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. 2019 Jan 25;15(1):e1007489.
doi: 10.1371/journal.ppat.1007489. eCollection 2019 Jan.

LANA oligomeric architecture is essential for KSHV nuclear body formation and viral genome maintenance during latency

Alessandra De Leo  1 , Zhong Deng  1 , Olga Vladimirova  1 , Horng-Shen Chen  1 , Jayaraju Dheekollu  1 , Abram Calderon  1 , Kenneth A Myers  2 , James Hayden  1 , Frederick Keeney  1 , Benedikt B Kaufer  3 , Yan Yuan  4 , Erle Robertson  5 , Paul M Lieberman  1
Affiliations

LANA oligomeric architecture is essential for KSHV nuclear body formation and viral genome maintenance during latency

Alessandra De Leo et al. PLoS Pathog. .

Abstract

The molecular basis for the formation of functional, higher-ordered macro-molecular domains is not completely known. The Kaposi's Sarcoma-Associated Herpesvirus (KSHV) genome forms a super-molecular domain structure during latent infection that is strictly dependent on the DNA binding of the viral nuclear antigen LANA to the viral terminal repeats (TR). LANA is known to form oligomeric structures that have been implicated in viral episome maintenance. In this study, we show that the LANA oligomerization interface is required for the formation of higher-order nuclear bodies that partially colocalize with DAXX, EZH2, H3K27me3, and ORC2 but not with PML. These nuclear bodies assemble at the periphery of condensed cellular chromosomes during mitotic cell division. We demonstrate that the LANA oligomerization interface contributes to the cooperative DNA binding at the viral TR and the recruitment of ORC to the viral episome. Oligomerization mutants failed to auto-regulate LANA/ORF73 transcription, and this correlated with the loss of a chromosome conformational DNA-loop between the TR and LANA promoter. Viral genomes with LANA oligomerization mutants were subject to genome rearrangements including the loss of subgenomic DNA. Our data suggests that LANA oligomerization drives stable binding to the TR and formation of an epigenetically stable chromatin architecture resulting in higher-order LANA nuclear bodies important for viral genome integrity and long-term episome persistence.

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Conflict of interest statement

Paul Lieberman is a founder of Vironika, LLC.

