Expression of the Epstein-Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice.

The latent membrane protein 1 (LMP1) of the Epstein-Barr virus has transforming properties in rodent fibroblasts and is expressed in most of the cancers associated with Epstein-Barr virus (EBV) infection including posttransplant lymphomas, Hodgkin's disease, nasopharyngeal carcinoma, and AIDS-related lymphomas. In this study, three lineages of LMP1 transgenic mice were established with LMP1 expressed under the control of the Ig heavy chain promoter and enhancer. Lymphoma developed in all three lineages, and the incidence of lymphoma increased significantly with age with lymphomas developing in 42% of transgenic mice over 18 months. The expression of LMP1 was detected at high levels in the lymphoma tissues but only at trace levels in normal lymphoid tissues. Gene rearrangement of the Ig heavy chain indicated monoclonality or oligoclonality in all lymphomas, some of the lymphoid hyperplastic spleens, and some histologically normal spleens. These data reveal that LMP1, without the expression of other EBV genes, is oncogenic in vivo and indicate that LMP1 is a major contributing factor to the development of EBV-associated lymphomas.

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.

Demonstration of developmental anomalies in mouse fetuses by transfer of murine cytomegalovirus DNA-injected eggs to surrogate mothers.

To study the potential consequences of sperm-mediated sexual cytomegalovirus (CMV) transmission, an in vitro model of microinjection of murine CMV (MCMV) DNA into uninfected fertilized murine ova was used. After microinjected ova were cultured to blastocysts and transferred to pseudopregnant mice, the effect of the DNA on implantation and development was analyzed. At various times, the sites of implantation in the endometrium were examined. Reductions in litter size, fetal growth retardation, resorption of embryos, and fetal maldevelopment, which often involved the central nervous system, were observed. The presence of MCMV DNA sequences in DNA derived from fetuses was detected by the polymerase chain reaction followed by oligonucleotide hybridization. By in situ DNA-DNA cytohybridization and indirect immunofluorescence assays the viral sequences and antigens were localized primarily to the brain, salivary gland, and skin of maldeveloped fetuses. These results establish the potential consequences of sperm-mediated CMV transmission and induction of fetal anomalies.

Oncogenic transformation by cellular DNA isolated from human cytomegalovirus-infected cells.

Delayed replication of human cytomegalovirus (CMV) was initiated in human embryonic fibroblasts using partially ultraviolet light-inactivated virus stock. Cellular [high molecular weight (HMW)] DNA extracted from CMV-infected cell cultures demonstrated a substantial increase in transforming activity after introduction into hamster embryo fibroblasts relative to HMW DNA extracted from mock-infected cells. The transforming activity of HMW DNA varied between 0.01 and 0.25 foci/micrograms DNA. HMW DNA isolated from CMV-infected cells after initiation of viral DNA synthesis demonstrated a significant decrease in the induction of morphologically transformed foci. The transforming activity of HMW DNA was unaffected by HindIII or XbaI endonuclease digestion, but it was sensitive to sonication and EcoRI endonuclease and DNase treatment. Six cell lines were established from the foci of morphologically altered cells. Cells of these lines demonstrated loss of contact inhibition, replication in semisolid agarose or in medium containing low serum, a high saturation density, and tumorigenicity when implanted into hamsters. Histopathological examination of the tumors identified the tissues as fibrosarcomas. In situ hybridization of cells isolated from foci of morphologically altered cells or Southern blot analysis of DNA isolated from either the cell lines or tumors did not demonstrate the presence of sequences with homology to viral DNA using the transforming region or the entire viral DNA as a probe. The lack of viral DNA sequences as well as the similar phenotypic characteristics of cell lines and tumors suggest that alteration(s) induced by CMV may occur in specific region(s) of cellular DNA.

Early-stage developmental abnormalities induced by murine cytomegalovirus.

The onset of the effects of murine cytomegalovirus (MCMV) infection on an early stage of embryonic development (nine days) in a mouse model was studied with the aid of scanning electron microscopy. MCMV was injected into the endometrial lumina of pregnant mice at the time of implantation. The mice were later killed, and sites of embryonic implantation were examined. Compared with uninfected mice and mice inoculated with heat-inactivated virus, litter sizes were reduced, and the incidence of abnormal fetuses was significantly increased among MCMV-infected animals. Scanning electron microscopy also revealed maldeveloped cranial regions characterized by an unclosed neural tube and severely underdeveloped head. Ectodermal abnormalities, including poxlike formations and ballooning cells, were observed in several embryos. Thus, early cytomegalovirus infection may not only result in fetal loss, but may also interfere with the process of morphogenesis.

Murine cytomegalovirus infection of mouse testes.

With the aim of illustrating a mechanism of cytomegalovirus (CMV) venereal transmission, we induced murine CMV infection in the mouse testes of immunologically competent mice. Using in situ cytohybridization, we were able to show that murine CMV-specific DNA was associated with spermatocytes and mature sperm. Electron microscopy studies also supported sperm infection. The virus could be reisolated from infected epididymal sperm by cocultivation with mouse embryo fibroblasts. We found no difference in either the sexual performance or the fertilization efficiency of the sperm between infected and uninfected males.

HCMV specific expression in HEL cells transformed by Xba I endonuclease fragmented HCMV-DNA.

The transfection of subconfluent monolayers of HEL cells with Xba I endonuclease fragmented HCMV-DNA resulted in a large number of morphologically transformed foci. The frequency of focus formation of Xba I fragmented DNA increased after TPA treatment of the cells. The morphologically altered cells showed specific reactivity with anti-HCMV serum pool in the nuclear and perinuclear region and in the cytoplasm. The HCMV-32P-labelled probe DNA has been specifically hybridized to the cells of morphologically altered foci. The autoradiographic grains were localized in both nucleic and cytoplasm of the cells showing a specific hybridization between the probe DNA and nuclear as well as polyribosomal RNA.

