Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral tether Margaret J. Granta, Matthew S. Loftusa, Aiola P. Stojaa, Dean H. Kedesa,b,1, and M. Mitchell Smitha,1

aDepartment of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA 22908; and bDepartment of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908

Edited by Donald E. Ganem, Novartis Institutes for Biomedical Research, Inc., Emeryville, CA, and approved March 2, 2018 (received for review December 13, 2017) By tethering their circular genomes (episomes) to host chromatin, qPCR to demonstrate a direct proportionality between the num- DNA tumor viruses ensure retention and segregation of their ber of LANA punctae and the amount of viral DNA, leading to genetic material during cell divisions. Despite functional genetic the conclusion that each nuclear dot represented a single viral and crystallographic studies, there is little information address- episome. Previous studies examining these punctae showed asso- ing the 3D structure of these tethers in cells, issues critical for ciation of LANA with mitotic at the resolution understanding persistent infection by these viruses. Here, we of epifluorescence microscopy (12, 27). Kelley-Clarke et al. (28) have applied direct stochastic optical reconstruction microscopy later suggested preferential localization of these LANA punc- (dSTORM) to establish the nanoarchitecture of tethers within cells tae to centromeric and telomeric regions on metaphase chromo- latently infected with the oncogenic human pathogen, Kaposi’s some. Such studies have provided a solid foundation for further sarcoma-associated herpesvirus (KSHV). Each KSHV tether com- inquiry into the nature of this tethering mechanism. prises a series of homodimers of the latency-associated nuclear Many fundamental features of full-length tethers in cells have antigen (LANA) that bind with their C termini to the tandem remained elusive due to the resolution constraints of epifluo- array of episomal terminal repeats (TRs) and with their N termini rescence microscopy. While X-ray crystallography has resolved to host chromatin. Superresolution imaging revealed that indi- the structures of an N-terminal 23 amino acid LANA peptide vidual KSHV tethers possess similar overall dimensions and, in bound to nucleosomes, and a C-terminal 139 amino acid peptide aggregate, fold to occupy the volume of a prolate ellipsoid. Using in complex with LBS1 (7, 15), questions remain as to the architec- with increasing numbers of TRs, we found that tethers tural features of a full-length tether. It is unknown how TR chro- display polymer power law scaling behavior with a scaling expo- matin folds and whether this is consistent among tethers, both nent characteristic of active chromatin. For plasmids containing a within and across cell lines. The close packing of nucleosomes two-TR tether, we determined the size, separation, and relative orientation of two distinct clusters of bound LANA, each corre- Significance sponding to a single TR. From these data, we have generated a 3D model of the episomal half of the tether that integrates and Kaposi’s sarcoma-associated herpesvirus propagates by attach- extends previously established findings from epifluorescent, crys- ing to host chromatin. This tether is essential for viral mainte- tallographic, and epigenetic approaches. Our findings also vali- nance, and its disruption represents a potential treatment for date the use of dSTORM in establishing novel structural insights persistent infection. However, fundamental questions remain, into the physical basis of molecular connections linking host and including how the underlying viral chromatin is folded, how pathogen genomes. the tether protein is organized, and how it is presented for host attachment. Using superresolution fluorescence microscopy, KSHV | LANA | terminal repeat | dSTORM | DNA bending we have visualized single tethers in cells and built a working model