Figures

Fig 1
Fig 1. LANA forms nuclear bodies that colocalize with DAXX and EZH2, but not PML.
(A-B) BCBL1 cells were imaged by indirect immunofluorescence with antibodies to LANA (red) and (A) DAXX (green), or (B) EZH2 (green) or merge with Dapi (blue). Scale bar = 10 μm. (C) Schematic of the RFP-LANA fusion integrated into the endogenous LANA of the KSHV BAC16 genome. (D-G) Stable iSLK cells with BAC16 expressing RFP-LANA (red) were imaged by indirect immunofluorescence with antibodies to (D) DAXX (green), (E) EZH2 (green), (F) H3K27me3 (green), or (G) PML (green). Cells were counter-stained with Dapi (blue) as shown in merge. Scale bar = 10 μm.
Fig 2
Fig 2. Dynamic organization of LANA nuclear bodies through mitotic cell division.
(A) Live cell imaging of RFP-LANA in iSLK cell line combined with Phase contrast during a 30 min interval through mitotic cell division. Scale bar = 10 μm. (B) Zoom and 3D reconstruction of confocal images of LANA bodies formed during live cell imaging in iSLK cells. Scale bar = 5 μm. (C) Confocal images of RFP-LANA from 3 orthogonal viewpoints at intervals across cell division. Relative time (min:sec) is indicated below each frame. Scale bar = 20 μm. (D) Confocal fixed images of mitotic RFP-LANA (red), α-tubulin (green) and Dapi (blue) in iSLK cells. Scale bar = 5 μm. (E) Deconvolved confocal images as shown in panel D with Dapi (i), α-tubulin (ii), RFP-LANA (iii), and merge (iv).
Fig 3
Fig 3. LANA oligomerization interface contributes to cooperative DNA binding at TR.
Rendering of LANA crystal structure decamer (A) and oligomeric interface (B). (C) Luciferase reporter with 1x (left panel) or 3x (right panel) LBS from TR was assayed in cells expressing FLAG-VP16-LANA DBD- WT or MT. ** p value < 0.01 using two-tailed student t-test. (D) The expression levels of FLAG-tagged VP16-LANA DBD- WT or MT as shown in (C) were assayed by Western blot with FLAG antibody, and β-tubulin control was shown below.
Fig 4
Fig 4. LANA oligomerization interface is required for LANA body formation and colocalization with DAXX and EZH2.
(A) Schematic of KSHV Bac-RFP-LANA WT and Bac-RFP-LANA MT (F1037A/F1041A). (B) Quantification of RFP-LANA nuclear body formation in iSLK stable clones for WT and MT LANA. P-value was determined by two-tailed student t-test. (C) Indirect immunofluorescence analysis of the association of RFP-LANA (red) with DAXX (green) in RFP-LANA WT1 and WT2, or MT1 or MT2 stable iSLK cell lines. Cells were counter-stained with Dapi (blue) as shown in merge. Scale bar = 10 μm. (D) The same as in panel C, except imaging with EZH2 (green). Scale bar = 10 μm.
Fig 5
Fig 5. LANA oligomerization is important for LANA-binding and ORC recruitment to KSHV TR.
(A) Schematic of ChIP-qPCR primer positions with relation to KSHV genes and loci. Red triangles indicate position of CTCF binding. (B) ChIP-qPCR analysis of LANA-RFP WT1, WT2, MT1, or MT2 stable iSLK cell lines using antibodies for LANA, ORC2, H3K27me3, or IgG control, as indicated. * p value < 0.05, ** p value < 0.01 using two-tailed student t-test. (C) RFP-LANA WT1 and WT2, or MT1 or MT2 stable iSLK cell lines were imaged with ORC2 (green) and Merge images were shown with Dapi (blue) staining. Scale bar = 10 μm.
Fig 6
Fig 6. LANA oligomerization controls ORF73 auto-regulation and TR chromosome conformation.
(A) RFP-LANA WT1, WT2, MT1, or MT2 stable iSLK cell lines were assayed by Western blot for LANA (top) and Actin loading control (lower). (B) RFP-LANA WT1, WT2, MT1, or MT2 stable iSLK cell lines were assayed by RT-PCR for expression of ORF71, ORF72, or LANA. mRNA was quantified relative to GAPDH. * p value <0.05, ** p value <0.01 using student t-test. (C) RFP-LANA WT1, WT2, MT1, or MT2 stable iSLK cell lines were assayed by 3C using anchor primer near TR (position 133872) and assayed at positions indicated on x-axis. 3C-qPCR relative to actin control is indicated. ** p value <0.01 using student t-test.
Fig 7
Fig 7. LANA oligomerization is important for viral genome integrity.
(A) RFP-LANA WT1, WT2, MT1, and MT2 stable iSLK cell lines were analyzed by qPCR for copy number variation using primers spanning KSHV genome, as indicated on X-axis. (B) RFP-LANA WT, MT1, and MT2 bacmid DNA was quantified by qPCR as in panel A. (C) Fold loss of KSHV DNA relative to bacmid control for MT1, MT2, and the average of WT1+WT2. Region of genetic instability are highlighted with salient viral genes and features indicated. * p value < .05, ** p value < .01 using student t-test. (D) BCBL1 or KSHV Bac RFP-LANA WT1, WT2, MT1, or MT2 were analyzed by agarose plug PFGE and Southern blot with KSHV bacmid probe (Episomes) or 18S DNA (Genomic DNA). Agarose plugs containing 106 cells were analyzed in duplicate. (E) Total genomic DNA from or KSHV Bac RFP-LANA WT1, WT2, MT1, or MT2 was digested with BamHI and assayed by PFGE and Southern blot with probes to TR (top) or KSHV genomic DNA between coordinates 2kb-18kb on the KSHV genome.
Fig 8
Fig 8. Model of LANA oligomerization induced changes in KSHV TR conformation and function.
(A) Depiction of TR bound to non-interacting, low affinity monomeric LANA or, (B) LANA oligomerization-induced conformational change in TR in the context of nucleosomes. Histone is shown in turquoise. (C) LANA oligomerization-induced conformation change in TR recruits additional factors, such as Daxx and ORC, and enables KSHV genome DNA loop interactions between TR and the LANA transcriptional regulatory region. LANA oligomerization also regulates the transcription and replication control necessary to maintain viral genome stability. Regions of viral genetic instability is indicated from OriLyt to TR. (D) LANA oligomerization is also required for the formation of LANA nuclear bodies (LNB) (red), distinct from PML-NBs (green), that can be transmitted as stable structures throughout mitosis.
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