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Journal of Virology, December 2008, p. 11516-11525, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01036-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Points of Recombination in Epstein-Barr Virus (EBV) Strain P3HR-1-Derived Heterogeneous DNA as Indexes to EBV DNA Recombinogenic Events In Vivo {triangledown}

Kazufumi Ikuta,1,2,{dagger} Shamala K. Srinivas,4,{ddagger} Tim Schacker,5,§ Jun-ichi Miyagi,1,2 Rona S. Scott,1,2,3 and John W. Sixbey1,2,3*

Center for Molecular and Tumor Virology,1 Department of Microbiology and Immunology,2 Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana,3 St. Jude Children's Research Hospital, Memphis, Tennessee,4 University of Washington, Seattle, Washington5

Received 16 May 2008/ Accepted 19 September 2008

Deletions and rearrangements in the genome of Epstein-Barr virus (EBV) strain P3HR-1 generate subgenomic infectious particles that, unlike defective interfering particles in other viral systems, enhance rather than restrict EBV replication in vitro. Reports of comparable heterogeneous (het) DNA in EBV-linked human diseases, based on detection of an abnormal juxtaposition of EBV DNA fragments BamHI W and BamHI Z that disrupts viral latency, prompted us to determine at the nucleotide level all remaining recombination joints formed by the four constituent segments of P3HR-1-derived het DNA. Guided by endonuclease restriction maps, we chose PCR primer pairs that approximated and framed junctions creating the unique BamHI M/B1 and E/S fusion fragments. Sequencing of PCR products revealed points of recombination that lacked regions of extensive homology between constituent fragments. Identical recombination junctions were detected by PCR in EBV-positive salivary samples from human immunodeficiency virus-infected donors, although the W/Z rearrangement that induces EBV reactivation was frequently found in the absence of the other two. In vitro infection of lymphoid cells similarly indicated that not all three het DNA rearrangements need to reside on a composite molecule. These results connote a precision in the recombination process that dictates both composition and regulation of gene segments altered by genomic rearrangement. Moreover, the apparent frequency of het DNA at sites of EBV replication in vivo is consistent with a likely contribution to the pathogenesis of EBV reactivation.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130. Phone: (318) 675-4272. Fax: (318) 675-5764. E-mail: jsixbe{at}lsuhsc.edu

{triangledown} Published ahead of print on 25 September 2008.

{dagger} Present address: Department of Microbiology, Fukushima Medical University, 1-1 Hikarigaoka, Fukushima 960-1295, Japan.

{ddagger} Present address: Research Programs Review Branch, NCI, NIH, DHHS, 6116 Executive Branch Blvd., Rm. 8133, MSC 8328, Bethesda, MD 20892.

§ Present address: Department of Medicine, Infectious Diseases Section, University of Minnesota, MMC 250, 516 Delaware Street, Minneapolis, MN 55455.

Journal of Virology, December 2008, p. 11516-11525, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01036-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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