No abstract

The DNAs of two herpesviruses, 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 for 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 sequences. 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.Marek disease virus (MDV), a herpesvirus, is the causative agent of Marek disease, a malignant lymphoma of chickens (for review, see refs. 1-3). From tumors of diseased animals, continuous lymphoblastoid T-cell lines can be established (4, 5) and these have been shown to harbor the MDV genome in a latent form (6-8). A serologically related herpesvirus of the turkey (HVT) (9-11) produces "microlymphomas" in its natural host and in chickens and, being essentially apathogenic for both, has been used as a vaccine against Marek disease (12).A somewhat similar "lymphotropic" biology is shared by several other members of the herpesvirus group. In humans, the Epstein-Barr virus (EBV) causes heterophile-positive infectious mononucleosis (for review, see refs. 13 and 14) and is considered to be a cofactor in two neoplastic diseases-Burkitt lymphoma and nasopharyngeal carcinoma (15)(16)(17) EBV, H. saimiri, and H. ateles also share certain peculiarities in the arrangement ofnucleotide sequences within their DNAs.All have genomes containing direct tandem repeats ofshort (Mr, 0.4-2.0 X 10') nucleotide sequences; H. saimiri and H. ateles carry a single sequence that is present in multiple copies at both extremities of the genome (23, 24) and EBV carries two nonhomologous sequences, one repeated at the extremities and a second at the interior of the genome (25)(26)(27) In light ofthe biological properties outlined above, one might expect the genomes of MDV and HVT to resemble those of EBV, H. saimiri, and H. ateles. The results ofthe study reported here do not, however, fulfill this expectation and show instead that the genomes of MDV and HVT contain inverted repeat sequences in the configuration found in HSV and BMV. A preliminary report of this work has been presented elsewhere. t MATERIALS AND METHODS Virus Strains, Cells, Virus Growth and Purification, and DNA Preparation. These have been described (8). Four strains of MDV were used in this study: the pathogenic GA and JM strains, the apathogenic Cal-i strain, and the attenuated CV1-988 strain. The HVT strain used was PBTHV1.Electron Microscopy. The spreading of DNA was carried out with a 50% formamide hyperphase and a 17% formamide hvpophase according to standard procedures (35). Single-stranded DNA was prepa...

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%o 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. (8, 41). Viral DNA in cell line 1670 is heavily 295 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from 296 KASCHKA-DIERICH ET AL.

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.