Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates the maintenance of episomal viral genomes in latently infected cells. The two central components of episome persistence are DNA replication with each cell division and the segregation of DNA to progeny nuclei. LANA self-associates to bind KSHV terminal-repeat (TR) DNA and to mediate its replication. LANA also simultaneously binds to TR DNA and mitotic chromosomes to mediate the segregation of episomes to daughter nuclei. The N-terminal region of LANA binds histones H2A and H2B to attach to mitotic chromosomes, while the C-terminal region binds TR DNA and also associates with chromosomes. Both the N-and C-terminal regions of LANA are essential for episome persistence. We recently showed that deletion of all internal LANA sequences results in highly deficient episome maintenance. Here we assess independent internal LANA regions for effects on episome persistence. We generated a panel of LANA mutants that included deletions in the large internal repeat region and in the unique internal sequence. All mutants contained the essential N-and C-terminal regions, and as expected, all maintained the ability to associate with mitotic chromosomes in a wild-type fashion and to bind TR DNA, as assessed by electrophoretic mobility shift assays (EMSA). Deletion of the internal regions did not reduce the half-life of LANA. Notably, deletions within either the repeat elements or the unique sequence resulted in deficiencies in DNA replication. However, only the unique internal sequence exerted effects on the ability of LANA to retain green fluorescent protein (GFP) expression from TR-containing episomes deficient in DNA replication, consistent with a role in episome segregation; this region did not independently associate with mitotic chromosomes. All mutants were deficient in episome persistence, and the deficiencies ranged from minor to severe. Mutants deficient in DNA replication that contained deletions within the unique internal sequence had the most-severe deficits. These data suggest that internal LANA regions exert critical roles in LANA-mediated DNA replication, segregation, and episome persistence, likely through interactions with key host cell factors.

Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence

“…We recently identified an internal 59-aa LANA region critical for efficient DNA replication and persistence (24,25). Here, we find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader (26), through this sequence to mediate viral DNA replication and episome persistence.…”

A short sequence immediately upstream of the internal repeat elements is critical for KSHV LANA mediated DNA replication and impacts episome persistence

“…N- and C-terminal LANA are essential for tethering KSHV DNA to mitotic chromosomes, and therefore are essential for segregation. We also recently showed that internal LANA residues 33-331 are critical for segregation(De Leon Vazquez et al, 2013). This work also showed that LANAΔ264-929 had an intermediate phenotype, consistent with amino acid 264 being near the border of sequence important for segregation.…”

“…LANA deleted for amino acids 332-929 (LANAΔ332-929) was 10.9 fold reduced for episome persistence efficiency, while LANA deleted for residues 264-929 (LANAΔ264-929) was reduced 228.6 fold (Fig. 1)(De Leon Vazquez et al, 2013). Here, we perform a targeted investigation of the region immediately upstream of the LANA internal repeat elements.…”

“…Further, a panel of large internal deletion mutants suggested that a small region, located immediately upstream of the internal repeat elements, may have a key role in episome persistence(De Leon Vazquez et al, 2013). Notably, this region overlaps with residues 275-331 that were reported to reorganize heterochromatin.…”

Identification of Novel Kaposi's Sarcoma-Associated HerpesvirusOrf50Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential

“…Another interesting promoter is the Orf73Li promoter that drives the transcription of the latency-associated protein LANA. This protein is essential for the establishment, maintenance, and control of viral latency (76)(77)(78)(79)(80). Two other unique transcripts that are more strongly upregulated by RTA isoform 2 than RTA isoform 1 are the Orf47 and Orf49 promoters, which drive the expression of transcription that are antisense to the Orf50 region (Fig.…”