Cytoplasmic isoforms of Kaposi sarcoma herpesvirus LANA recruit and antagonize the innate immune DNA sensor cGAS

Guigen Zhanga, Baca Chanb, Naira Samarinaa, Bizunesh Aberea, Magdalena Weidner-Glundea, Anna Bucha, Andreas Pichc, Melanie M. Brinkmanna,b, and Thomas F. Schulza,1

aInstitute of Virology, Hannover Medical School, 30625 Hannover, Germany; bHelmholtz Centre for Infection Research, 38124 Braunschweig, Germany; and cInstitute of Toxicology, Hannover Medical School, 30625 Hannover, Germany

Edited by Patrick S. Moore, University of Pittsburgh Cancer Institute, Pittsburgh, PA, and approved December 31, 2015 (received for review August 25, 2015) The latency-associated nuclear antigen (LANA) of Kaposi sarcoma LANA, one of the major proteins expressed in KSHV latently herpesvirus (KSHV) is mainly localized and functions in the nucleus infected cells, represses IFN-β production by competing with IRF3 of latently infected cells, playing a pivotal role in the replica- to bind the IFN-β promoter (15). The processed forms of LANA tion and maintenance of latent viral episomal DNA. In addition, resulting from caspase cleavage blunt apoptosis and caspase N-terminally truncated cytoplasmic isoforms of LANA, resulting 1–mediated inflammasome in KSHV-infected cells exposed to from internal translation initiation, have been reported, but their oxidative stress (24). LANA is also involved in the modulation of function is unknown. Using coimmunoprecipitation and MS, we adaptive immunity by inhibiting antigen presentation of both found the cGMP-AMP synthase (cGAS), an innate immune DNA major histocompatibility complex class I (MHC I) and class II sensor, to be a cellular interaction partner of cytoplasmic LANA (MHC II) (25–28). Meanwhile, host restriction factors inhibit isoforms. By directly binding to cGAS, LANA, and particularly, a KSHV infection by activating immune responses. KSHV infection cytoplasmic isoform, inhibit the cGAS-STING–dependent phos- of human primary naïve B cells induces rapid activation-induced phorylation of TBK1 and IRF3 and thereby antagonize the cGAS- cytidine deaminase (AID) expression, which plays a role in the mediated restriction of KSHV lytic replication. We hypothesize that innate immune defense against KSHV (29). cytoplasmic forms of LANA, whose expression increases during It is well established that LANA localizes to the nucleus of lytic replication, inhibit cGAS to promote the reactivation of the infected cells, where the known functions of LANA involve KSHV from latency. This observation points to a novel function of binding both the viral episome and cellular , and the cytoplasmic isoforms of LANA during lytic replication and ex- recruitment of -associated proteins such as BRD2, tends the function of LANA from its role during latency to the lytic BRD4, and MeCP2 (30–34). In addition, a recent publication replication cycle. reported that lower-molecular-weight LANA isoforms can be generated by the use of noncanonical internal translation initi- KSHV | cytoplasmic LANA | cyclic GMP-AMP synthase ation sites within the N-terminal domain and are localized to the cytoplasm, because they lack a nuclear localization signal (35). aposi sarcoma herpesvirus (KSHV, also known as HHV-8) is The generation of LANA isoforms lacking part of the N-terminal Kthe causative agent of Kaposi sarcoma, multicentric Castle- domain by caspase cleavage has also been recently reported (24). man’s disease (MCD), and primary effusion lymphoma (PEL). However, the functions of these cytoplasmic isoforms of LANA KSHV-encoded latency-associated nuclear antigen (LANA), orig- are still unknown. inally identified in KSHV-infected PEL cell lines and encoded by Here we report the identification of cellular proteins inter- KSHV orf73 (open reading frame 73), is constitutively expressed in acting with KSHV LANA using coimmunoprecipitation and MS. all forms of KSHV-associated malignancies (1–5). LANA is essential for latent KSHV replication and maintenance of latency by tethering Significance the viral episome to cellular chromosomes during cell division (6, 7). As a multifunctional protein, LANA is involved in many cellular In addition to the well-characterized main nuclear latency- processes, such as regulation of cellular and viral , cell – associated nuclear antigen (LANA) protein of Kaposi sarcoma growth, angiogenesis, and immune modulation (8 15). herpesvirus (KSHV), cytoplasmic LANA isoforms are known to Innate immunity is the first line of defense against incoming exist, but their function has thus far been unknown. Here we pathogens. KSHV efficiently inhibits the host innate immune re- show that N-terminally truncated cytoplasmic isoforms of sponse by targeting several pattern recognition receptors (PRRs) LANA play a role in antagonizing the innate response trig- signaling, such as Toll-like receptors (TLRs), RIG-I-like receptors gered, by means of cGMP-AMP synthase (cGAS) and stimulator (RLRs), and the DNA sensor cGMP-AMP synthase (cGAS). of interferon (STING), during the reactivation of KSHV Several KSHV-encoded proteins, such as viral IFN regulatory from latency. By directly interacting with cGAS, cytoplasmic factor 1 (vIRF1), vIRF2, vIRF3, K8 (k-bZIP), LANA, ORF45, LANA variants inhibit the cGAS-STING–dependent induction of ORF64, ORF75, and RTA (replication and transcription activa- interferon and thereby promote the reactivation of KSHV from tor)/ORF50 are known to modulate the innate immune response γ – latency. These findings extend the roles of a -herpesvirus la- (16 21). RTA inhibits the TLR-mediated innate immune re- tent protein into the lytic replication cycle. sponse by down-regulating the expression of TLR2 and TLR4 (19). The KSHV deubiquitinase encoded by ORF64 inhibits the Author contributions: G.Z. and T.F.S. designed research; G.Z., B.C., N.S., and B.A. per- RIG-I–mediated innate immune response by reducing ubiquiti- formed research; M.W.-G., A.B., and A.P. contributed new reagents/analytic tools; G.Z., nation of RIG-I, a crucial step in the activation of RIG-I (20). M.M.B., and T.F.S. analyzed data; G.Z. and T.F.S. wrote the paper. vIRF1 targets STING and ORF52 inhibits cGAS enzymatic ac- The authors declare no conflict of interest. tivity to prevent the cGAS-mediated DNA sensing (21, 22). Re- This article is a PNAS Direct Submission. cently, two oncogenes of DNA tumor viruses, including E7 of 1To whom correspondence should be addressed. Email: [email protected]. human papillomavirus and E1A of adenovirus, were reported to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. block cGAS-STING signaling pathway by binding to STING (23). 1073/pnas.1516812113/-/DCSupplemental.

