Using simultaneous immune fluorescence to detect LANA and fluorescent in situ hybridization to detect KSHV DNA, LANA was shown to colocalize with KSHV episomes along metaphase chromosomes in KSHV latently infected cells (Ballestas et al., 1999; Cotter and Robertson, 1999). This finding suggested LANA had a role in KSHV episome persistence analogous to EBNA1 of Epstein-Barr virus (EBV) (Reedman and Klein, 1973; Grogan et al., 1983; Yates et al., 1984; Harris et al., 1985), and in fact, LANA expressing cells were shown to allow persistence of plasmids containing KSHV TR DNA (Ballestas et al., 1999; Ballestas and Kaye, 2001). This work led to a model in which LANA bridges KSHV DNA to chromosomes during mitosis through concomitantly binding to the cis-acting TRs sequence in the KSHV genome and to mitotic chromosomes. Therefore, LANA tethers KSHV episomes to mitotic chromosomes during cell division to ensure segregation of the viral genome to the nuclei of daughter cells.

Both N- and C-terminal regions of LANA are involved in mitotic chromosome binding. N-terminal LANA residues 5–22 were originally reported by Piolot et al. (2001) to target chromatin during interphase and mitosis. N-terminal LANA also encodes a nuclear localization signal, which is bipartite, located within amino acids 24 to 30 and 41 to 47. Cherezova et al. (2011) consistent with its role in tethering KSHV DNA to mitotic chromosomes, the LANA N-terminal chromosome association region was later shown to be essential for LANA mediated episome persistence. Of interest, the N-terminal region was also found to be critical for efficient LANA mediated DNA replication, despite having no role in binding KSHV DNA (Barbera et al., 2004; Lim et al., 2004). In addition, the N-terminal LANA chromosome binding region also has effects on LANA transcriptional regulation (Wong et al., 2004). Other than microscopy, biochemical evidence also supports N- and C-LANA interactions with chromatin (Viejo-Borbolla et al., 2003; Wong et al., 2004).

N-terminal LANA binds directly to the surface of the nucleosome to attach to chromosomes. X-ray crystal structure of N-terminal LANA complexed with a nucleosome core particle showed that the first 23 amino acids of LANA forms a hairpin that binds exclusively to the acidic patch at the interface of histones H2A and H2B (Barbera et al., 2006a) (Figure 3A). The nucleosome binding property of N-terminal LANA determines its broad distribution across mitotic chromosomes in the absence of episomal DNA (Piolot et al., 2001; Barbera et al., 2004, 2006a; Wong et al., 2004; Chodaparambil et al., 2007). Whether or not histone epigenetic modifications exert a modulatory role on N-terminal LANA binding to the nucleosome surface is not currently known.

C-terminal LANA contains an independent chromosome binding domain, which consists of amino acids 996–1139 (Krithivas et al., 2002; Kelley-Clarke et al., 2007a, 2009). The LANA C-terminal domain is conserved among the LANA orthologs from different gamma-2 herpesviruses, including ORF73s from Retroperitoneal fibromatosis herpesvirus (RFHV) (Burnside et al., 2006), Rhesus rhadinovirus (RRV), Herpesvirus saimiri (HVS) (Calderwood et al., 2004), Herpesvirus ateles (HVA), and murine gammaherpesivrus 68 (MHV68) (Grundhoff and Ganem, 2003; Paden et al., 2012).

C-terminal LANA binds chromatin in a unique pattern in the absence of episomal DNA, concentrating to punctate foci at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes (Krithivas et al., 2002; Kelley-Clarke et al., 2007a). This pattern contrasts sharply with the diffuse chromosome distribution of N-terminal LANA. Self-association of LANA C-terminal region is required for LANA C-terminal chromosome binding (Komatsu et al., 2004; Kelley-Clarke et al., 2009). Of other episome maintenance proteins, EBV EBNA1 does not localize to pericentromeric regions of sister chromatids during mitosis (Szekely et al., 1999; Kanda et al., 2007; Nanbo et al., 2007). The binding pattern of human papillomavirus (HPV) E2 episome maintenance protein to chromosomes can be pericentromeric or not, depending on the virus type (Oliveira et al., 2006).

