Expansion of a unique region in the Marek's disease virus genome occurs concomitantly with attenuation but is not sufficient to cause attenuation

Abstract

Pathogenic Marek's disease viruses (MDVs) have two head-to-tail copies of a 132-bp repeat. As MDV is serially passaged in cell culture, the virus becomes attenuated and the number of copies of the 132-bp repeat increases from 2 to often more than 20 copies. To determine the role of the repeats in attenuation, we used five overlapping cosmid clones that spanned the MDV genome to reconstitute infectious virus (rMd5). By mutating the appropriate cosmids, we generated clones of infectious MDVs that contained zero copies of the 132-bp repeats, rMd5(Delta132); nine copies of the 132-bp repeats, rMd5(9-132); and nine copies of the 132-bp repeats inserted in the reverse orientation, rMd5(rev9-132). After two passages in cell culture, wild-type Md5, rMd5, and rMd5(Delta132) were stable. However, rMd5(9-132) and rMd5(rev9-132) contained a population of viruses that contained from 3 to over 20 copies of the repeats. A major 1.8-kb mRNA, containing two copies of the 132-bp repeat, was present in wild-type Md5 and rMd5 but was not present in rMd5(Delta132), rMd5(9-132), rMd5(rev9-132), or an attenuated MDV. Instead, the RNAs transcribed from the 132-bp repeat region in rMd5(9-132) and rMd5(rev9-132) closely resembled the pattern of RNAs transcribed in attenuated MDVs. When inoculated into susceptible day-old chicks, all viruses produced various lesions. Thus, expansion of the number of copies of 132-bp repeats, which accompanies attenuation, is not sufficient in itself to attenuate pathogenic MDVs.

FIG. 1.

Overlapping cosmid clones of Md5. The MDV-1 viruses contain two copies of a 132-bp repeat, located in two separate parts of the genome. One pair of repeats is in the terminal repeat region (TRL) adjacent to the unique long region (UL). The other pair of repeats is inverted and located in the internal repeat (IRL) at the other end of the UL. The five overlapping cosmid clones are shown below the MDV genome and are aligned with the regions of the MDV genome from which they were cloned. The small bar in SN5 and A6 represents the location of the 132-bp repeats in these cosmids and their corresponding location in the TRL and IRL.

FIG. 2.

Generation of deletion in cosmid clones. The figure shows an overview of the RARE cleavage procedure used to delete the 132-bp repeats in SN5 and A6. The short lines numbered 1 and 2 represent oligonucleotides 1 and 2, used to protect two TaqI sites. The two TaqI sites shown flank a 411-bp fragment of cosmid DNA that contains two copies of the 132-bp repeat.

FIG. 3.

HindIII digestion of cosmids. The DNA from the cosmids was extracted, digested with HindIII, and separated on an agarose gel. The arrows point to the 4.7-kb fragment in SN5 and A6 that contains two copies of the 132-bp repeat. Numbers at left of each panel indicate molecular size in kilobases.

FIG. 4.

Southern blot of recombinant MDV. DNA from MDV-infected DEF was digested with TaqI, Southern blotted, and probed with the 132-bp repeats. Viral names that are followed by an A, B, or C represent separate independently generated recombinants. Positions of two, three, and nine copies of the 132-bp repeat are shown on the right. Numbers at left are molecular sizes in kilobases.

FIG. 5.

Northern blot of RNA from infected DEF. RNA extracted from MDV-infected DEF was Northern blotted and probed with a labeled probe from the 132-bp repeat region (A), the gB gene (UL27) (B), or an 834-bp fragment that spans the 132-bp repeat region (C). The arrows in panels A and C point to the 1.8-kb RNA. Numbers at left of panel A and at right of panels B and C are molecular sizes in kilobases.

FIG. 6.

Growth curve of recombinant MDVs in DEF. Fresh DEF were infected with the indicated viruses. On days 0, 1, 2, 3, 5, and 7, cells were trypsinized and plated on fresh DEF. The resulting plaques were counted 7 to 8 days later. Each point represents the average from three plates.

FIG. 7.

Southern blot of DNA extracted from tumors. Upon death or termination of the animal experiment, DNA was extracted from gross tumors and digested with TaqI. A Southern blot of the RE-digested DNA was probed with a [³²P]dCTP-labeled 132-bp repeat fragment. Each lane is labeled with the name of the inoculated virus, followed by the bird number and the tumor source. The positions of two and nine copies of the 132-bp repeat are shown on the right. Numbers at left are molecular sizes in kilobases.