Kaposi's Sarcoma Herpesvirus Genome Persistence

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) has an etiologic role in Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. These diseases are most common in immunocompromised individuals, especially those with AIDS. Similar to all herpesviruses, KSHV infection is lifelong. KSHV infection in tumor cells is primarily latent, with only a small subset of cells undergoing lytic infection. During latency, the KSHV genome persists as a multiple copy, extrachromosomal episome in the nucleus. In order to persist in proliferating tumor cells, the viral genome replicates once per cell cycle and then segregates to daughter cell nuclei. KSHV only expresses several genes during latent infection. Prominent among these genes, is the latency-associated nuclear antigen (LANA). LANA is responsible for KSHV genome persistence and also exerts transcriptional regulatory effects. LANA mediates KSHV DNA replication and in addition, is responsible for segregation of replicated genomes to daughter nuclei. LANA serves as a molecular tether, bridging the viral genome to mitotic chromosomes to ensure that KSHV DNA reaches progeny nuclei. N-terminal LANA attaches to mitotic chromosomes by binding histones H2A/H2B at the surface of the nucleosome. C-terminal LANA binds specific KSHV DNA sequence and also has a role in chromosome attachment. In addition to the essential roles of N- and C-terminal LANA in genome persistence, internal LANA sequence is also critical for efficient episome maintenance. LANA's role as an essential mediator of virus persistence makes it an attractive target for inhibition in order to prevent or treat KSHV infection and disease.

Keywords: DNA binding; KSHV; chromosome; latency-associated nuclear antigen; viral persistence.

FIGURE 1

Schematic diagram of Kaposi’s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA). Indicated are the proline-rich region (P), and the repetitive regions (DE, QEP, QREP, QDE, L, EQE). The black areas indicate the bipartite N-terminal nuclear localization signal (NLS) within amino acids 24 to 30 and 41 to 47. Amino acids 5 to 13 mediate chromosome association through interaction with histones H2A/H2B. Amino acids 996 to 1139 contain TR DNA binding, self-association, and chromosome association functions.

FIGURE 2

Schematic representation of the circularized KSHV genome. The terminal repeats (TRs), in blue, comprise around 20% of the genome. KSHV open reading frames (ORF) are in brown, miRNAs are in dark gray and non-coding long RNA PAN is in light gray. Alternative protein names are written in parenthesis. Arrows indicate transcription direction. Although KSHV persistence is the focus of this article, most KSHV genes are shown. This map was generated from the HHV-8 complete genome sequence (Genbank accession AF148805) strain GK18 using SerialCloner software.

FIGURE 3

Latency-associated nuclear antigen associates with chromosomes. (A) X-ray crystal structure of N-terminal LANA complexed with the nucleosome. A space-filling representation is shown. (histone H2A, yellow; H2B, red; H3 light blue; H4, green; LANA, dark blue; DNA, silver). From PDB 1zla (Barbera et al., 2006a). (B) LANA associates with cellular chromosomes in a broad distribution in the absence of episomes, and concentrates to dots on chromosomes at sites of episomes. Cells expressing LANA were arrested in metaphase with colcemid. LANA (green) was detected with antibody directed against LANA. DNA was counterstained with propidium iodide (red) (overlay of red and green generates yellow.) White arrows indicate LANA dots at sites of KSHV episomes.

FIGURE 4

Latency-associated nuclear antigen binds specific sequence in KSHV TR DNA. (A) TR sequence with LBS sequences indicated by arrows and highlighting. (B) C-terminal LANA residues. Residues which impact DNA binding by EMSA (Kelley-Clarke et al., 2007b; Han et al., 2010) are highlighted in blue and those involved in oligomerization (Correia et al., 2013; Domsic et al., 2013; Hellert et al., 2013, 2015; Ponnusamy et al., 2015) of LANA dimers are highlighted in orange. (C) Crystal structure of C-terminal LANA complexed with LBS1 (Hellert et al., 2015) PDB:4UZB. One LANA monomer is shown in blue, and the other in green.

FIGURE 5

Higher-order crystal structures of the KSHV LANA DNA binding domain. (A) Ring form structure of the KSHV LANA DNA binding domain composed of 5 dimers (from PDB:4K2J.) (Domsic et al., 2013). (B) Non-ring form tetramer structure of KSHV LANA DNA binding domain (from PDB:5A76.) (Ponnusamy et al., 2015).

FIGURE 6

Schematic representation of episome persistence through the cell cycle. LANA tethers the episome to the cellular chromosome. During S phase, the viral episome replicates once in concert with cell DNA (Verma et al., 2007). A potential model is shown of two replicated episomes attaching to adjacent sister chromatids and segregating to daughter nuclei through tethering to chromatids.

FIGURE 7

Model of LANA tethering the KSHV genome to a chromosome. N-terminal LANA (N) binds to core histones H2A/H2B. C-terminal LANA (C) self-associates, binds to KSHV TR DNA in the KSHV genome, and binds to a putative protein (X) that associates with the chromosome.