In the absence of KSHV episomes, full length LANA broadly distributes on mitotic chromosomes (Ballestas et al., 1999; Piolot et al., 2001). However, LANA’s distribution is uneven, and not diffuse, when expressed at physiological levels (Kelley-Clarke et al., 2009). Since N-terminal LANA diffusely paints mitotic chromosomes through binding to histones H2A/H2B, and since C-terminal LANA concentrates to pericentromeric and peri-telomeric regions of mitotic chromosomes (as described above), the result of combining these two localization targeting mechanisms in full length LANA effects broad distribution over mitotic chromosomes, but with more intense staining at peri-telomeric and pericentromeric regions.

In stark contrast to its broad chromosomal distribution in the absence of episomes, LANA concentrates to dots in the presence of KSHV episomal DNA (Figure 3B), both in interphase and along mitotic chromosomes (Ballestas et al., 1999; Cotter and Robertson, 1999; Sakakibara et al., 2004). The concentration to dots occurs at the sites of KSHV episomal genomes, as shown by simultaneous immune fluorescent detection of LANA and fluorescent in situ hybridization with KSHV DNA (Ballestas et al., 1999; Cotter and Robertson, 1999). The strong concentration of LANA to dots at sites of episomal DNA is likely a result of the higher affinity that C-terminal LANA has for its DNA binding site in TR DNA [Kd of binding to LANA adjacent binding sites 1 and 2 ~13.7 nM (Garber et al., 2002; Ponnusamy et al., 2015)] compared to a lower affinity for N-terminal LANA binding to the nucleosome [Kd ~184 nM (Beauchemin et al., 2014)]. Further, each KSHV genome contains ~40 TR copies, and each TR contains three adjacent LANA binding sites (Garber et al., 2002; Hellert et al., 2015). Therefore, each KSHV genome contains ~120 LANA binding sites within its TR elements, to which a LANA dimer binds at each site, resulting in ~240 LANA molecules binding to TR DNA per KSHV genome. LANA bound at TR DNA and simultaneously binding to nucleosomes within mitotic chromosomes results in tethering of the viral genome to mitotic chromosomes. What is less clear, however, is whether or not a subset of LANA molecules bound at TR DNA bind nucleosomes within the KSHV episome; such binding would not result in tethering to mitotic chromosomes but instead would result in LANA doubly bound to the episome: through direct TR DNA binding and also through nucleosomal attachment. If such binding occurs, it would potentially compete with binding to chromosomes, and perhaps could serve as a regulatory mechanism.

Although both N- and C-terminal LANA contain independent chromosome binding regions, N-terminal LANA appears to be the primary effector. Alanine substitution of key chromosome attachment residues in N-terminal LANA abolished LANA’s chromosome association and its ability to mediate episome persistence (Barbera et al., 2004). In contrast, alanine substitutions that dramatically impair C-terminal LANA’s ability to bind mitotic chromosomes did not reduce full length LANA’s association with chromosomes or its ability to mediate episome persistence (Kelley-Clarke et al., 2009). It is important to note, however, that these experiments could not use LANA that was completely abolished for C-terminal chromosome binding. Such null chromosome binding mutations also impaired other critical C-terminal LANA functions, such as DNA binding. Therefore, it remains possible that N-terminal LANA may have “rescued” the impaired (but not abolished) C-terminal LANA chromosome binding, possibly through a cooperative effect. In fact, when N-terminal LANA was mutated so as to reduce (but not abolish) N-terminal LANA chromosome association, the C-terminal chromosome binding mutations resulted both in a reduction of full length LANA binding to mitotic chromosomes and also in a reduction of LANA’s ability to mediate episome persistence (Kelley-Clarke et al., 2009).

Altogether, these data suggest that N-terminal LANA is the dominant effector for chromosome binding, and that C-terminal LANA exerts an auxiliary role. It is tempting to consider that C-terminal LANA was the original chromosome tether, and that N-terminal LANA evolved subsequently as a more efficient alternative. Consistent with this possibility, C-terminal LANA is the most highly conserved region among LANA orthologs.

