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Visualization of Molecular Biology: the LANA Tether Vaibhav Jaina and Rolf Rennea,B,C,1

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Visualization of Molecular Biology: the LANA Tether Vaibhav Jaina and Rolf Rennea,B,C,1 COMMENTARY COMMENTARY Visualization of molecular biology: The LANA tether Vaibhav Jaina and Rolf Rennea,b,c,1 The latency-associated nuclear antigen (LANA) plays we focus on the role of LANA with respect to ge- a central role in the biology and pathogenesis of nome persistence during latency. The first evidence Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV). that KSHV LANA, like EBNA1 from the related hu- Both classical and endemic KS in HIV-infected individ- man tumor virus Epstein–Barr virus (EBV), is re- uals and two lymphoproliferative diseases are associ- sponsibleforgenomesegregationcamein1999, ated with KSHV. During the latent phase of the viral when it was demonstrated that plasmids containing TR life cycle in dividing tumor cells, the LANA protein en- sequences were stably segregated in cells expressing sures that viral genomes persist by supporting both LANA (9, 10). Multiple groups identified the TR se- the initiation of DNA replication and segregation of quences as cis-regulatory elements essential for both viral episomes (nonintegrated circular viral genomes) the initiation of DNA replication and the segregation into daughter cells. Arguably, interrupting these com- of TR-containing plasmids during mitosis. The LANA plex LANA-dependent processes could be one of the C-terminal domain was mapped and shown to bind most promising antiviral and antitumor therapeutic to two LANA binding sites (LBS1 and LBS2) in a co- strategies. Hence, studying the molecular and cell bio- operative manner (11). Next, elegant structural and ge- logical details of LANA’s host/viral protein and chro- netic approaches demonstrated that an 18-aa-long matin interactions has been a focus in a number of N-terminal peptide specifically interacts with the H2A/ laboratories since 1996. In PNAS, Grant et al. (1) pro- H2B histone interface, and that this interaction is re- pose an intriguing model of the architectural super- quired for episomal segregation (12). The model that structure of LANA multimers bound to the terminal arose from these molecular studies is that LANA binds repeats (TRs) of KSHV genomes by applying superre- to the viral TR sequences via its C-terminal DNA binding solution microscopy in combination with computational domain in a highly sequence-specific manner, while modeling. tethering viral episomes to host chromatin through in- teraction of the LANA N-terminal domain with histones. A Brief History In other words, LANA forms a “tether” or “bridge” Shortly after KSHV was discovered in KS lesions and between viral and host chromatin. As described above, primary effusion lymphoma (PEL) cells had been many molecular details are now known. identified as a source for KS virus, reports described characteristic nuclear speckles that were observed by The Challenges of Unraveling the LANA Tether immunofluorescence when staining PEL cells with sera When imaging the structure of the LANA tether in from patients who were PCR-positive for KSHV (2–4). the context of infected cells, many challenges and Soon after, cloning and sequencing of the complete hurdles exist. For one, only recently have X-ray crys- KSHV genome and identification of the major KSHV tallographic data revealed the structure of the C- latency-associated genes, in combination with trans- terminal DNA binding domain (less than 25% of fection experiments, revealed that LANA encoded by LANA), either bound or unbound to TR sequences. ORF73 is the antigen that reacts with KSHV-positive These data revealed a third, previously overlooked, patient antisera to give rise to “LANA speckles” (5, binding site for LANA, termed LBS3, adjacent to 6). To date, detection of LANA speckles is the gold LBS1 and LBS2 (13–16). To the best of our knowl- standard for KSHV diagnostics (7). LANA is a large edge, no crystals have been obtained for full-length 220- to 240-kDa nuclear protein that interacts with LANA. Second, KSHV-infected cells contain many many host cellular proteins involved in DNA replication episomes, each containing between 21 and 45 801-bp and transcriptional regulation (8). For this discussion, TR sequences; each TR contains three LBSs. Third, it is aDepartment of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610; bUF Health Cancer Center, University of Florida, Gainesville, FL 32610; and cUF Genetics Institute, University of Florida, Gainesville, FL 32610 Author contributions: V.J. and R.R. wrote the paper. The authors declare no conflict of interest. Published under the PNAS license. See companion article on page 4992. 1To whom correspondence should be addressed. Email: [email protected]. Published online April 24, 2018. 