory proteins.

Analysis of cytomegalovirus gene expression by transfection.

Human cytomegalovirus (HCMV), a human herpesvirus, is the leading viral cause of birth defects (1,2). In acquired immune deficiency syndrome (AIDS) patients, HCMV can cause severely debilitating colitis and retinitis; it is now an increasingly common cause of life-threatening pneumonitis in transplant patients (3-5). HCMV has a severely restricted host range in culture. It grows efficiently only in primary human diploid fibroblasts (HFF) and a few selected human cell lines (6-9). Monocytes serve as a major reservoir of latent HCMV in humans and HCMV can be grown in vitro in primary monocyte-derived macrophages (M/M) (10-14).

The latex allergen Hev b 5 transcript is widely distributed after subcutaneous injection in BALB/c mice of its DNA vaccine.

BACKGROUND: DNA vaccines reduce IgE responses to selected allergens, but severe reactions to the expressed antigen may limit the usefulness of the technique in allergen immunotherapy. OBJECTIVE: We sought to determine the extent of spread of an injected DNA vaccine in mice. METHODS: We placed the gene encoding the potent Hevea latex allergen Hev b 5 in a mammalian expression vector and injected this DNA vaccine subcutaneously into BALB/c mice. At several times after injection, the presence of Hev b 5 transcript was determined in multiple tissues by RT-PCR. The identity of the amplification product was confirmed by Southern hybridization and restriction analyses. RESULTS: Hev b 5 RNA appeared at the injection site and in the lymph nodes, spleen, and lungs within 1 day after injection and persisted for at least 14 days. Hev b 5 RNA was also identified in the blood and tongue 14 days after injection. Antibody and cell-mediated responses to Hev b 5 were also noted in the immunized animals at later time points. As expected, animals injected with the identical plasmid containing the Hev b 5 DNA in the antisense orientation mounted no immune response to Hev b 5. CONCLUSIONS: The rapid and widespread appearance of the Hev b 5 transcript in the injected mice confirms that DNA is translocated from the injection site, transcribed, and expressed in immune and nonimmune tissues after injection. Controlling the extent and degree of expression in specific target tissues may allow therapeutic DNA vaccination with plasmids that encode potentially toxic allergens.

The acidic domain of pUL37x1 and gpUL37 plays a key role in transactivation of HCMV DNA replication gene promoter constructions.

Transient complementation of human cytomegalovirus (HCMV) oriLyt DNA replication in permissive human diploid cells expressing replication genes under native promoters requires its UL36-38 gene products. Two of the immediate early (IE) proteins encoded by this locus, pUL37x1 and, to a lesser extent, gpUL37, activated expression of HCMV early gene promoter constructions. The other IE protein encoded by the UL36-38 locus, pUL36, and the early product, pUL38, did not transactivate the HCMV early promoter constructions under similar conditions. The acidic domain, common to both pUL37x1 and gpUL37, is required for activation of HCMV early promoter constructions. Conversely, gpUL37 sequences downstream of amino acid 199 are not required for transactivation of viral early promoters. Taken together, these results suggest that the requirement for UL36-38 products for HCMV DNA replication results, at least in part, from the requirement of the transactivation of HCMV early DNA replication promoters by pUL37x1 and, to a lesser extent, by gpUL37 and that the acidic domain is critical for this activity.

PCR detection of human cytomegalovirus DNA in clinical specimens using novel UL37 exon 3 and US3 primers.

The sensitivity and specificity of novel UL37 exon 3 (UL37x3) and US3 immediate-early (IE) gene PCR primers to detect human cytomegalovirus (HCMV) DNA in clinical specimens are comparable to those of HCMV DNA polymerase (UL54) primers. The use of these IE primers increases the diagnostic performance of HCMV PCR.

The human cytomegalovirus UL37 immediate-early regulatory protein is an integral membrane N-glycoprotein which traffics through the endoplasmic reticulum and Golgi apparatus.

