Supporting Information

Zhang et al. 10.1073/pnas.1516812113

A 1 329 931 1162 pSERS-eGFP-LANA GFP N terminal domain internal repeat C terminal domain (LANA-FL) 1 329/931 1162

pSERS-eGFP-LANAΔ329-931 GFP N terminal domain C terminal domain (LANA-NC)

B C

BCBL1 pSERSeGFP pSERSeGFP pSERSeGFP LANA-NC LANA-FL

GFP GFP-LANA-NC GFP-LANA-FL BCBL1

GFP

Sorting of GFP+ cells

DAPI preparation of cell lysates

IP α-GFP Ab or GFP trap

SDS-PAGE Coomassie staining

Mass spectrometry

Data analysis

Fig. S1. Identification of cGAS as an interaction partner of LANA in BCBL-1 cells. (A) Schematic representation of LANA constructs used for coimmunopre- cipitation and MS analysis of BCBL-1 cells stably expressing full-length LANA (LANA-FL), LANA Δ329–931 (LANA-NC), or GFP. (B) BCBL-1 cells transduced with GFP-LANA-FL or GFP-LANA-NC were fixed and GFP expression analyzed by microscopy. Nuclei were stained with DAPI. (C) Workflow of the coimmunopre- cipitation and MS analysis.

Zhang et al. www.pnas.org/cgi/content/short/1516812113 1of5 HuAR2T.rKSHV

controlRTA+SB RTA SB M.W. (kD) - 250

α-LANA -150

α-p-TBK1 - 75

α-p-IRF3 - 50

α-K-bZIP - 37

α-actin

Fig. S2. Lytic replication induction of KSHV activates the cGAS-dependent signaling pathway. HuAR2T.rKSHV.219 was treated with a baculovirus expressing the KSHV RTA protein and 1.5 mM Na-butyrate, the baculovirus expressing the KSHV RTA alone, or Na-butyrate alone. The cell lysates were analyzed by immunoblot using antibodies to p-IRF3, p-TBK1, and the KSHV K-bZIP protein.

Zhang et al. www.pnas.org/cgi/content/short/1516812113 2of5 A

cGAS GFP RFP GFP RFP GFP RFP siRNA

-

+

No pre-treatment /no RTA+SB No pre-treatment /RTA+SB 2‘3‘cGAMP /RTA+SB

-

+

3‘3‘cGAMP /RTA+SB di-UMP /RTA+SB IFN-β /RTA+SB

B STING siRNA - + - + - + - + - + - + Scrambled siRNA + - + - + - + - + - + - 2‘3‘cGAMP - - - - + + ------3‘3‘cGAMP ------+ + - - - - di-UMP ------+ + - - IFN-β ------+ + RTA+SB - - + + + + + + + + + + -250

-150 α-LANA

-100

α-K-bZIP -37

- α-STING 37

α-actin

Fig. S3. Knockdown of cGAS or STING increases KSHV reactivation. (A) Representative images of HuAR2T.rKSHV.219 cells pretreated with siRNA to cGAS or control siRNA before the activation of the lytic replication cycle with recombinant RTA and Na-butyrate and treated with 2′3′cGAMP, 3′3′cGAMP, diUMP, or IFN-β, as described in Fig. 2D. GFP channel, cells harboring latently infected KSHV; RFP channel, KSHV reactivated cells. (B) HuAR2T.rKSHV.219 cells were transfected with STING or scrambled siRNAs. Twenty-four hours later, cells were treated with 2′3′-cGAMP, 3′3′-cGAMP, di-UMP, or IFN-β, and about 12 h later, the lytic cycle was induced with baculovirus exprssing RTA and Na-butyrate. Forty-eight hours later, cells were lysed and analyzed by immunoblotting.

Zhang et al. www.pnas.org/cgi/content/short/1516812113 3of5 ISD -4h8h GFP △161 GFP △161 GFP △161

α-LANA -150 -100

α-p-TBK1 -75

α-p-IRF3 -50 α-GFP

α-actin

2‘3‘cGAMP n.d. n.d.1.1 nM n.d.

Fig. S4. The cytoplasmic isoform of LANA inhibits 2′3′cGAMP production. HeLa MZ cells transduced with lentiviruses expressing LANAΔ161 or GFP were stim- ulated by transfection with ISD. Four and 8 h after stimulation, 2′3′-cGAMP was quantified by RP-HPLC/MS. Cells were lysed and the activation of the cGAS-STING pathway was verified by immunoblotting with antibodies to phosphorylated TBK1 and IRF3. Low levels of 2′3′-cGAMP could only be detected after 8 h of ISD stimulation in cells expressing GFP, but not in cells expressing LANAΔ161. Amounts of 2′3′-cGAMP (nM) measured are shown below the actin immunoblot.

