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Superresolution Microscopy Reveals Structural Mechanisms Driving the Nanoarchitecture of a Viral Chromatin Tether Margaret J

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Superresolution Microscopy Reveals Structural Mechanisms Driving the Nanoarchitecture of a Viral Chromatin Tether Margaret J Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin 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 chromosomes 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- plasmids 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 j LANA j terminal repeat j dSTORM j 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 genes (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 j PNAS j May 8, 2018 j vol. 115 j no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1721638115 Downloaded by guest on September 26, 2021 A per mAb (Supporting Information), up to six mAbs bound per LANA dimer (see The 2TR Images Support the Prediction of a SEE COMMENTARY LANA Coiled-Coil Domain), and up to three LANA dimers per TR. Since all of the episomes from the same virus have the same number of TRs (17, 35), and each TR is 801 bp, we asked whether the polymer architectures revealed by dSTORM would be sim- ilarly consistent across multiple tethers. We examined 65 indi- vidual LANA tethers from BCBL-1 cells (a KSHV-positive PEL line) and 34 from SLKp/Caki-1p cells, a human epithelial line BCD exogenously infected with KSHV derived from BCBL-1 (36, 37). We found that, while each tether presented a unique shape, their overall dimensions were similar in the two cell lines (Fig. 2 A and B). To quantify this similarity, we aligned clusters along their three principal axes and calculated their dimensions and radii of gyration, Rg (see Supporting Information), shown in Fig. 2 C and D. The median Rg for tethers in BCBL1 cells was 341 nm, 95% CI [309, 353] and in SLKp cells it was 368 nm, 95% CI [323, 405]. There was no statistical difference between the Rg of BCBL-1 and SLKp tethers (P = 0:26, Mood’s Median Test). The similar- EGIFHJity of these parameters in a single viral strain in two different cell types shows that the polymer folding of KSHV LANA tethers is likely to be intrinsic to the viral episome itself, rather than being a result of the cellular milieu. LANA Occupancy Scales with the Number of TRs. We postulated Fig. 1. dSTORM offers improved image resolution of LANA tethers. (A) Schematic depiction of the anatomy of a KSHV LANA tether. LANA dimers that, if the tethers visualized by dSTORM reflect the underlying (shown in yellow) bind via their N termini to histones H2A/B (gray). The C termini bind with sequence specificity to three LBSs located on each TR. (B) An anti-LANA stained BCBL-1 cell imaged by differential interference con- trast microscopy. (C) The boxed region in B viewed by conventional epifluo- A rescence microscopy. (D) The same field of view as in C imaged by dSTORM microscopy. (Scale bars for B–D, 2 µ m.) (E, G, and I) The three LANA tethers boxed in C are enlarged for better visualization. (F, H, and J) The three LANA tethers boxed in D are enlarged for comparison with their paired standard epifluorescence images (E, G, and I). (Scale bars for E–J, 250 nm.) and presence of heterochromatin protein 1 (HP1) at TRs sup- ports heterochromatin conformation, whereas epigenetic, repli- B cation, and transcription studies suggest active chromatin (14, 29–33).
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