Structure of nonintegrated, circular Herpesvirus saimiri and Herpesvirus ateles genomes in tumor cell lines and in vitro-transformed cells.

Nonintegrated, circular DNA molecules of Herpesvirus saimiri and Herpesvirus ateles were found in five lymphoid cell lines originating from tumor tissues or established by in vitro immortalization of T lymphocytes. The arrangement of unique (L) and repetitive (H) DNA sequences in circular viral genomes was analyzed by partial denaturation mapping followed by visualization with an electron microscope. Three types of circular viral DNA structures were found. (i) The virus-producing cell line RLC, which is derived from an H. ateles-induced rabbit lymphoma, contains circular viral genomes which consist of a single L-DNA and a single H-DNA region, both the same length as in virion DNA. (ii) The circular viral genomes of the nonproducer cell lines H1591 and A1601, in vitro transformed by H. saimiri and H. ateles, respectively, have deletions in the unique L-DNA region and larger H-DNA regions. Cell line A1601 lacks about 8% of virion L-DNA, and H1591 cells lack about 40% of viral L-DNA information. (iii) The nonproducing H. saimiri tumor cell lines 1670 and 70N2 harbor viral genomes with two L-DNA and two H-DNA regions, respectively. Both types of circular molecules have a long and a short L-segment. The sequence arrangements of circular DNA molecules from H. saimiri-transformed cell lines were compared with those of linear virion DNA by computer alignment of partial denaturation histograms. The L-DNA deletion in cell line H1591 was found to map in the right half of the virion DNA. Comparison of the denaturation patterns of both L regions of cell lines 1670 and 70N2 identified the short L regions as subsets of the long L regions. Thus, circular viral DNA molecules of all four nonproducer cell lines represent defective genomes.

Inverted repeat nucleotide sequences in the genomes of Marek disease virus and the herpesvirus of the turkey.

The DNAs of two herpesvirus, the oncogenic Marek disease virus and the serologically related herpesvirus of the turkey, were studied by electron microscopy. On the basis of fold-back molecules observed in single-stranded DNA from both viruses, structures have been derived from the overall nucleotide sequence arrangement in their genomes. Although differing in molecular weight, the genomes of Marek disease virus and turkey herpesvirus are both constructed according to the same plan--two regions of unique nucleotide sequence, each enclosed by inverted repeat sequence. The genome structure of these viruses therefore closely resembles that of herpes simplex virus rather than the biologically more similar herpesvirus Epstein--Barr virus, H. saimiri, and H. ateles.

Relationship between Herpesvirus ateles-associated nuclear antigen (HATNA) and the number of virus genome equivalents in HVA-carrying lymphoid lines.

A DNA-binding antigen (HATNA) was demonstrated in 7 out of 14 cell lines carrying Herpesvirus ateles (HVA) by the acid-fixed nuclear-binding technique. The seven HATNA-positive lines had means of 95, 96, 103, 177, 240, 343 and 326 virus genome equivalents/cell. For the seven HATNA-negative lines, the figures were 4, 8, 10, 33, 39, 72 and 110. This indicates a relationship between the number of HVA genome equivalents/cell and the detectability of HATNA. This association was independent of the virus producer status of the lines.

Episomal and nonepisomal herpesvirus DNA in lymphoid tumor cell lines.

Tumor cell lines derived from Herpesvirus saimiri (H. saimiri)- and Herpesvirus ateles (H. ateles)-induced lymphomas of New World primates and rabbits contain multiple copies of viral genomes. Partial denaturation mapping and blot hybridizations of episomal DNA from lymphoid tumor cell line No. 1670 showed that a 12.5md-fragment is missing which represents the EcoRI D- and H-fragments of virion L-DNA. However, the missing piece can be demonstrated in total cellular DNA by reassociation kinetics, possibly because it persists in integrated form. Both episomal and nonepisomal H-DNA are heavily methylated in a number of the lymphoid cell lines, and methylation may be reduced by conventional methylation inhibitors (S-adenosyl homocystein, SIBA) as well as by the tumor promoting phorbol ester TPA.

Studies on the temperature-dependent DNA replication of the herpesvirus of the turkey in chicken embryo fibroblasts.

The development of cytopathogenic changes in chicken embryo fibroblasts infected with the herpesvirus of the turkey, strain PB-THVI, and the release of virus particles into the supernatant of infected cultures is accelerated at temperatures higher than 37 degrees C and is fastest at 41 degrees C, the normal body temperature of chickens. The growth rate of HVT in CEF cultures was followed by determination of the number of virus genome equivalents within infected cells at various time intervals p.i. A temperature-dependent increase in the amount of virus DNA per infected cell could be detected, which is highest at 41 degrees C. At all temperatures tested (34, 36, 41 and 43 degrees C) the number of virus genome equivalents per infected cell ultimately reaches the same level. In the course of infection, virus DNA in CEF cultures at 37 and 41 degrees C becomes associated with the cellular DNA, as determined by neutral CsCl gradients of the total cellular DNA and hybridization of each fraction with 32P-labelled virus-specific complementary RNA. Association of virus to cellular DNA occurs earlier at 41 than at 37 degrees C. However, the same proportion (45%) of the total virus DNA appears ultimately to be associated with cellular DNA at both temperatures. A temperature-shift of CEF cultures infected with PB-THVI from 41 to 37 degrees C 24 h p.i. resulted in the same replication kinetics of virus DNA as was found at 41 degrees C.