Murine cytomegalovirus-induced congenital defects and fetal maldevelopment.

The effects of congenital infection with cytomegalovirus on fetal development were studied by injection of murine cytomegalovirus (MCMV) into the endometrial lumina of mice in the early stages of pregnancy. The mice were later killed, and the sites of embryonic implantation were examined. Litter size was reduced and the incidence of abnormal fetuses was significantly increased among MCMV-infected animals. Morbid, moribund, severely growth-retarded, asynchronously developed, and malformed fetuses were found. Virus was reisolated from infected mothers, placentas, and fetal tissues but not from control animals that had received either heat-inactivated virus or culture medium. The recovered virus was identical to the original virus, as verified by restriction enzyme analysis. MCMV-specific DNA was detected in experimental tissue sections by in situ nucleic-acid hybridization. Seroconversion occurred in infected female mice. Thus, the presence of MCMV in the genital tract at the time of embryonic implantation causes not only embryonic infection but also fetal maldevelopment.

Human cytomegalovirus and herpes simplex type 2 virus in normal and adenocarcinomatous prostate glands.

Normal prostate, benign prostate hypertrophy (BHP), and prostate adenocarcinoma (ACP) biopsy specimens were analyzed for the presence of human cytomegalovirus (HCMV) and herpes simplex virus type 2 (HSV-2)-specific macromolecules. HCMV DNA homologous sequences were detected in 2 of 13 normal prostate, 2 of 9 BHP, and 3 of 10 ACP tissues, and HSV-2 DNA homologous sequences were found in 1 normal prostate tissue and 2 ACP tissues. In situ DNA-RNA hybridizations indicated HCMV-specific RNA in 3 of 8 BHP and 4 of 9 ACP tissues. No positive in situ hybridization for HCMV RNA was observed in normal prostate tissues. Parallel in situ DNA-RNA hybridization localized HSV-2-specific RNA in only 1 of 8 tumor sections. No HSV-2-specific RNA was observed in sections of normal and BHP tissues. Anticomplement immunofluorescence (ACIF) tests of BHP and ACP specimens showed specific HCMV immunofluorescence in 3 of 8 BHP and 4 of 9 ACP tissues. ACIF results correlate closely with in situ hybridization results and imply some degree of HCMV association with prostatic abnormality. The results also suggest that latent HCMV may be harbored by the human prostate gland.

Cytomegalovirus infection of murine testicular interstitial Leydig cells.

We studied the susceptibility of mouse testicular interstitial Leydig cells to cytomegalovirus both in vivo and in vitro. The in vivo studies included intratesticular and intraperitoneal infection of 6-week-old mice with murine cytomegalovirus (MCMV); the in vitro studies involved an MCMV-Leydig cell interaction using a Leydig tumor cell line (I-10). MCMV-specific antigens were detected in interstitial Leydig cells in sections of MCMV-inoculated testes by an indirect immunofluorescence test. MCMV DNA was also localized in the same testes cells derived from mice, which received intratesticular and intraperitoneal MCMV inoculations, respectively, by in situ DNA-RNA hybridization. Cytopathic effects were seen in MCMV-infected I-10 cell cultures 2 or more days after exposure to MCMV. The infected cells showed intranuclear inclusions characteristic of cytomegalovirus when stained with May-Grunwald-Giemsa stain. The indirect immunofluorescence test was also positive with MCMV-infected I-10 cells. MCMV DNA was detected in these cells by in situ DNA-RNA cytohybridization, and the presence of viral particles in MCMV-infected I-10 cells was confirmed by electron microscopy. Thus, we conclude that the interstitial Leydig cell is susceptible to MCMV infection both in vivo and in vitro.

Reduction of fertility of mice by the intrauterine injection of prostaglandin antagonists.

The intrauterine injection of 7-oxa-13-prostynoic acid, 18,18,20-trimethyl PGE-2, and meclofenamic acid in mice at the expected times of implantation significantly reduced the number of implantation sites. Indomethacin was ineffective possibly because it was exposed to high pH during the preparation of the solutions for injection. It is suggested that these prostaglandin antagonists exert their antifertility action at multiple sites involving both the embryo and mother.

Inhibition of hatching of mouse blastocysts in vitro by various prostaglandin antagonists.

Hatching of mouse blastocysts in vitro is inhibited by 18,18,20-trimethyl PGE-2, 17,17-dimethyl PGE-2, 8,12-epiPGE-2 and N-dimethylamide PGF-2 alpha, whose activity is between that of 7-oxa-13-prostynoic acid and meclofenamic acid.

Infection of mouse ectoplacental cone cells with murine cytomegalovirus.

In order to understand the mechanism of congenital cytomegalovirus (CMV) infection, we studied the effect of murine cytomegalovirus (MCMV) on murine ectoplacental cone (EPC) cells in vitro. Cytopathic effects (c.p.e.) were seen in many MCMV-infected EPC cell cultures 5 to 7 days after exposure to MCMV. The infected cells showed intranuclear inclusions characteristic of CMV infection when stained with May-Grunwald-Giemsa. Culture fluids collected from MCMV-infected EPC cells after 4 or more days of culture caused c.p.e. when co-cultivated with mouse embryo fibroblasts (MEF). Employment of the anti-complement immunofluorescence (ACIF) test detected MCMV-specific antigens, in situ hybridization localized MCMV DNA and electron microscopy detected the presence of the viral particles in the MCMV-infected EPC cells. Thus, exposure of EPC cells to MCMV in vitro resulted in a productive infection.