of their structure. The folding of the viral chromatin mimics that of active chromatin, driven by nucleosome posi- aposi’s sarcoma-associated herpesvirus (KSHV) is the etio- tioning and DNA bending. Furthermore, tether proteins are Klogical agent of Kaposi’s sarcoma, an inflammatory tumor arranged in ordered clusters projected outward from the viral of the skin and mucous membranes, as well as two B-cell tumors, chromatin axis. These principles are likely to be applicable to primary effusion lymphoma (PEL) and multicentric Castleman’s the tethers of other DNA tumor viruses. disease (1–3). KSHV infection is marked by long periods of latency when the virus expresses only a small number of essen- Author contributions: M.J.G., D.H.K., and M.M.S. designed research; M.J.G., M.S.L., A.P.S., tial (4). Among these is ORF73 that encodes LANA (5, 6), and M.M.S. performed research; M.J.G. and M.M.S. contributed new reagents/analytic a protein of 1,162 amino acids with multiple functions, includ- tools; M.J.G., M.S.L., A.P.S., D.H.K., and M.M.S. analyzed data; M.J.G., D.H.K., and M.M.S. wrote the paper; M.J.G. and D.H.K. codesigned experiments; D.H.K. and M.M.S. con- ing the tethering of viral episomes to host chromatin. The N ter- tributed to the conception of the project; M.M.S. conducted and analyzed the dSTORM mini of LANA dimers bind to histones H2A/B and to chromatin- microscopy; and M.S.L. and A.P.S. contributed to determining the BCBL-1 TR copy number associated bromodomain proteins (7–10). The C termini of three and the two-TR nucleotide sequence, respectively. LANA dimers bind to three adjacent 20-bp LANA binding sites The authors declare no conflict of interest. (LBSs 1, 2, and 3) located in each 801-bp terminal repeat (TR) This article is a PNAS Direct Submission. (Fig. 1A) (11–15). There are an estimated 35 to 60 tandem TRs Published under the PNAS license. per viral episome (16, 17), arranged head to tail at a single loca- Data deposition: The DNA sequence of the TR region of p2TR reported in this paper has tion on the circular episome. The resulting tether, comprising been deposited in the GenBank database (accession no. MG963161). this cluster of TRs and bound LANA dimers, is essential for epi- See Commentary on page 4816. somal maintenance within dividing cells and, presumably, per- 1 To whom correspondence may be addressed. Email: [email protected] or kedes@ sistence in patients (12, 18–21). Initial epifluorescence studies virginia.edu. identified LANA as nuclear punctae recognized by serum anti- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. bodies from KSHV-infected patients (22–25). Ten years later, 1073/pnas.1721638115/-/DCSupplemental. Adang et al. (26) used a combination of flow cytometry and Published online April 2, 2018.

4992–4997 | PNAS | May 8, 2018 | vol. 115 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1721638115 Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 lnswti asinlclzto ouefo ahconju- molecules each dye three from approximately at volume present molecule localization dye gated Gaussian a within fluorophore-labeled blinks from 1 emissions (Fig. individual mAbs anti-LANA of cluster dot a LANA into epifluorescent reconstruction different each optical two resolved stochastic (dSTORM) in direct microscopy isolate that found viral examined We same episome, types. we the cell viral alternatives, of of the these morphology tether state among to the distinguish intrinsic the To features of by both. by primarily packaging or chromatin, determined the host be DNA, the could episome chromatin and TR nucleosomes host Consis- Types. both and Cell Uniform Different Are Across Tethers tent LANA of Dimensions Nanoscale The Results architectural generating of tethers. 