E1034–E1043 | PNAS | Published online January 25, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1516812113 Downloaded by guest on September 28, 2021 PNAS PLUS Among these is cGAS, an innate DNA sensor, which, on rec- benzonase IP A -+ M ognition of dsDNA or RNA:DNA hybrids in the cytoplasm, IgG α-LANA input M.W. (kD) generates 2′3′ cGMP-AMP (2′3′cGAMP) (36–41). cGAMP then -250

binds to stimulator of IFN genes (STING, also known as -150 TMEM173, MITA, ERIS, or MPYS), which recruits and acti- α-LANA vates TANK-binding kinase 1 (TBK1) and IFN regulatory factor -100 3 (IRF3) to induce the expression of type I IFNs, which in turn α-cGAS - IgG heavy induce expression of IFN-stimulated genes (ISGs). cGAS was α-actin chain reported to inhibit replication of DNA viruses such as Murid herpesvirus 68 (MHV-68), vaccinia virus, and herpes simplex B virus 1 (HSV-1) (42, 43). In this study, we find that the cyto- input IP benzonase plasmic isoforms of KSHV LANA interact with cGAS and an- - +M GFP-LANA - + - + tagonize its function in type I IFN signaling, thereby promoting GFP + - + - cGAS + + + + M.W. (kD) the reactivation of KSHV from latency. -250 Results -150 α-LANA cGAS Is a Cellular Binding Partner of LANA. LANA, a multifunctional -100 -75 protein, is expressed in all KSHV-infected cells. LANA consists of α-cGAS an amino terminal domain, an extended internal repeat region, α-actin and a carboxy terminal domain involved in the binding to viral episomal DNA (4, 5, 25, 44–46). The internal repeat region α-GFP is required for the maintenance of viral episomes (47–49).