The human cytomegalovirus (HCMV) UL37 immediate-early gene is predicted to encode a type I membrane-bound glycoprotein, gpUL37. Following expression of the UL37 open reading frame in vitro, its signals for translocation and N-glycosylation were recognized by microsomal enzymes. Its orientation in the microsomes is that of a type I protein. gpUL37 produced in HCMV-infected human cells was selectively immunoprecipitated by rabbit polyvalent antiserum generated against the predicted unique domains of the UL37 open reading frame and migrated as an 83- to 85-kDa protein. Tunicamycin treatment, which inhibits N-glycosylation, increased the rate of migration of the UL37 protein to 68 kDa, verifying its modification by N-glycosylation in HCMV-infected cells. Consistent with this observation, gpUL37 was found to be resistant to digestion with either endoglycosidase F or H but sensitive to peptide N-glycosidase F digestion. These results suggested that gpUL37 is N-glycosylated and processed in both the endoplasmic reticulum (ER) and the Golgi apparatus. Direct demonstration of passage of gpUL37 through the ER and the Golgi was obtained by confocal microscopy. gpUL37 colocalized with protein disulfide isomerase, a protein resident in the ER, and with a Golgi protein. Subcellular fractionation of HCMV-infected cells demonstrated that gpUL37 is an integral membrane protein. Taken together, our results demonstrate that the HCMV gpUL37 immediate-early regulatory protein is a type I integral membrane N-glycoprotein which traffics through the ER and the Golgi network.

The acidic domain of the human cytomegalovirus UL37 immediate early glycoprotein is dispensable for its transactivating activity and localization but is not for its synergism.

The product of the human cytomegalovirus (HCMV) immediate early (IE) UL37 gene, gpUL37, is predicted to be a type I membrane-bound glycoprotein. Typically for HCMV IE proteins, gpUL37 transactivates nuclear gene expression and acts synergistically with other IE proteins. We have initiated mutational analysis of the gpUL37 domains to determine which are required for its transactivating activity. The acidic domain, a feature notably required for the activity of many nuclear transcription factors, was deleted from gpUL37. Similar to wild-type gpUL37, the mutant retained a dose responsive transactivating activity in transiently transfected HeLa cells. Transactivating activity of the mutant was also observed in permissive human diploid fibroblasts when it was cotransfected with IE1. However, the gpUL37 acidic domain mutant is defective for synergism with another HCMV IE protein, pUS3. We found that wild-type gpUL37 and its acidic domain mutant (delta aa53-140) are nonnuclear proteins and are indistinguishable in localization. Confocal microscopy of human cell types coexpressing both HCMV IE regulatory proteins, IE1 and gpUL37, showed gpUL37 does not colocalize with the IE1 nuclear protein. Taken together, our results establish that gpUL37 is a nonnuclear protein that requires its acidic domain for synergism with pUS3 but not for its transactivating activity or its localization.

The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.

Developmental analysis of the cytomegalovirus enhancer in transgenic animals.

The major immediate-early promoter (MIEP) of human, cytomegalovirus (HCMV) constitutes a primary genetic switch for viral activation. In this study, regulation of the enhancer-containing segment (nucleotides -670 to +54) of the HCMV MIEP attached to the 1acZ reporter gene was examined in the developing embryos of transgenic mice to identify temporal and tissue-specific expression. We find that the transgene reporter is first detected as a dorsal stripe of expression in the neural folds of embryos at day 8.5 postcoitum (p.c.). A broad expression pattern is exhibited in embryos at day 9.5 p.c. This pattern becomes more restricted by day 10.5 p.c. as organogenesis progresses. By day 14.5 p.c., prominent expression is observed in a subpopulation of central nervous system cells and spinal ganglia, endothelial cells, muscle, skin, thyroid, parathyroid, kidney, lung, liver, and gut cells, and the pancreas and submandibular and pituitary glands. This distribution pattern is discussed in relation to human congenital HCMV infection. These results suggest that the transcriptional activity of the HCMV MIEP may determine in part, the ability of the virus to specifically target developing fetal tissues in utero.