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MICROBIOLOGY SEE COMMENTARY TR architecture, then their structural parameters should change To address the conformational state directly, we took advan- systematically with different numbers of TR elements. To chal- tage of the fact that the radius of gyration, Rg , of a chromatin c lenge that hypothesis, we examined synthetic tethers with differ- polymer follows the power law relationship, Rg ∝ L , where L ent numbers of TRs. We cotransfected BJAB cells, a KSHV- is the DNA length in base pairs and c is the scaling expo- negative human B-cell line, with two plasmids, one encoding nent. This scaling parameter is a sensitive measure of canon- LANA and the other containing two, five, seven, or eight tan- ical active, inactive, and repressed states of mammalian chro- dem TRs arranged head to tail as in the native episome (Fig. S1 matin (40). By plotting Rg against L, we determined the power A and B). In addition, we infected BJAB cells with BAC16 virus law scaling exponent for the LANA-bound TR chromatin to be that we determined contains approximately 21 TRs (Fig. S1 C c = 0.36 ± 0.07 (Fig. 3E). This exponent approximated the value and D). Transfection with a LANA-encoding plasmid and a plas- of 0.37 ± 0.02 found by Boettiger et al. (40) for active chro- mid lacking any functional TR sequence (p0TR) resulted only matin and is distinct from values for inactive (c = 0.30 ± 0.02) in diffuse nuclear staining by epifluorescence (Fig. S2A), consis- or repressed (c = 0.22 ± 0.02) chromatin. We obtained similar tent with earlier work (12, 38). In contrast, individual tethers in results for TR plasmid scaling in COS-7 cells (Fig. S3A). This cells harboring plasmids with 2, 8, or 21 TRs showed a progres- finding clarifies the long-standing paradox surrounding the chro- sive increase in size (Fig. 3 A–C). Accompanying this increase matin folding state of the TR region. was a corresponding linear rise in the number of dSTORM flu- orophore emissions and, therefore, anti-LANA Ab binding (Fig. Adjacent TRs Are Arranged with a Specific Asymmetric Architec- 3D). Our observation that an increase in TR number results in a ture. Closer analysis of the dSTORM emissions from individual proportional increase in LANA binding argues against a plateau p2TR tethers revealed that they frequently gave rise to two dis- for TR occupancy per plasmid or episome, at least over the range tinct, resolvable clusters (Fig. 3A), and alignment of the com- of 1 to 21 TRs. plete set of 28 p2TR data (see Supporting Information) rein- forced this separation (Fig. 4A). Individual clusters had an Rg Tether Polymer Conformation Has the Characteristics of Active Chro- of 90 nm, 95% CI [82, 96], which is similar to the Rg of 86 nm matin. At present, there is conflicting evidence regarding the predicted by extrapolation of the power law function to a sin- conformation of TR chromatin, which exhibits features of both gle TR (Fig. 3E). Thus, we interpreted the two clusters as the repressed and active conformations. On one hand, TR nucle- signals emanating from two adjacent TRs. We obtained simi- osomes are tightly packed in a typically “closed” conformation lar results for p2TR dSTORM images acquired in COS-7 cells (31). Furthermore, LANA recruits the histone H3 methyltrans- (Fig. S3B). The p2TR tethers, individually or as an aligned com- ferase SUV39H1 and establishes HP1 binding to the TR chro- posite, showed striking asymmetry. They comprised flat pairs of matin (30). In contrast, in support of an active conformation, TR prominently elongated ellipsoids whose centroids were spaced chromatin is marked by histone hyperacetylation (29, 31, 32) asso- apart by 174 nm, 95% CI [152, 185] (Fig. 4B). Moreover, the ciated with bromodomain protein BRD4 (9, 39), and the TRs long axes of the two clusters intersected to form a 46° angle (Fig. have both promoter and origin of replication functions (14, 33). 4B), demonstrating a specific differential placement of the indi- vidual TRs within the p2TR structure. These parameters pro- vided strong constraints on the underlying folding of the p2TR AB Cchromatin tether.

