Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3855-3859, May 1993 Biochemistry Retroviral insertions into a herpesvirus are clustered at the junctions of the short repeat and short unique sequences (Marek disease virus/reticuloendotheliosis virus/long terminal repeat/insertional mutagenesis/retroviral integration) DAN JONES*, ROBERT ISFORTt, RICHARD WITTERt, RHONDA KoST*, AND HSING-JIEN KUNG*§ *Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106; tGenetic Toxicology Section, Human and Environmental Safety Division, The Procter and Gamble Company, Miami Valley Laboratories, Cincinnati, OH 45239; and tU.S. Department of Agriculture Agricultural Research Service, Avian Disease and Oncology Laboratory, 3606 East Mount Hope, East Lansing, MI 48823 Communicated by Frederick C. Robbins, January 11, 1993 (receivedfor review November 3, 1992) ABSTRACT We previously described the integration of a integrations are associated with large structural changes in nonacute retrovirus, reticuloendotheliosis virus (REV), into the MDV genome in both the Us and Rs regions (11). the genome of a herpesvirus, Marek disease virus (MDV), By coinfection of duck embryo fibroblasts (DEFs) with following both long-term and short-term coinfection in cul- both REV and MDV, we demonstrated that this process of tured fibroblasts. The long-term coinfection occurred in the retroviral integration can also occur within several passages course of attenuating the oncogenicity ofthe JM strain ofMDV of cocultivation (10). The structure of the inserted REV and was sustained for >100 passages. The short-term coinfec- sequences resembles those of cellular integrated proviruses, tion, which spanned only 16 passages, was designed to recreate with loss of the terminal nucleotides of the retrovirus and a the insertion phenomenon under controlled conditions. We 5-bp duplication ofthe MDV sequence at the site ofinsertion. found that REV integrations into MDV were common and We have extended these analyses and constructed a de- could occur within the first passage following coinfection. Now tailed insertional map of REV sequences in the MDV ge- we have mapped the integration sites. After 5-16 passages in nome. Although no distinct sequence is found at insertion vitro, 17 out of 19 REV junction sites are clustered in two sites, REV integrations in both experiments cluster at the 1-kilobase regions at thejunctions ofthe short unique and short junctions of Us. We report that these junction regions are repeat region of MDV. In the long-term cocultivation experi- heterogeneous in several MDV strains examined.$ ment, 6 out of 10 insertions also mapped in this region. In both cases, integrated proviruses are unstable and undergo subse- MATERIALS AND METHODS quent recombinative deletion, often leaving a solitary long terminal repeat. The long terminal repeat sequences are, Virus Propagation. The MDV clone JM/102W (12) and the however, stably maintained for many rounds of passaging in CS strain ofREV (13) were used for coinfection experiments. vitro. This clustering of insertions presumably is influenced by Other MDV strains used were CU2 (14), Md5 (15), and Mdll selection for viable and fast-growing viruses, and occurs in a (16). Viruses were propagated in DEFs or chicken embryo region of the MDV genome which shows significant size het- fibroblasts as described (17). erogeneity in several strains. Polymerase Chain Reaction (PCR). Total genomic DNA from MDV-infected cells or MDV DNA purified by pulsed- Marek disease virus (MDV) is an avian herpesvirus which field gel electrophoresis (18) was cut with a variety of causes malignant transformation of T cells in chickens. The restriction enzymes (e.g., EcoRI, Sal I, orAva II) and ligated genome structure ofMDV is similar to that ofHerpes simplex in a 100-,ul volume. REV-containing fragments were ampli- virus (HSV), with long and short unique regions (UL and Us) fied by inverse PCR using LTR primers (U3, 5'-CCGAGA- each bounded by inverted repeats (TRL, IRL, IRs and TRs). AATGATATCAGCG-3'; U5, 5'-GGGTGGGGTAGGGA- Although the gene organization is similar to that of HSV (1), TCCGG-3' or 5'-CCGATTCGAATCTGTAATAAAAGC- novel open reading frames in both the Us region and the two TTTTTCTTC-3') which would extend into the flanking repeats have been reported (2, 3). sequences (19). Ten to twenty discrete bands between 0.2 and The disease phenotype of MDV can be altered by pro- 3 kb were seen on ethidium-stained gels with each amplifi- longed passage of the virus in fibroblast culture. With such cation. Products were cloned by using the EcoRV, BamHI, long-term passage both the virulence and the oncogenicity of or HindIll sites present within the primers. Genomic regions MDV are attenuated and changes in the genome are detected. of JM-Lo and Mdll strains were cloned by direct PCR using Most studies have