Functional roles of immediate early proteins encoded by the human cytomegalovirus UL36-38, UL115-119, TRS1/IRS1 and US3 loci.

Human cytomegalovirus (HCMV) encodes multiple regulatory proteins at immediate early (IE) times of infection. Ancillary IE proteins are encoded by the UL36-38, UL115-119, TRS1/IRS1 and US3 loci. In contrast to the major IE nuclear proteins, several of the ancillary IE proteins are type I integral membrane N-glycoproteins. Nonetheless, all of the ancillary proteins examined to date have the ability to regulate nuclear gene expression and to interact cooperatively. Significantly, products from the UL36-38 and TRS1/IRSI IE loci as well as products from the MIE locus are required for HCMV ori-Lyt DNA replication. Moreover, the products of the UL36 and UL37 IE genes are essential for HCMV growth in human cells. Finally, one ancillary IE glycoprotein, gpUS3, is known to have a nonregulatory function; that is, gpUS3 binds and retains major histocompatibility complex class I heavy chains in the endoplasmic reticulum, thereby inhibiting antigen presentation. Thus, the functional presence of multiple IE proteins is required during HCMV replication both in vitro and in vivo to orchestrate necessary events for HCMV replication as well as for the survival of the infected host cell.

The human cytomegalovirus US3 immediate-early protein lacking the putative transmembrane domain regulates gene expression.

Through alternative transcript splicing, the human cytomegalovirus (HCMV) US3 immediate-early (IE) locus encodes multiple products including potential membrane-bound glycoproteins. To characterize the US3 products and determine which encode regulatory activity, individual cDNAs were cloned and expressed. Three transcript species were confirmed through the isolation of cDNAs; an unspliced transcript, a transcript spliced once from exon 3 to exon 5 and a transcript spliced both at exon 1 to exon 3 and at exon 3 to exon 5. The predicted signal sequences and N-linked glycosylation sites in the US3 products were confirmed using expression in reticulocyte lysates containing microsomal membranes. Regulatory activity of the individual US3 products was demonstrated using transient transfection assays. The unspliced cDNA and the cDNA containing the exon 3 to exon 5 splice, encoded products which increased expression of the human heat shock protein 70 (hsp70) promoter, while the product of the doubly-spliced US3 cDNA did not. Transactivation was synergistically increased by coexpression with the HCMV UL37 protein. We conclude that the first 132 amino acids common to the unspliced and the singly-spliced US3 gene products are sufficient for hsp70 transactivation; while the amino-terminal 28 amino acids, encoded by the doubly-spliced US3 cDNA, are not. These results demonstrate that a US3 IE protein lacking the putative transmembrane domain has regulatory activity.

Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression.

We have established the ability of the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) gene products to alter gene expression in human cells by using transient transfection assays. The cellular heat shock protein 70 (hsp70) promoter was transactivated following cotransfection with the HCMV IE regions in nonpermissive HeLa cells by UL36-38, US3, or IE1 and in permissive human diploid fibroblasts (HFF) by IE1 or IE2. Moreover, hsp70 expression was synergistically increased in HeLa cells cotransfected with US3 and UL36, with US3 and UL37, or with US3 and UL37x1. The synergistic transactivation of hsp70 expression by US3 and UL36-38 was not observed in HFF cells. Synergy was also not observed in HeLa cells between US3 and UL38, an early gene product encoded by the UL36-38 IE locus. Synergistic transactivation of hsp70 expression in HeLa cells required the syntheses of UL36-38 and US3 IE proteins, since nonsense mutants were not functional. hsp70 expression increased with increasing amounts of transfected US3 and UL37 DNA and occurred at the level of stable hsp70-promoted RNA. In contrast to the broad hsp70 response, promoters from the HCMV UL112 early gene and another cellular gene, brain creatine kinase, both responded strongly only to singly transfected IE2 in HeLa cells. Nevertheless, IE2 transactivation of the UL112 promoter was further stimulated by cotransfection of IE1 or of UL36-38 in both HeLa and HFF cells. Thus, different patterns of promoter transactivation and interactions between HCMV IE gene products in transactivation were found in HFF cells and in HeLa cells. These results establish the ability of the HCMV US3 and UL36-38 proteins to alter cellular and viral gene expression and are consistent with involvement of cellular transcription factors in HCMV IE regulation of gene expression.