The 2TR Images Support the Prediction of a LANA Coiled-Coil Domain. To begin to interpret the 2TR tether images at the molecular level, we evaluated the structure of LANA dimers N-terminal to the LBS binding domains, for which previous X-ray crystal struc- tures are known (10, 15, 39). Examination of the primary pro- tein sequence of LANA with Paircoil2 (41) revealed a segment strongly predicted to form a coiled coil (Fig. S4A; P < 0.025). To D E test for the presence of the coiled coil, we measured the number of single-molecule emissions detected by dSTORM for LANA tethers stained with LN53-A647, which recognizes the tetrapep- tide epitope EQEQ (42). There are 22 such epitopes within the LANA protein sequence, and this number of mAbs would yield over 18,000 dSTORM emissions per tether (see Supporting Infor- mation). However, 19 of the most C-terminal of these epitopes would be embedded within the putative coiled-coil region of LANA, occluding mAb recognition. The remaining three epi- topes just N-terminal to the coiled coil would yield on the order of only 2,500 emissions per tether. Indeed, we routinely recov- ered fewer than 2,000 emissions per tether, consistent with only two to three functional epitopes per LANA protein, providing Fig. 3. LANA tethers show polymer-like scaling as a function of the number strong empiric data for the predicted coiled coil. Furthermore, of TRs. (A–C) The 3D projections of representative LANA tethers from a cell the positioning of these available epitopes at the N-terminal transfected with (A) p2TR, (B) p8TR, or (C) BAC16 with 21 TRs are shown end of a rigid coiled-coil domain is supported by the separation at identical scales as described in Fig. 2. (Scale bar for A–C, 250 nm.) (D) between the two emissions clusters in the 2TR images. We gen- The linear relationship between the median number of emissions and the erated a molecular model of LANA dimers incorporating these number of TRs per tether is shown for p2TR (n = 28), p5TR (n = 39), p7TR features (Fig. S4). (n = 13), p8TR (n = 17), and BAC16 (n = 32) (R2 = 0.99). (E) The log of median Rg data for the tethers in D and BCBL-1 KSHV episomes (43 TRs, n = 65) are shown as a function of the log of the tether DNA lengths (top axis) or the LANA-Imposed DNA Bending and Nucleosome Translational Position- log of the TR number (bottom axis). The regression (solid red line) through ing Are Predicted to Direct TR Chromatin Folding. Although the tethers with known numbers of TRs (blue circles) was used to calculate the DNA of each TR is known to be bent by LANA dimer bind- scaling exponent c = 0.36 ± 0.07 (R2 = 0.94). Error bars represent 95% CIs. ing (15, 43, 44), and to be occupied by four nucleosomes (31),

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MICROBIOLOGY SEE COMMENTARY Discussion reflect such assemblies. Further, our current model has some Until now, LANA tethers have appeared as poorly defined limitations. For example, it is unlikely that the fixed packing of nuclear punctae via epifluorescence microscopy. The localiza- tetranucleosomes we adapted from the crystallographic struc- tion data from this study offer insights into the architecture ture reflects the full range of conformations adapted by TR of the tethers linking KSHV episomes to human chromatin. nucleosomes in vivo. Unlike the KSHV tether histones, the his- First, we found that the folding of tether chromatin was intrin- tones present in the tetranucleosome crystal structure lacked any sic to the viral episome and independent of the cellular envi- posttranslational modifications. The presence of such modifica- ronment. Consistent shapes and emission counts were mea- tions might alter the tetrasome structure, potentially loosening sured for comparable TR episomes within the B-cell–derived or tightening the histone packing, and thereby impact our model lines BCBL-1 and BJAB, the nonlymphocytic epithelial cell line and its fit to our data. SLKp, and even the monkey kidney fibroblast cell line COS- Recently, Chiu et al. (46) described the clustering of a sub- 7. Second, regardless of the number, TRs were proportionally set of KSHV episomes. In our study, we focused on individ- occupied by LANA dimers. This argues for a relatively uniform ual TR structures at single-molecule localization precision (10 chromatin environment across the terminal repeats wherein nm to 30 nm) and excluded any ambiguous structures from our LBS sites are equally accessible to LANA binding in all TRs. analyses. We occasionally noted single bright LANA dots by Third, we provided physical proof that TR chromatin folds with epifluorescence, but, most often, dSTORM resolved these into parameters characteristic of active chromatin, likely driven by distinct LANA tethers. In these cases, the N termini of the nucleosome-free regions in the TRs, helping to clarify conflicting LANA tethers might have also clustered episomes