4816–4818 | PNAS | May 8, 2018 | vol. 115 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1804797115 Downloaded by guest on September 30, 2021 important to note that both viral DNA and host DNA are fully chro- domain folded as a coiled-coil, only three of these epitopes matinized and additionally carry specific epigenetic modifications should be accessible for antibody binding, which was confirmed that dictate both transcriptional status and chromatin accessibility. by the observed dSTORM signals. Putting it all together, Grant Facing these challenging circumstances, the Kedes laboratory et al. (1) simulate a large number of different models by varying focused on applying high-resolution imaging techniques to LANA occupancy across LBS1, LBS2, and LBS3; the linkage gather structural insight about the LANA tether. They began in between the LANA N terminus and C terminus as a coiled-coil 2006, using flow cytometry analysis of LANA epifluorescence in domain; and the X-ray crystal structures observed for the C ter- primary infected B cells in combination with qPCR to demonstrate minus, and, importantly, the angle between LANA tethers in the that each LANA speckle is composed of LANA molecules bound two-TR model by phasing 10 nucleotides along 360° of the DNA to a single viral episome, thereby taking out one of the many helix. The calculations result in a model with full LANA occu- stoichiometric variables (17). In this collaborative study between pancy at each TR and phase 8 for the two-TR model. In- Kedes and Smith and their coworkers (1), direct stochastic optical terestingly, when analyzing infected cells with multiple TRs, the reconstruction microscopy (dSTORM) is applied to generate a 3D phasing varies between different TR tethers. In summary, as in- architectural model of one-half of the LANA tether consisting of dicated in the title, the combination of dSTORM and computa- LANA molecules bound to different numbers of TR sequences tional modeling resulted in a beautiful model that integrates either in the context of viral infection or in cells transfected with dSTORM data with many previous in vitro observations, and TR-containing plasmids and a LANA expression construct. Using therefore lets us “see” the underlying molecular biology of the this platform in combination with elegant genetic tools, they make LANA tether. several key observations that are based on imaging of photons emitted from specific antibodies that stain full-length LANA. First, What’s Next? they show that each tether has specific dimensions in two different This working model provides a significant step toward addressing cell types infected with the same virus strain. Imaging cells additional open questions. For example, how can these studies transfected with different copy numbers of TRs (2, 8, or 21) extend to the other half of the LANA tether to host chromatin? showed linear scaling with LANA binding, suggesting that all TRs Additionally, a number of proteins, including BRD4, have been are occupied by LANA. By integrating previously published data proposed to be part of the tether, and recent imaging data on on the dimensions of active versus transcriptionally suppressed chromatin, they determine that those TR regions between occu- episomes have suggested diversity or clustering during cell divi- pied LANA binding sites that are nucleosome-associated show sions between EBV and KSHV episomes (14, 16, 19, 20). These the characteristics of active chromatin. This latter finding is in issues could be addressed by applying dSTORM to EBV-infected agreement with studies demonstrating active chromatin region at cells. Furthermore, dSTORM can be extended to multiple color TRs, but in contrast to studies that identified repressive chromatin channels, thereby allowing the simultaneous staining of LANA remodelers at TRs (11, 18). To construct an overall model of the plus BRD4, and by using modalities to stain viral DNA, for ex- LANA tether in infected cells, many observations based on the ample, with custom-designed zinc fingers. Additional structural two-TR tether structure are incorporated and integrated with insight may come from applying cryoelectron microscopy, many of the above-described molecular details, such as the whose resolution is constantly improving, and which recently has bending of DNA by LANA, and the proposed coil-coil domain of resolved molecular interactions within viral capsids and even the central domain, which will have an impact on the overall tether ribonucleotide protein complexes of large RNA virus polymer- architecture. To structurally interrogate this domain, a LANA- ases (21, 22). We are confident that we will have the opportunity specific antibody recognizing 22 potential epitopes within the to see the next generation of the LANA tether in action in the central region was used for imaging. However, if the central LANA near future. 1 Grant MJ, Loftus MS, Stoja AP, Kedes DH, Smith MM (2018) Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin tether. Proc Natl Acad Sci USA 115:4992–4997. 2 Chang Y, et al. (1994) Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 266:1865–1869. 3 Kedes DH, et al. (1996) The seroepidemiology of human herpesvirus 8 (Kaposi’s sarcoma-associated herpesvirus): Distribution of infection in KS risk groups and evidence for sexual transmission. Nat Med 2:918–924.
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