To identify novel cellular proteins interacting with the N- and C(i) GST pull-down D LANA C-terminal domains or the internal repeat region of LANA, we GST C N BJAB lysate + ++M.W. input transduced the BCBL-1 PEL cell line with lentiviral vectors -75 262-320 288-320 306-320 α-cGAS α-cGAS FL Δ Δ Δ expressing a fusion protein of GFP with full-length LANA cGAS ++ + + -75 α-actin (LANA-FL) or a LANA mutant lacking the internal repeat α-cGAS Δ – α-GST

region (LANA-NC, LANA 329 931) (Fig. S1A). Both GFP- T7 - -25 α LANA-FL and GFP-LANA-NC proteins localized to the typical α-LANA LANA speckles in KSHV-infected BCBL-1 cells (Fig. S1B). IP: As shown in the experimental workflow (Fig. S1C), BCBL-1 (ii) cells expressing GFP-LANA-FL or GFP-LANA-NC were lysed, GST pull-down α-LANA GST N input and GFP-LANA proteins were immunoprecipitated with either GFP-cGAS ++ 1.67% -100 beads conjugated with an anti-GFP antibody or GFP-trap, a α-cGAS input small GFP-binding protein, coupled to agarose beads. Immu- α-cGAS -75 noprecipitates were analyzed by MS. Cellular partners of GFP- α-GST α-actin LANA-FL and GFP-LANA-NC identified with high frequency -25 (at least three times in four MS runs of GFP-LANA-FL or GFP- LANA-NC after subtracting the hits from the control groups) are Fig. 1. cGAS interacts with the N-terminal domain of LANA. (A) Coimmu- shown in Table S1. Several of the cellular proteins identified noprecipitation of endogenous cGAS and LANA in BCBL-1 cells. LANA was here have previously been reported to interact with LANA. immunoprecipitated from benzonase-treated lysates of BCBL-1 cells with an antibody to the CR2 region of LANA (Fig. 5A) and immunoprecipitates Examples include lysine-specific demethylase 3A (KDM3A), stained on Western blots with antibodies to LANA (Upper, central panel) or death domain-associated protein 6 (Daxx), core histone macro- cGAS (Lower, central panel). The presence of LANA and cGAS in the cellular H2A.1 (H2AFY), FACT complex subunit SSRP1 (SSRP1), lysates is shown on the Left and the degradation of cellular DNA by ben- nuclear mitotic apparatus protein 1 (NUMA1), and centro- zonase on the Right.(B) HEK293-T cells were cotransfected with an expres- mere protein F (CENPF), as reviewed in ref. 9. Interestingly, sion for cGAS (pUNO1-cGAS) together with pSERSeGFP-LANA FL or we found Daxx to be the binding partner of LANA-FL, but not pSERSeGFP control vector. Cellular lysates were treated with benzonase and of LANA-NC, which is in line with a previous study which subsequently subjected to immunoprecipitation with a GFP specific anti- reported that the deletion of the internal repeat region of body. (C)(i) Pull-down assay with GST fusion proteins consisting of the LANA abrogates Daxx binding (50). Among the newly iden- C-terminal domain of LANA (LANA-C), the N-terminal domain (LANA-N), and BJAB cell lysates. Samples were analyzed by immunoblotting with antibodies tified LANA binding proteins was the DNA sensor cGAS, specific for cGAS and GST. (ii) Pull-down assay with GST LANA-C or LANA-N which was found in three of four LANA-FL and four of four fusion proteins and in vitro transcribed/translated GFP-cGAS recombinant LANA-NC immunoprecipitates. protein. Samples were analyzed by immunoblotting with antibody specific for cGAS and GST (Ci). (D) HEK293-T cells were cotransfected with expression The N-Terminal Domain of LANA Interacts with cGAS. To confirm the for cGAS together with N-terminally T7 epitope tagged full length interaction between endogenous LANA and cGAS, we performed LANA (LANA-FL) or the LANA mutants Δ262–320, Δ288–320, or Δ306–320. a coimmunoprecipitation (Co-IP) in the KSHV-positive PEL cell Forty-eight hours later, cells were lysed and subjected to immunoprecipita- line BCBL-1, as well as a Co-IP in HEK 293T cells transiently tion with a T7-specific antibody. Western blots were stained with anti-LANA transfected with LANA and cGAS. cGAS was coprecipitated with (CR2-3), anti-cGAS, or for actin. LANA from cell lysates treated with benzonase to exclude an involvement of cellular DNA in this interaction (Fig. 1 A and B). N-terminal domain of LANA and an in vitro transcribed and To identify which of the three domains of LANA (N- or C-terminal domain or internal repeat region) interact with cGAS, translated GFP-tagged cGAS protein. This assay confirmed that we performed GST pull-down assays with GST proteins fused to the N-terminal domain of LANA was sufficient for the in- theC-orN-terminaldomainsofLANAandlysatesofBJAB teraction of LANA and cGAS (Fig. 1 C, ii). To further confirm cells. This assay showed the N-terminal domain of LANA was that this region of LANA is necessary for the interaction with

