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-⌬LNA, 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-⌬LNA 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-⌬LNA 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-⌬LNA. These results conclusively indicate that LNA is essential for the establishment and persistence of KSHV episomes in mammalian cells.

Kaposi's sarcoma herpesvirus-encoded latency-associated nuclear antigen stabilizes intracellular activated Notch by targeting the Sel10 protein

“…It has been shown that LANA autoactivates its own promoter; thus, the expression of LANA is maintained consistently at a high level by the regulation of this positive-feedback loop (33). High expression of LANA in KSHV-infected cells is one strategy for the virus to establish and maintain latent infection because down regulation of LANA expression results in disruption of viral latency (34)(35)(36).…”

Bovine herpesvirus 4 ORF73 is dispensable for virus growth in vitro, but is essential for virus persistence in vivo

“…For example, bovine herpesvirus 4 (BoHV-4) encodes orthologues of ORF71 and ORF73, whereas ORF72 is absent (Zimmermann et al, 2001 pORF73 is the latency-associated nuclear antigen 1 (LANA-1) of HHV-8. LANA-1 associates with mitotic chromosomes to enable episome tethering during division of latently infected cells and its subsequent partitioning between daughter cells (Ballestas et al, 1999; Barbera et al, 2006;Ye et al, 2004). This crucial role in latency is reinforced by the ability of LANA-1 to modulate cellular pathways implicated in cell growth and survival by targeting transcriptional regulators or co-regulators such as p53, pRB, CBP and sin3A (Friborg et al, 1999;Krithivas et al, 2000;Lim et al, 2001;Radkov et al, 2000).…”

Increased tumorigenicity of cells carrying recombinant human herpesvirus 8

“…Recently, a recombinant bacterial artificial chromosome (BAC) containing the entire HHV-8 genome (HHV-8 BAC) was generated [30]. Using such technology, essential in vitro roles for RTA (ORF50) in viral reactivation, LANA (ORF73) in viral episome maintenance, and glycoprotein B (ORF8) for viral egress were clearly demonstrated, highlighting the usefulness of the HHV-8 BAC [14,27,28]. The lack of suitable animal models allowing the study of the tumorigenic potential of cells carrying WT HHV-8 prompted us to tackle this issue.…”