Disruption of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen leads to abortive episome persistence.

ABSTRACT: Latent nuclear antigen (LNA) is implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) episome persistence. LNA colocalizes with KSHV episomes on chromosomes in metaphase, and it maintains the stability and replication of KSHV terminal repeat-containing plasmids. In this study, we examined the function of LNA in episome persistence in the context of full-length KSHV genome by mutagenesis analysis. We generated a KSHV mutant, BAC36-DeltaLNA, with LNA disrupted by transposon-based mutagenesis with a KSHV BAC clone, BAC36, as a template. Immunofluorescence antibody staining revealed that the insertion of a transposon cassette into LNA disrupted its expression but had no effect on the expression of two adjacent genes, the vCyclin and vFLIP genes. Using a green fluorescent protein (GFP) cassette as a tracking marker for the KSHV episome, we found 8.7-fold-fewer GFP-positive cells in BAC36-DeltaLNA cultures than in wild-type BAC36 cultures at the early stage following episome delivery into 293 cells by transfection, which could be partially rescued by cotransfection with a LNA expression plasmid but not a control plasmid. Cells harboring BAC36-DeltaLNA with or without transient complementation rapidly lost episomes and became virus-free after 2 weeks of culture based on GFP expression and Gardella gel analysis and quantitative PCR assays for detecting KSHV genomes. In contrast, BAC36 episomes were stably maintained during the same period. Stable cultures with close to 100% of cells harboring KSHV episomes were readily established by hygromycin selection for BAC36 but not for BAC36-DeltaLNA. These results conclusively indicate that LNA is essential for the establishment and persistence of KSHV episomes in mammalian cells.

SUBMITTER: Ye FC 

PROVIDER: S-EPMC521844 | biostudies-other | 2004 Oct

REPOSITORIES: biostudies-other