sufficient for the interaction with cGAS (Fig. 1 C, i). We also cGAS, different internal deletion mutants in the N-terminal do- MICROBIOLOGY performed a GST pulldown assay with GST protein fused to the main of full-length LANA (LANA Δ262–320, LANA Δ288–320,

Zhang et al. PNAS | Published online January 25, 2016 | E1035 Downloaded by guest on September 28, 2021 LANA Δ306–320) and full-length LANA (LANA FL) were A B *** cotransfected with cGAS (pUNO1-hcGAS) in HEK293T cells, cGAS siRNA - + - + and coimmunoprecipitations were performed. cGAS was copre- scrambled siRNA + - + - Δ – Δ – RTA+SB - - + + M.W. (kD) cipitated with LANA 288 320 and LANA 306 320 mutants, -250 α-LANA

Δ – IU/ml but not with LANA 262 320 (Fig. 1D). This observation con- -150 firmed the results of the GST pull-down assay and points to the -100 – -75 region of aa 262 320, within the N-terminal domain, as being α-cGAS

required for the interaction with cGAS. α-p-IRF3 cGAS siRNA -+ - + -50 scrambled siRNA +- +- α-actin KSHV Reactivation Activates the cGAS-STING Axis. To investigate the RTA+SB -- ++ role of cGAS in the KSHV lytic replication cycle, we silenced cGAS C expression using siRNA in HuAR2T.rKSHV.219, a conditionally cGAS siRNA - + - + scrambled siRNA + - + - immortalized endothelial cell line (HuAR2T) persistently infected RTA+SB - - + + M.W. (kD) with recombinant KSHV.219 (51). We activated the lytic replication -250 cycle by treatment with recombinant RTA and sodium butyrate, or α-LANA -150 -100 -75 recombinant RTA alone, and analyzed IRF3 phosphorylation as α-cGAS an indicator of the activation of the cGAS-STING-IRF3 signaling pathway. We found that IRF3 and TBK1 phosphorylation was α-K-bZIP -37 efficiently induced on lytic reactivation (Fig. S2). When cGAS α-actin expression was silenced in HuAR2T.rKSHV.219 cells, IRF3 - α-ORF45 75 phosphorylation was not observed after induction of the lytic ←unspecific cycle (Fig. 2A). These results indicate that the cGAS-STING- bands IRF3 signaling pathway is activated on KSHV reactivation in HuAR2T.rKSHV.219. D cGAS siRNA - + - + - + - + - + - + 2‘3‘cGAMP - - - - + + ------3‘3‘cGAMP ------+ + - - - - di-UMP ------+ + - - Both cGAS and STING Inhibit KSHV Reactivation. Because we ob- IFN-beta ------+ + served that the cGAS-STING-IRF3 pathway is activated during RTA+SB - - + + + + + + + + + +