Latency-associated nuclear antigen has been described to undergo several types of post translational modifications. LANA is a phosphoprotein and is the target of several host cell kinases. Pim-1 and Pim-3 phosphorylate LANA serine residues 205 and 206 to allow lytic reactivation (Bajaj et al., 2006; Cheng et al., 2009). RSK1 and ERK1/2 interact with LANA along with GSK-3 and phosphorylate LANA residues located within 246–258 (Fujimuro et al., 2005; Liu et al., 2007). Notably, RSK3 phosphorylates LANA serine 13 and threonine 14, within the N-terminal chromosome binding region. RSK small molecule inhibition decreased LANA binding to histone H2B as well as LANA protein levels. Treatment of KSHV infected PEL cells with the RSK inhibitor resulted in growth cessation and a decrease in viable cell numbers. In contrast, treatment of uninfected BJAB B cell lymphoma cells resulted in growth cessation, but there was not a decrease in the number of viable cells. These findings show that inhibition of RSKs can impact KSHV persistence (Woodard et al., 2012). DNA-PK was also shown to phosphorylate LANA in at least two sites, one located between amino acids 31–52, and a second between amino acids 91–340, although the specific residues were not identified. The phosphorylation between residues 31–52 may have reduced LANA mediated DNA replication (Cha et al., 2010). C-terminal LANA is also phosphorylated at serine and threonine residues between amino acids 951–1107, and this is mediated by a kinase recruited by RING3 (Brd2) (Platt et al., 1999).

In addition to phosphorylation, N-terminal LANA arginine residue 20, located near the chromosome attachment region, was shown to be methylated by protein arginine methyltransferase 1 and to have a role in antagonizing viral lytic reactivation (Campbell et al., 2012). LANA is also acetylated, and sodium butyrate (a histone deacetylase inhibitor) treatment, reduced LANA’s ability to co immunoprecipitate with histones H2A/H2B (Lu et al., 2006). LANA is also a target of poly (ADP-ribose) polymerase 1 (PARP-1) and is poly (ADP-ribosyl) ated (Ohsaki et al., 2004). The sites of acetylation or poly (ADP-ribosyl) ation within LANA are not currently known. In addition, C-terminal LANA is SUMOylated at lysine 1140 (Cai et al., 2013).

Latency-associated nuclear antigen binds TR DNA to mediate KSHV DNA replication and to tether episomes to mitotic chromosomes. The finding that LANA acted on KSHV TR associated DNA to mediate episome persistence in the absence of other virus genes led to the hypothesis that LANA can directly interact with TR DNA. Subsequently, a 20 bp sequence in the TR was identified as a LANA binding site (LBS1) and was bound by the LANA C-terminal domain, also referred to as the DNA binding domain (DBD) (Ballestas and Kaye, 2001; Cotter et al., 2001; Garber et al., 2001). Mutagenesis of the LANA binding site revealed that the ₆CCC₈ nucleotides of LBS1 are critical for LANA interaction. Substitutions of these nucleotides severely impacted LANA binding. In contrast, mutations within other nucleotides had only modest effects on binding (Srinivasan et al., 2004). A second LBS (LBS2) was identified downstream of LBS1 through electrophoretic mobility shift assay (EMSA) and DNase I footprinting experiments (Garber et al., 2002). Compared to LBS1, LBS2 is a low affinity LANA binding site. However, since LBS2 is adjacent to LBS1, LBS1 is capable of facilitating cooperative binding of LANA to LBS2 (Garber et al., 2002). Recently, a third LBS (LBS3) was identified, also adjacent to LBS1, but with inverse orientation compared to LBS1 and LBS2 (Hellert et al., 2015) (Figure 4A). Similar to LBS2, LBS3 is a low affinity binding site with about a 100-fold lower LANA binding affinity compared to LBS1. Interestingly, LANA DBD binding to LBS1 and LBS3, or to LBS2–LBS1–LBS3 proceeds in a biphasic manner, with LANA binding to LBS1 (or cooperatively to LBS2–LBS1) in the first phase, and then binding LBS3 in the second phase (Ponnusamy et al., 2015). The biphasic binding to LBS3 could be due to the slightly greater distance between LBS1 and LBS3 as compared to LBS1 and LBS2 (Ponnusamy et al., 2015). LBS3 was previously recognized as a 32 bp sequence essential for LANA mediated replication of TR DNA, and had been termed the replication element (Hu and Renne, 2005). Although all three LBSs share homologous sequence, both LBS2 and LBS3 harbor a cytosine to guanine transversion at position 8 (Hellert et al., 2015). Considering the critical role of this cytosine for LBS1 binding affinity (Srinivasan et al., 2004), it is very likely that the low binding affinities for LANA of LBS2 and LBS3 arises from this cytosine to guanine substitution at position 8. The spacing of 22 bp between each LBS results in location of the LBS’s on the same face of DNA, thereby allowing adjacent LANA dimers to bind cooperatively (Garber et al., 2002; Hu and Renne, 2005; Hellert et al., 2015). In addition to binding viral DNA, LANA is capable of binding host genomic DNA. Chip-seq data shows that host LANA-binding sites are generally found within transcriptionally active promoters with sequence homology to the characterized LBS’s within TR DNA (Lu et al., 2012; Hu et al., 2014; Mercier et al., 2014). A novel LANA binding motif (TCCAT)₃, which is only present in host LANA-binding sites, and to which LANA binds with low affinity, similar to LBS2, was also confirmed by gel shift analysis (Hu et al., 2014).

