Visualization of molecular biology: The LANA tether

The latency-associated nuclear antigen (LANA) plays a central role in the biology and pathogenesis of Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV). Both classical and endemic KS in HIV-infected individuals and two lymphoproliferative diseases are associated with KSHV. During the latent phase of the viral life cycle in dividing tumor cells, the LANA protein ensures that viral genomes persist by supporting both the initiation of DNA replication and segregation of viral episomes (nonintegrated circular viral genomes) into daughter cells. Arguably, interrupting these complex LANA-dependent processes could be one of the most promising antiviral and antitumor therapeutic strategies. Hence, studying the molecular and cell biological details of LANA’s host/viral protein and chromatin interactions has been a focus in a number of laboratories since 1996. In PNAS, Grant et al. (1) propose an intriguing model of the architectural superstructure of LANA multimers bound to the terminal repeats (TRs) of KSHV genomes by applying superresolution microscopy in combination with computational modeling.

A Brief History

Shortly after KSHV was discovered in KS lesions and primary effusion lymphoma (PEL) cells had been identified as a source for KS virus, reports described characteristic nuclear speckles that were observed by immunofluorescence when staining PEL cells with sera from patients who were PCR-positive for KSHV (2–4). Soon after, cloning and sequencing of the complete KSHV genome and identification of the major KSHV latency-associated genes, in combination with transfection experiments, revealed that LANA encoded by ORF73 is the antigen that reacts with KSHV-positive patient antisera to give rise to “LANA speckles” (5, 6). To date, detection of LANA speckles is the gold standard for KSHV diagnostics (7). LANA is a large 220- to 240-kDa nuclear protein that interacts with many host cellular proteins involved in DNA replication and transcriptional regulation (8). For this discussion, we focus on the role of LANA with respect to genome persistence during latency. The first evidence that KSHV LANA, like EBNA1 from the related human tumor virus Epstein–Barr virus (EBV), is responsible for genome segregation came in 1999, when it was demonstrated that plasmids containing TR sequences were stably segregated in cells expressing LANA (9, 10). Multiple groups identified the TR sequences as cis-regulatory elements essential for both the initiation of DNA replication and the segregation of TR-containing plasmids during mitosis. The LANA C-terminal domain was mapped and shown to bind to two LANA binding sites (LBS1 and LBS2) in a cooperative manner (11). Next, elegant structural and genetic approaches demonstrated that an 18-aa-long N-terminal peptide specifically interacts with the H2A/H2B histone interface, and that this interaction is required for episomal segregation (12). The model that arose from these molecular studies is that LANA binds to the viral TR sequences via its C-terminal DNA binding domain in a highly sequence-specific manner, while tethering viral episomes to host chromatin through interaction of the LANA N-terminal domain with histones. In other words, LANA forms a “tether” or “bridge” between viral and host chromatin. As described above, many molecular details are now known.

The Challenges of Unraveling the LANA Tether

When imaging the structure of the LANA tether in the context of infected cells, many challenges and hurdles exist. For one, only recently have X-ray crystallographic data revealed the structure of the C-terminal DNA binding domain (less than 25% of LANA), either bound or unbound to TR sequences. These data revealed a third, previously overlooked, binding site for LANA, termed LBS3, adjacent to LBS1 and LBS2 (13–16). To the best of our knowledge, no crystals have been obtained for full-length LANA. Second, KSHV-infected cells contain many episomes, each containing between 21 and 45 801-bp TR sequences; each TR contains three LBSs. Third, it is important to note that both viral DNA and host DNA are fully chromatinized and additionally carry specific epigenetic modifications that dictate both transcriptional status and chromatin accessibility.

Facing these challenging circumstances, the Kedes laboratory focused on applying high-resolution imaging techniques to gather structural insight about the LANA tether. They began in 2006, using flow cytometry analysis of LANA epifluorescence in primary infected B cells in combination with qPCR to demonstrate that each LANA speckle is composed of LANA molecules bound to a single viral episome, thereby taking out one of the many stoichiometric variables (17). In this collaborative study between Kedes and Smith and their coworkers (1), direct stochastic optical reconstruction microscopy (dSTORM) is applied to generate a 3D architectural model of one-half of the LANA tether consisting of LANA molecules bound to different numbers of TR sequences either in the context of viral infection or in cells transfected with TR-containing plasmids and a LANA expression construct. Using this platform in combination with elegant genetic tools, they make several key observations that are based on imaging of photons emitted from specific antibodies that stain full-length LANA. First, they show that each tether has specific dimensions in two different cell types infected with the same virus strain. Imaging cells transfected with different copy numbers of TRs (2, 8, or 21) showed linear scaling with LANA binding, suggesting that all TRs are occupied by LANA. By integrating previously published data on the dimensions of active versus transcriptionally suppressed chromatin, they determine that those TR regions between occupied LANA binding sites that are nucleosome-associated show the characteristics of active chromatin. This latter finding is in agreement with studies demonstrating active chromatin region at TRs, but in contrast to studies that identified repressive chromatin remodelers at TRs (11, 18). To construct an overall model of the LANA tether in infected cells, many observations based on the two-TR tether structure are incorporated and integrated with many of the above-described molecular details, such as the bending of DNA by LANA, and the proposed coil-coil domain of the central domain, which will have an impact on the overall tether architecture. To structurally interrogate this domain, a LANA-specific antibody recognizing 22 potential epitopes within the central region was used for imaging. However, if the central LANA domain folded as a coiled-coil, only three of these epitopes should be accessible for antibody binding, which was confirmed by the observed dSTORM signals. Putting it all together, Grant et al. (1) simulate a large number of different models by varying LANA occupancy across LBS1, LBS2, and LBS3; the linkage between the LANA N terminus and C terminus as a coiled-coil domain; and the X-ray crystal structures observed for the C terminus, and, importantly, the angle between LANA tethers in the two-TR model by phasing 10 nucleotides along 360° of the DNA helix. The calculations result in a model with full LANA occupancy at each TR and phase 8 for the two-TR model. Interestingly, when analyzing infected cells with multiple TRs, the phasing varies between different TR tethers. In summary, as indicated in the title, the combination of dSTORM and computational modeling resulted in a beautiful model that integrates dSTORM data with many previous in vitro observations, and therefore lets us “see” the underlying molecular biology of the LANA tether.

What’s Next?

This working model provides a significant step toward addressing additional open questions. For example, how can these studies extend to the other half of the LANA tether to host chromatin? Additionally, a number of proteins, including BRD4, have been proposed to be part of the tether, and recent imaging data on episomes have suggested diversity or clustering during cell divisions between EBV and KSHV episomes (14, 16, 19, 20). These issues could be addressed by applying dSTORM to EBV-infected cells. Furthermore, dSTORM can be extended to multiple color channels, thereby allowing the simultaneous staining of LANA plus BRD4, and by using modalities to stain viral DNA, for example, with custom-designed zinc fingers. Additional structural insight may come from applying cryoelectron microscopy, whose resolution is constantly improving, and which recently has resolved molecular interactions within viral capsids and even ribonucleotide protein complexes of large RNA virus polymerases (21, 22). We are confident that we will have the opportunity to see the next generation of the LANA tether in action in the near future.