Human cytomegalovirus UL36-38 and US3 immediate-early genes: temporally regulated expression of nuclear, cytoplasmic, and polysome-associated transcripts during infection.

During permissive in vitro infection, the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) regions give rise to multiple distinct species of RNA in a temporally regulated manner. We have compared the temporally regulated expression of the UL36-38 and US3 regions with that of the well-characterized major IE (MIE) region. Northern (RNA) blot hybridizations with antisense RNA probes were used to examine RNA isolated from infected cells at IE, early, and late times after infection and from cells infected in the presence of anisomycin (used to block de novo viral protein synthesis) or in the presence of phosphonoformate (used to block HCMV DNA synthesis). Different US3 region transcripts were expressed in the cytoplasm during the IE and late phases of infection, with kinetics similar to those of the MIE region. In contrast, various cytoplasmic transcripts from the UL36-38 region were expressed during each of the IE, early, and late phases of infection, including some expressed from IE through late times. The levels of steady-state RNA from the US3 and MIE regions were increased dramatically by infection in the presence of anisomycin, predominantly because of an increase in multiply spliced transcripts. Two of the three UL36-38 IE transcripts were largely unaffected by anisomycin and were expressed abundantly throughout infection, but a third, multiply spliced UL36-38 IE transcript was abundant only during infection in the presence of anisomycin. Nuclear, cytoplasmic, and polysome-associated transcripts from the three IE regions were not significantly different qualitatively or quantitatively. These results suggest that posttranscriptional controls at the levels of nuclear retention or polysome exclusion of transcripts are not operative for the IE region genes. Overall, these results indicate common features of expression of US3, MIE, and UL36-38, in addition to distinctive expression of the UL36-38 region during all temporal phases of expression.

A sensitive method for the detection of beta-galactosidase in transfected mammalian cells.

A sensitive method has been developed for the detection of E. coli beta-galactosidase in transfected HeLa cells. The chromogenic substrate, CPRG (chlorophenol red-beta-D-galactopyranoside), was compared with ONPG (o-nitrophenyl-beta-D-galactopyranoside) by kinetic analysis with purified beta-galactosidase. The Km for CPRG was 1.35 mM and the Vmax was 21.4, whereas the Km for ONPG was 2.42 and the Vmax was 41.1. CPRG at 8.0 mM (6-fold Km) gave 86% of the Vmax and was used as the standard concentration for quantitation of enzyme levels. The Vmax for CPRG was half that for ONPG, and chlorophenol red has an extinction coefficient that is 21-fold higher than o-nitrophenol; these factors make CPRG about 10-fold greater in sensitivity for the quantitation of enzyme levels. The use of Nonidet P-40 to lyse the cells and the use of CPRG as substrate permitted the rapid detection of low levels of enzyme production from transfected human cells that could not be detected using ONPG.

Expression of the human cytomegalovirus UL36-38 immediate early region during permissive infection.