Mutagenesis has also defined residues within the LANA DBD as important for DNA binding (Figure 4B). LANA mutants which are deficient for, or lack DNA binding ability, are also deficient or abolished for the ability to mediate KSHV DNA replication or episome persistence (Komatsu et al., 2004; Han et al., 2010). The inability of LANA abolished for DNA binding to mediate episome persistence is due both to an inability to replicate KSHV DNA as well as loss of the ability to tether KSHV episomes to mitotic chromosomes to segregate episomes to daughter nuclei. C-terminal LANA also mediates LANA’s self-association, and self-association is necessary for DNA binding; deletions in C-terminal LANA that result in the loss of LANA self-association also result in loss of DNA binding and episome persistence (Schwam et al., 2000; Komatsu et al., 2004). As noted above, C-terminal LANA concentrates to percicentromeres and peri-telomeres of a subset of mitotic chromosomes in addition to binding KSHV TR DNA. Alanine scanning mutagenesis of C-terminal LANA revealed that different subsets of residues are important for binding to TR DNA versus mitotic chromosomes (Kelley-Clarke et al., 2007b; Han et al., 2010). Therefore, C-terminal LANA does not appear to attach to mitotic chromosomes through recognition of its TR DNA binding sequence.

The x-ray crystal structures of the KSHV and MHV68 LANA DNA binding domains were recently solved (Correia et al., 2013; Domsic et al., 2013; Hellert et al., 2013, 2015; Ponnusamy et al., 2015). These revealed an alpha-beta fold that assembles as a dimer, and that is structurally similar to the EBV EBNA1 and papillomavirus E2 DBDs. The hydrophobic interface of LANA DBD dimerization interface is mediated by an eight-strand anti-parallel intermolecular β-barrel to which each monomer contributes four β-sheets (Domsic et al., 2013; Hellert et al., 2013, 2015). However, in contrast to EBNA1 and E2, a unique feature of LANA is a positive electrostatic patch on the surface opposite of the DNA binding surface. This positive patch serves to interact with BET proteins, although BET proteins also interact with a second region (residues 1125–1129) in C-terminal LANA (Hellert et al., 2013). Mutation of the positive patch reduced LANA’s ability to mediate DNA replication and episome persistence, and mutation of the peripheral region of the positive patch generated the most severe effects, compared to mutation of other portions of the patch (Correia et al., 2013; Domsic et al., 2013; Hellert et al., 2013; Li et al., 2015). These deficiencies in replication and maintenance were unrelated to BET protein binding (Correia et al., 2013; Li et al., 2015). Mutation of the analogous positive patch in MHV68 LANA impacted the ability of MHV68 to establish latent infection in mice (Correia et al., 2013; Hellert et al., 2013).

