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

Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus-8) is aeti ologically associated with Kaposi's sarcoma and several other lymphoprolife rative disorders. The latent nuclear antigen (LNA) encoded by KSHV ORF73 ha s important functions in virus latent infection and shows molecular polymor phism. Sequence variations were identified in the internal repeat domain (I RD) 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 mu tations located mainly in repeat region 2, the glutamine-rich region of ORF 73 IRD, compared with ORF73 of BC-1 KSHV. Similar sequence variations of OR F73 were also identified in two other isolates. None of the sequence variat ions caused any translational frame-shift in these four KSHV isolates exami ned, suggesting that LNA has a conservative function in virus latent infect ion. 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, indi cating that in vivo KSHV infection was established with single predominate genotypes, which was further supported by the presence of invariable genoty pes in multifocal lesions from individual KS patients. Four KSHV subtypes w ere classified based on the RFLP patterns that represent the patterns of DN A sequence variations in the ORF73 IRD. PCR-RFLP genotyping is capable of i dentifying LNA genetic variations and differentiating individual KSHV isola tes, and thus may be useful for KSHV molecular epidemiology studies.