We have characterized the expression of human cytomegalovirus immediate early (IE) genes encoding the UL36-38 open reading frames. Single-stranded RNA hybridization probes, transcribed from UL36-38 cDNAs or smaller exon-specific clones, were used in analyses of RNA isolated during various phases of infection in the presence and absence of inhibitors of protein and HCMV DNA synthesis. There are three IE transcripts that arise by the use of alternative promoters and splicing signals. Different patterns of expression are observed for each of the three IE RNAs during permissive infection. Comparison of nucleotide sequences of UL36-38 IE cDNAs with those of the genomic sequence verified the use of polyadenylation and splice signals. An additional abundant early class RNA, totally encoded within the UL36-38 region, was found to be expressed from a different promoter than the IE RNAs. In vitro transcription and translation of the IE cDNAs were used to express the UL36-38 IE proteins. These studies define the structure of the UL36-38 gene products and demonstrate that expression from the region is both qualitatively and temporally complex.

Induction of cellular hsp70 expression by human cytomegalovirus.

Expression of the cellular heat shock protein 70 gene (hsp70) is transiently induced by human cytomegalovirus (HCMV) infection of permissive human diploid fibroblasts. Induction of the cellular heat shock response during critical times of infection had previously been reported to alter the growth of HCMV in vitro. Thus, a potential interaction between heat shock proteins and HCMV expression was indicated. HCMV dramatically increased expression of hsp70 RNA within 8 h of infection. hsp70 RNA remained elevated at 24 and 48 h postinfection and decreased to low levels of 72 h postinfection. Induction of HSP70 protein occurred more slowly; inducible HSP70 protein encoded by this RNA increased within 16 h postinfection and continued to increase throughout infection until 72 h postinfection, when the highest abundance of inducible HSP70 protein was observed. Cells that received both heat (43 degrees C for 70 min) treatment and HCMV infection expressed hsp70 RNA to levels above the sum of levels present in cells given either treatment alone. Furthermore, hsp70 RNA induction occurred earlier and remained elevated longer than in cells infected with HCMV alone or in cells treated with heat alone, respectively. Nevertheless, the pattern of HCMV immediate-early transcript expression at 2, 4, and 6 h postinfection appeared to be unchanged by this prior heat treatment. Our results suggest that heat shock treatment and HCMV infection can act additively in stimulating hsp70 RNA expression. The previously reported stimulation of HCMV growth in vitro following the heat shock response apparently does not result from alterations in the steady-state expression of HCMV immediate-early transcripts.

RNA analysis and isolation of cDNAs derived from the human cytomegalovirus immediate-early region at 0.24 map units.

We have examined transcription from the human cytomegalovirus immediate-early genes that are located at approximately 0.24 map units of the viral genome. Upon infection of permissive cells, nonoverlapping transcripts of 1.65 and 1.7 kb were abundant 8 h after infection and at lower levels at later times of infection or in the presence of cycloheximide. A transcript of approximately 3.4 kb that spanned the entire region was less abundant than the smaller RNAs at 8 h after infection, but was not decreased in abundance with cycloheximide treatment. These RNA species appear to correspond to the immediate-early transcripts from this region. Polyadenylated RNA was isolated from human cytomegalovirus infected cells 8 h after infection and used to synthesize directly a cDNA library. cDNAs corresponding to the abundant 1.65- and 1.7-kb transcripts of this region were isolated and characterized.

Selective induction of chromosomal gene expression by human cytomegalovirus.

During permissive infection, human cytomegalovirus (HCMV) increases the activities of certain cellular enzymes. In this report, we show that induction of these cellular genes occurs at the level of RNA. The abundance of several additional growth-regulated cellular transcripts in HCMV-infected human foreskin fibroblasts (HFF) was also compared to that in serum-stimulated HFF cells. HCMV infection of HFF cells resulted in increased abundance of ornithine decarboxylase, thymidine kinase, heat-shock protein 70 (hsp70), and brain creatine kinase transcripts. In contrast to serum treatment, infection with HCMV did not result in dramatic increases in c-myc, beta-actin, or metallothionein IIA RNA. The induction of hsp70 RNA occurred during the earliest stage of HCMV infection of HFF cells, preceding its induction in serum-stimulated HFF cells by several hours. Thus, a role for the earliest events in HCMV infection in the induction of hsp70 RNA is suggested.