Another notable finding of the LANA structure was an oligomerization interface between LANA dimers. This ability of adjacent LANA dimers to interact underlies LANA’s cooperative binding to adjacent DNA binding sites. Disruption of this interface results in loss of LANA cooperative binding to LANA binding sites such as LBS1 and LBS2, deficient DNA replication, and deficient episome persistence (Domsic et al., 2013; Hellert et al., 2013). Importantly, the hydrophobic interface that mediates the dimer–dimer interaction results in a bend and also is a flexible pivot point that allows rotation and bending between two adjacent dimers (Ponnusamy et al., 2015). Variation in the degree of bend between adjacent dimers can result in LANA DBD dimers oligomerizing to form ring structures consisting of different numbers of dimers (e.g., four or five dimers) (Figure 5A). In these LANA structures, the DNA binding surface of each dimer is on the exterior face (Domsic et al., 2013; Hellert et al., 2013, 2015). In addition, LANA dimers can form non-ring structures (Figure 5B) as well as a spiral (Hellert et al., 2015; Ponnusamy et al., 2015). MHV68 LANA also forms oligomers through interactions between adjacent dimers, which mediates cooperative binding to adjacent binding sites in MHV68 TR DNA. However, in contrast to KSHV LANA, MHV68 LANA forms a rigid, linear conformation compared to KSHV LANA’s bent, flexible configuration (Hellert et al., 2013; Ponnusamy et al., 2015).

A recent x-ray crystal structure of the KSHV LANA DBD bound to the high affinity LBS1 recognition site was solved, revealing insight into LANA’s recognition of DNA (Hellert et al., 2015) (Figure 4C). To obtain the structure, point mutations were introduced in the DBD in order to obtain well-behaved complexes with DNA. Notably, and in contrast to EBNA1 or E2’s recognition of DNA (Hegde et al., 1992; Bochkarev et al., 1996), the LANA DNA dimer binds LBS1 with remarkable asymmetry, with the primary sequence recognition occurring within G5 to G10 of the 20 bp LBS1. These findings are consistent with mutagenesis studies of LBS1 as discussed above, which revealed that alteration of LBS1 ₆CCC₈ nucleotides resulted in severe deficiency of LANA binding as compared to other mutations (Srinivasan et al., 2004). The N-terminal arm residues of one monomer, and helices 1 and 2 of the second monomer make sequence specific contacts with base pairs G5 to G10. The corresponding residues of the other monomer within the dimer make contact only with the DNA backbone. Deletion (LANAΔ1007–1021) or point mutations (R1013, Y1014) within the N-terminal arm of the LANA DBD ablates TR DNA binding, replication and episome maintenance (Komatsu et al., 2004; Domsic et al., 2013). A bend of 25° is introduced in DNA by the bound LBS1 DNA, which is less than the previously reported bend of 57°, but could be due to crystal packing constraints (Wong and Wilson, 2005).

In addition to sequence specific binding, the LANA DBD was also proposed to bind DNA in a sequence independent manner (Hellert et al., 2015). In both the ring forms (Figure 5A) and spiral structures (as described above) formed by oligomerizing LANA dimers, the sequence specific binding surface is located on the outside of the rings or spiral, and the positive electrostatic patch is located on the inside of the rings or circle, to form a continuous, positively charged inner surface. The inner diameter of the rings or spiral is large enough to accommodate double stranded DNA. In fact, negative staining electron microscopy demonstrated that LANA DBDs mutated for sequence specific binding, were able to form complexes on an arbitrary dsDNA fragment. Mutations of the oligomerization surface or of the positive electrostatic patch abolished the LANA DBD’s ability to complex with the DNA fragment (Hellert et al., 2015). It is possible that related, higher order structures of LANA may account for the LANA dots observed when LANA concentrates at episomes in cells.