reactivation, we next analyzed whether activation of this pathway α-LANA has an effect on the KSHV lytic replication cycle. To measure the level of reactivation in HuAR2T.rKSHV.219 cells, the ex- α-cGAS pression of an early lytic viral protein K-bZIP, as well as a viral α-K-bZIP tegument protein ORF45, was measured by Western blot. Ad- ditionally, the number of cells expressing lytic viral genes was α-actin ascertained by monitoring the expression of RFP, which is driven 1 2 3 4 5 6 7 8 9 10 11 12 by a KSHV lytic promoter (PAN promoter) in the recombinant Fig. 2. Silencing of cGAS increases KSHV reactivation. (A)HuAR2T.rKSHV.219 rKSHV.219 (52), which is present in the HuAR2T.rKSHV.219 cells were transfected with an siRNA targeting cGAS or scrambled siRNA cell line. We also measured the virus titer in the culture me- as control and 24 h hours later the lytic replication cycle of KSHV was dium of HuAR2T.rKSHV.219 cells. We observed an increase induced by addition of baculovirus expressing the KSHV RTA protein in the release of infectious KSHV progeny from reactivated and 1.5 mM Na-butyrate. Forty-eight hours later, supernatants were col- HuAR2T.rKSHV.219 cells after treatment with cGAS siRNA lected for analysis of viral titers, and cells were lysed and subjected to (Fig. 2B), as well as an enhanced expression level of K-bZIP and immunoblotting analysis. (B) Supernatants collected as described in A were ORF45 (Fig. 2 C and D, compare lanes 3 and 4), and an increased titered on HEK293T cells. Infectious units per milliliter are shown. (C) HuAR2T.rKSHV.219 cells were transfected with an siRNA targeting cGAS number of RFP-positive cells (Fig. S3A). These results suggest that or scrambled siRNA as control, and 24 h hours later, the lytic repli- the cGAS-STING-IRF3 signaling pathway is important to suppress cation cycle of KSHV was induced by addition of baculovirus expressing KSHV reactivation from latency in KSHV-infected cells. the KSHV RTA protein and 1.5 mM Na-butyrate. The cell lysates were sub- To confirm that the cGAS-mediated DNA sensing pathway is jected to immunoblotting analysis using antibodies to cGAS, the early lytic important for the control of viral reactivation, we assessed the protein K-bZIP, and the ORF45-endoded viral tegument protein. (D) effect of cGAMP and IFN-β on KSHV reactivation. 2′3′cGAMP, HuAR2T.rKSHV.219 cells were transfected with an siRNA targeting cGAS or a unique class of the 2′-5′linked second messenger molecule, is scrambled siRNA. Twenty-four hours later, cells were treated with 2′3′- ′ ′ β produced by cGAS on DNA binding and then binds to and ac- cGAMP, 3 3 -cGAMP, di-UMP, or IFN- , and 12 h later, the lytic replication cycle of KSHV was induced by addition of baculovirus expressing the KSHV tivates STING. It has a higher affinity for human STING than – ′ ′ ′ ′ ′ ′ RTA protein and 1.5 mM Na-butyrate (lanes 3 12) or cells were left un- 3 3 cGAMP (38, 40). Unlike 2 3 cGAMP and 3 3 cGAMP, cyclic treated (lanes 1–2). Cells were lysed 48 h later and subjected to immuno- di-uridine monophosphate (c-di-UMP) is not able to bind blotting analysis. STING. When we pretreated HuAR2T.rKSHV.219 cells with 2′3′cGAMP, 3′3′cGAMP, c-di-UMP, or IFN-β before induction of the lytic cycle, we found that 2′3′cGAMP is able to inhibit KSHV reactivation. Similarly to cGAS, we found that knock- KSHV reactivation in HuAR2T.rKSHV.219 cells, in which down of STING enhanced KSHV reactivation, as measured by the cGAS had been silenced by siRNA, as assessed by expression of increased level of K-bZIP in HuAR2T.rKSHV.219 (Fig. S3B). After the early protein K-bZIP and the RFP marker (Fig. 2D, compare knockdown of STING, pretreatment with 2′3′cGAMP, which ′ ′ lanes 4 and 6, and Fig. S3A). Pretreatment with 3 3 cGAMP only binds to STING and induces STING trafficking and signal- moderately inhibited K-bZIP expression, and pretreatment with β ing, had no effect on viral reactivation as expected, nor did c-di-UMP had no effect, whereas pretreatment with IFN- ′ ′ β completely repressed KSHV reactivation (Fig. 2D and Fig. S3A). 3 3 cGAMP or di-UMP. In contrast, pretreatment with IFN- , The above results indicate that cGAS and its second messenger which acts downstream of STING, was still able to block viral molecule 2′3′cGAMP prevent KSHV reactivation and play a role reactivation (Fig. S3B). These results confirm that the activation in the maintenance of viral latency. of the cGAS-STING-IRF3 signaling pathway and the resulting Next, we tested whether STING, a downstream mediator of production of IFN-β prevent KSHV reactivation and help to cGAS and 2′3′cGAMP, also contributes to inhibition of lytic maintain KSHV latency.