as Chiu et al. evidence pertaining to the chromatin state (14, 29–33). Fourth, suggest. the ability of dSTORM to resolve single TR units within 2TR Our current working model captures major features of the clusters demonstrates that the episomal half of LANA dimers KSHV tether, detailing the likely stoichiometry and relative posi- must be well ordered. If LANA dimers radiated randomly from tions of its major molecular components emanating from the TR the LBSs or were highly unstructured in their episomal half, region of the viral genome within the cellular milieu. The model we would be unable to separate discrete clusters in the anal- also provides a platform for future experimentation that could yses of plasmids containing two TRs. Finally, our data argue include determining the nanoarchitecture of tethers with greater that all three LBS sites in individual TRs are likely to be fully numbers of TRs. This approach has broad applicability to a wide occupied by LANA dimers. The discrete dimensions and posi- variety of persistent viral pathogens, thus potentially contributing tional asymmetry of the dye emission clusters in the 2TR data to our ability to target them therapeutically. are not well accounted for if only two LBS sites are occupied, because the axis through the epitopes of each TR is shortened Materials and Methods and there is less bending of the LBS DNA. Furthermore, mod- Cell Lines. BCBL-1 cells have been in the laboratory of D.H.K., who was a els with three LANA dimers per TR consistently performed coauthor on the study first describing the line (47). BJAB cells (48) were a gift better than models with only two LANA dimers per TR in from Don Ganem, Novartis Institutes for Biomedical Research, Emeryville, CA. SLKp/Caki-1p cells (37) were a gift from Adam Grundhoff, Heinrich Pette explaining dSTORM data for 26 independent 2TR datasets. The Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany, linear relationship between LANA dye emissions and TR num- and Don Ganem (36). iSLK-BAC16 cells (49) were a gift from Rolf Renne, Uni- ber suggests that this is true for complete KSHV episomes versity of Florida, Gainesville, FL. COS-7 cells were a gift from Jim Casanova, as well. University of Virginia, Charlottesville, VA. We identified several key features of tether structure by mod- eling the 2TR data. First, the modeling revealed that the internal Plasmids. The pcDNA3-LANA plasmid (50) was a gift from Rolf Renne, Uni- repeats of LANA likely comprise a coiled-coil domain for the versity of Florida. The p8TR plasmid (12) was produced by the laboratory of LANA dimer. This domain was first predicted by in silico anal- Kenneth Kaye, Harvard Medical School, Boston, and was a gift from Paul ysis, and then supported by quantitative analysis of LN53 mAb Lieberman, The Wistar Institute, Philadephia. The construction of plasmids binding, and masks all but the two to three N-terminal most mAb containing zero, two, five, and seven terminal repeats is described in SI Materials and Methods. binding sites. Second, the modeling highlighted an impact of translational positioning of the nucleosomes for each TR. Mov- Antibodies. The anti-LANA antibody LN53 (rat, monoclonal) (51) was con- ing each tetranucleosome along the nine possible phases of the jugated using an Alexa Fluor 647 succinimidyl ester kit (Thermo Fisher DNA helix alters the trajectory of the output TR DNA. This, in Scientific). turn, greatly impacts the relative positions of the LBSs on adja- cent TRs and, hence, their associated anti-LANA fluorophore dSTORM Microscopy. The instrumentation, sample preparation, image emissions. The architecture driven by nucleosome positioning acquisition, and data analysis for dSTORM imaging are described in SI Mate- and LBS DNA bending positions the LANA N-terminal histone rials and Methods. binding domains in a way that facilitates exploration of the sur- ACKNOWLEDGMENTS. We thank Barbara Knowles, Samuel Hess, Travis rounding environment, promoting their primary function of teth- Gould, and Mudalige Gunewardene for an introduction to superresolu- ering to host chromatin. tion microscopy and advice on instrumentation, and Stefan Bekiranov for While the 2TR model describes most of the tethers well, a discussions. This work was supported, in part, by the University of Vir- few datasets are exceptions mainly due to their having relatively ginia Cancer Center Grant P30CA044579 (to D.H.K. and M.M.S.). D.H.K. R acknowledges support of the National Institute of Dental and Craniofa- larger g values. Others have proposed large multi-LANA dimer cial Research Award R01DE022291. M.M.S. acknowledges support of the complexes based on crystallography of the C-terminal binding National Institute of General Medical Sciences Awards RC1GM091175 and domain (10, 15, 39), and it is possible these 2TR exceptions R01GM116994.

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