Journal of General Virology

Hot-spot variations of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen and application in genotyping by PCR–RFLP

Yan-Jin Zhang,¹ Jian-Hong Deng,¹ Charles Rabkin² and Shou-Jiang Gao¹

¹ Departments of Pediatrics and Microbiology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
² National Cancer Institute, Bethesda, Maryland, USA

Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus-8) is aetiologically associated with Kaposi's sarcoma and several other lymphoproliferative disorders. The latent nuclear antigen (LNA) encoded by KSHV ORF73 has important functions in virus latent infection and shows molecular polymorphism. Sequence variations were identified in the internal repeat domain (IRD) of ORF73. DNA sequencing of ORF73 from one KSHV-infected cell line, PK-1, revealed that there were 558 bp (30.2 %) deletions and 66 (3.6 %) point mutations located mainly in repeat region 2, the glutamine-rich region of ORF73 IRD, compared with ORF73 of BC-1 KSHV. Similar sequence variations of ORF73 were also identified in two other isolates. None of the sequence variations caused any translational frame-shift in these four KSHV isolates examined, suggesting that LNA has a conservative function in virus latent infection. The frequent sequence variations in repeat region 2 of ORF73 IRD were also identified by PCR–RFLP genotyping in 26 KSHV isolates, suggesting that this region is a 'hot-spot' for genetic variations. Each Kaposi's sarcoma lesion sample contained one virus genotype with a unique RFLP pattern, indicating that in vivo KSHV infection was established with single predominate genotypes, which was further supported by the presence of invariable genotypes in multifocal lesions from individual KS patients. Four KSHV subtypes were classified based on the RFLP patterns that represent the patterns of DNA sequence variations in the ORF73 IRD. PCR–RFLP genotyping is capable of identifying LNA genetic variations and differentiating individual KSHV isolates, and thus may be useful for KSHV molecular epidemiology studies.

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© 2000 SGM

This article is now available in the August 2000 print issue of JGV (vol. 81, 2049–2058). The complete issue of the journal may be seen in electronic form on JGV Online.