No homology detectable between Marek's disease virus (MDV) DNA and herpesvirus of the turkey (HVT) DNA.

The relation of four different strains of MDV and two strains of HVT was analyzed by gel electrophoresis of viral DNA digested by various restriction endonucleases and by filter hybridization of viral DNA with complementary RNA. The four MDV strains showed fragment patterns completely different from those of HVT upon digestion of the viral DNA with Bam H I, Eco R I, Hind III, Hpa, I, and Xho and separation of fragments on agarose gels. The cleavage patterns of the four MDV strains showed great similarities among each other as well as some differences between the individual strains. In the cleavage patterns of HVT a similar close relationship was observed between the two HVT strains with slight divergence between both. Filter hybridizations of viral DNA with labelled complementary RNA prepared from the DNA of the GA strain of MDV or from the DNA of the PH-THV1 strain of HVT revealed no cross-hybridization between the MDV and the HVT strains. cRNA prepared from the DNA of an MDV strain hybridized only to restriction enzyme fragments of the MDV strains transferred to nitrocellulose filters, but not to fragments of HVT DNA, and vice versa.

Human lymphoblastoid cell lines derived from individuals without lymphoproliferative disease contain the same latent forms of Epstein-Barr virus DNA as those found in tumor cells.

The physical state of the intracellular Epstein-Barr virus DNA was characterized in four human lymphoblastoid cell lines, F-265, NC-37, U-303 L, and PG-1, derived from individuals without lymphoproliferative disease. For comparison, a previously investigated Burkitt lymphoma line, Raji, and a more recently established cell line of that origin, Rael, were also studied. The techniques employed were CsCl density gradient centrifugation, glycerol gradient centrifugation, and ethidium bromide-CsCl density gradient centrifugation in combination with nucleic acid hybridization, as well as electron microscopy contour length measurements of purified circular EBV DNA. All six cell lines contained multiple copies of covalently closed circular EBV DNA-molecules of the same size, as well as viral DNA with the properties of integrated DNA. No differences could be detected between the forms of EBV DNA present in cell lines derived from non-malignant sources and those present in lymphoma lines.

An immunoperoxidase method for the identification of B lymphocytes by light microscopy.

Peroxidase-conjugated anti-human immunoglobulin was used to determine the percentage of B lymphocytes in the mononuclear cell fraction of peripheral blood of 100 healthy persons (blood donors). Peroxidase activity was revealed by incubation with the usual mixture of 3'3 diaminobenzidine and hydrogen peroxide. Cells which were peroxidase-positive after incubation with benzidine solution alone were considered to be monocytes. The number of T lymphocytes was estimated by the formation of E rosettes. 19.6% mononuclear cells could be shown to be B cells, 4.1% were monocytes and 57.0% T cells. The use of peroxidase-conjugated anti-human immunoglobulin gives rise to the same percentage of B cells as the use of fluorescein-conjugated anti-human immunoglobulin. The advantage of the method described here is that B lymphocytes can be counted by conventional light microscopy.

Circular Epstein-Barr virus genomes of reduced size in a human lymphoid cell line of infectious mononucleosis origin.

Circular Epstein-Barr virus (EBV) DNA molecules have been purified and characterized from a human lymphoid cell line derived from a case of heterophile antibody-positive, blood transfusion-induced infectious mononucleosis, 883L. The circular EBV DNA in three cell lines obtained by transformation of human umbilical cord blood leukocytes with a strain of EBV originally derived from 883L was also studied. As estimated from sedimentation velocity data and electron microscopy, the circular EBV DNA molecules are 10 to 15% smaller than either the circular EBV DNA previously found intracellularly in several other types of EBV-transformed cells or the linear EBV DNA present extracellularly in virus particles. In addition, the EBV-transformed cord blood cell lines studied here differed from other EBV-transformed cells in that integrated virus DNA sequences could not be detected.

Intracellular forms of Epstein-Barr virus DNA in human tumour cells in vivo.

Tumour biopsies from Burkitt lymphoma patients, as well as human nasopharyngeal carcinoma cells growing in athymic mice, contain Epstein-Barr virus DNA as covalently closed circular DNA. In addition integrated viral DNA sequences seem to be present.