Zhang et al. www.pnas.org/cgi/content/short/1516812113 4of5 Table S1. Cellular proteins interacting with GFP-LANA-FL or GFP-LANA-NC (LANA Δ329–931) were identified by MS LANA-FL LANA-NC No. LANA-interacting proteins proteins Times Score No. of peptide Times Score No. of peptide

1 Lysine-specific demethylase 3A GN = KDM3A 4 705.64 15 4 391.41 8 2 Death domain-associated protein 6 GN = DAXX 4 546.34 9 3 Helicase-like factor GN = HLTF 4 329.48 7 4 557.17 12 4 Sister chromatid cohesion protein PDS5 homolog A 4 232.01 5 3 238 5 GN = PDS5A 5 FACT complex subunit SPT16 GN = SUPT16H 4 122.89 3 4 408.91 9 6 Core histone macro-H2A.1 GN = H2AFY 4 94.27 2 4 182.28 3 7 FACT complex subunit SSRP1 GN = SSRP1 4 89.26 2 4 166.94 3 8 DNA repair protein complementing XP-C cells 4 84.4 2 2 217.01 5 GN = XPC 9 Telomere-associated protein RIF1 GN = RIF1 3 412.85 7 4 979.53 19 10 Mediator of DNA damage checkpoint protein 1 3 275.37 7 4 958.41 19 GN = MDC1 11 Isoform 2 of Nuclear mitotic apparatus protein 1 3 239.12 5 4 1,489.34 29 GN = NUMA1 12 Tyrosine-protein kinase BAZ1B GN = BAZ1B 3 215.21 4 4 463.57 9 13 SWI/SNF complex subunit SMARCC1 GN = SMARCC1 3 185.34 4 3 309.42 8 14 AT-rich interactive domain-containing protein 1A 3 133.87 3 3 309.49 8 GN = ARID1A 15 Centromere protein F GN = CENPF 3 130.02 3 4 667.49 14 16 DNA replication licensing factor MCM6 GN = MCM6 3 112.59 2 2 123.32 3 17 DNA repair protein RAD50 GN = RAD50 3 108.28 3 4 163.56 4 18 DNA 2-α GN = TOP2A 3 93.39 2 4 460.74 10 19 cGMP-AMP synthase GN = MB21D1 3 358.11 8 4 391.26 9 20 Metastasis-associated protein MTA1 GN = MTA1 3 201.35 4 2 143.84 3 21 SWI/SNF-related matrix-associated actin-dependent 3 189.98 4 2 256.92 6 regulator of chromatin subfamily E member 1 GN = SMARCE1 22 Targeting protein for Xklp2 GN = TPX2 3 184.51 5 3 203.16 4 23 PC4 and SFRS1-interacting protein GN = PSIP1 3 132.27 3 3 148.42 3 24 Transcriptional repressor p66-alpha GN = GATAD2A 3 130.53 3 1 89.37 2 25 Putative oxidoreductase GLYR1 GN = GLYR1 3 120.92 2 3 119.51 2 26 Torsin-1A-interacting protein 1 GN = TOR1AIP1 3 71.4 2 1 173.45 6 27 Nucleosome-remodeling factor subunit BPTF 2 535.27 11 3 550.94 12 GN = BPTF 28 Ras GTPase-activating protein-binding protein 2 2 350.51 8 3 401.69 8 GN = G3BP2 29 DNA topoisomerase 2-beta OS = Homo sapiens 2 370.91 8 3 242.56 5 GN = TOP2B 30 Remodeling and spacing factor 1 GN = RSF1 1 272.61 6 3 212.41 4 31 Partitioning defective 3 homolog GN = PARD3 1 450.03 10 3 168.1 4 32 Actin-like protein 6A GN = ACTL6A 2 202.72 4 3 112.8 2 33 Heterogeneous nuclear ribonucleoprotein D0 3 111.93 2 (Fragment) GN = HNRNPD 34 Centrosomal protein of 170 kDa GN = CEP170 2 168.23 3 3 104.13 2 35 Src substrate cortactin OS = Homo sapiens 2 230.8 4 3 495.72 11 GN = CTTN 36 26S proteasome non-ATPase regulatory subunit 11 1 102.16 2 3 111.26 2 GN = PSMD11 37 Ewing’s tumor-associated antigen 1 GN = ETAA1 2 151.61 3 3 78.36 2 38 SWI/SNF-related matrix-associated actin-dependent 2 290.1 7 3 201.21 5 regulator of chromatin subfamily A-like protein 1 GN = SMARCAL1

List contains proteins with high frequency (≥3 times in four independently repeated LANA-FL or LANA-NC precipitations).

Zhang et al. www.pnas.org/cgi/content/short/1516812113 5of5