focused on the expansion of a repeat a Us primer for each junction (+394, 5'-ATGGCAGTTTG- sequence in RL (4-6). However, other, undefined structural AGGTTCATG-3'; + 11139, 5'-GACATAACACTCATATT- changes have been noted in the Rs/Us region as well (7, 8). AAGGG-3') or an Rs primer (-30, 5'-GCGGTATGAGATG- During prolonged passage ofthe JM strain ofMDV, viruses CACG-3') with an upstream Rs primer (-862, 5'-TAGCTC- were obtained with improved in vitro growth properties but GAGCCAAAAGGGAA-3') and directly sequenced or blunt- no detectable replication in chickens (9). We have recently end cloned. reported these high-passage JM viruses contain integrated DNA Sequence Analysis. PCR products were cloned into an retroviral sequences which resulted from accidental coinfec- M13 vector and sequenced on both strands by using tion of the MDV-infected fibroblasts with reticuloendotheli- [a-[35S]thio]dATP and T7 polymerase (Sequenase; United osis virus (REV), a chicken retrovirus (10). Even though States Biochemical) (20). Direct DNA sequencing of PCR infectious retrovirus is no longer present in the culture, REV products was also done using thefmol system (Promega) with long terminal repeat (LTR) sequences have been stably maintained in these MDV viruses for >100 passages. These Abbreviations: DEF, duck embryo fibroblast; HSV, herpes simplex virus; LTR, long terminal repeat; MDV, Marek disease virus; REV, reticuloendotheliosis virus. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" $The sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L09061). 3855 Downloaded by guest on October 2, 2021 3856 Biochemistry: Jones et al. Proc. Natl. Acad. Sci. USA 90 (1993) 0 5 7 9 11 13 16 appear by passage 5 following coinfection, and by passage 16 X M.i kb two prominent bands (12 and 1.6 kb) and many minor bands are evident. While this experiment in itself does not distin- guish insertions into the host or MDV genome, we note that 12 REV infection alone should give rise to only the 1.6-kb band, an internal viral fragment (22), and a background smear representing the randomly integrated proviruses. The pres- ence of other bands suggests that there may be specific REV insertions in the MDV genome and that MDV genomes harboring certain REV insertions are the dominant popula- FIG. 1. Detection of LTR in- tion at late passages. sertions in short-term coinfection. Clustered Insertions at the Rs/Us Junctions. To effectively Total genomic DNA from REV/ screen a large number of distinct insertion sites, we used 1 6 MDV-infected DEF monolayers inverse PCR (10). Total genomic DNA collected at each was isolated, digested with passage after coinfection was digested, ligated, and amplified and .w BamHI, blot-hybridized to the by using primers specific for REV LTR. To guard against b REV LTR Sac I-BamHI probe selective amplification of only certain insertion sites, several (10). Lane numbers (0, 5, 7, 11, 13, different and 16) refer to the serial restriction enzymes were used to generate the initial passage linear fragments of the MDV genome prior to ligation. REV 1ITR numbers from the time of REV/ MDV coinfection. As a control for Products representing REV integrants into MDV were ob- 0 5 7 9 11 13 16 MDV copy number, the same blot tained in every passage examined, including several in the was probed with the BamHI N first passage following coinfection. fragment from the UL region of Fig. 2 (upper arrows) shows the distribution of 19 inde- MDV (21). Only a small portion of pendent REV insertions obtained after PCR amplification of MDV Barrn N the autoradiograph is shown. DNA collected from passages 5-16. Remarkably, most ofthe [a-32P]GTP-labeled primers. DNA sequence analysis was insertions obtained are confined to two regions of the virus. carried out with the IBI MACVECTOR program. The left-hand region contains 3 insertions that map in the first Pulsed-Field Gel Electrophoresis. The electrophoresis con- 50 bp of Us; the right side contains 3 insertions that map in ditions and preparation of the DNA plugs were as described the last 700 bp ofUs. Eleven integrants are located in the last (18). The DNA was electrophoresed in a 1% agarose gel in 400 bp of the repeat, so whether they were present on both 0.5 x TBE (TBE is 0.09 M Tris base/0.09 M boric acid) for 20 sides could not be determined in most cases. The remaining hr at 200 V with a 50- to 90-sec switch gradient (Bio-Rad two sites are located in the reported coding region of the gD CHEF-DR II system). MDV minichromosomes from passage glycoprotein in Us (2). To confim the results obtained from 16 DNA were extracted from the gel, digested with EcoRI the inverse PCR, MDV DNA from passage 16 cells was (which does not cleave REV DNA), and ligated into the A purified through a pulsed-field gel and used to construct DASH vector (Stratagene) for construction of the phage phage libraries.