E1036 | www.pnas.org/cgi/doi/10.1073/pnas.1516812113 Zhang et al. Downloaded by guest on September 28, 2021 The Cytoplasmic Isoforms of LANA Interact with cGAS. LANA is cGAS with an N-terminally truncated and therefore cytoplasmic PNAS PLUS known to be a nuclear protein forming nuclear speckles in LANA mutant, LANAΔ161 (aa161–1162) (Fig. 4 A and B). On KSHV-infected cells. A recent study reported the existence of cotransfection of cGAS and LANAΔ161 in HEK293T cells and cytoplasmic isoforms of LANA, which are mainly generated by coimmunoprecipitation with a LANA antibody, we could con- noncanonical internal translation initiation sites localized within firm that LANAΔ161, which only localizes to the cytoplasm (Fig. the LANA N-terminal domain (35). Another recent report 4 B and C, Left, and F, Upper), interacted with cGAS (Fig. suggested the existence of N-terminally truncated LANA forms 4C, Right). resulting from caspase cleavage (24). In a complementary ex- Among the HeLa cell lines of different origins tested in our periment to that shown in Fig. 1A (coimmunoprecipitation of laboratory, HeLa MZ cells were found to express both cGAS and cGAS by LANA with a LANA-specific antibody), we immuno- STING. The cGAS-STING-IRF3 pathway can be efficiently precipitated cGAS with a cGAS specific antibody from BCBL-1 activated in HeLa MZ cells on IFN stimulatory DNA (ISD) cells and found that mainly lower-molecular-weight isoforms of transfection, as indicated by the detection of IRF3 phosphory- LANA associated with cGAS [Fig. 3A; compare immunopreci- lation (Fig. 4D and Fig. S4). To test whether LANAΔ161 can pitated LANA bands (Right) with LANA bands in the input cell counteract cGAS activity, we generated HeLa MZ cell lines lysate (Left)]. This result suggests that cGAS might preferentially stably expressing LANAΔ161 and full-length LANA (LANA FL). interact with the short cytoplasmic isoforms of LANA. To verify As shown in Fig. 4B,LANAΔ161 is localized in the cytoplasm, this hypothesis, we fractionated lysates of BCBL-1 cells into whereas LANA FL is found in the nucleus. We induced the cytoplasmic and nuclear extracts. cGAS is known to localize to cGAS-STING-IRF3 signaling pathway in HeLa cell lines by the cytoplasm, and as expected, we detected its expression in transfection with ISD and found that the presence of LANAΔ161 the cytosolic extract, but not in the soluble nuclear extract (Fig. strongly inhibited both IRF3 and TBK1 phosphorylation, compared 3B, Left). In the cytoplasmic extract we detected mainly the with the activation seen in parental and LANA FL-expressing short isoforms of LANA, whereas full-length LANA was pre- HeLa MZ cells (Fig. 4D and Fig. S4). dominantly in the nuclear extract (Fig. 3B, Left). When we used Next, we transiently transfected HeLa MZ-LANAΔ161 or an antibody recognizing an epitope within the internal repeat LANA FL with an IFN-β promoter luciferase reporter plasmid region of LANA for coimmunoprecipitations with the BCBL-1 and found that both LANA FL and the cytoplasmic LANA cytoplasmic and nuclear fractions and analyzed the immuno- mutant LANAΔ161 were able to repress the transcriptional ac- precipitates with an antibody to LANA by Western blotting, we tivation of the IFN-β promoter (Fig. 4E). This observation is in observed short LANA isoforms in the cytoplasm (Fig. 3B, line with the recently published screen of KSHV ORFs, which Right). Interestingly, cGAS coprecipitated with the short iso- showed that full-length LANA can inhibit the activation of an forms of LANA in the cytoplasm (Fig. 3B, Right). These results IFN-β reporter in response to cGAS stimulation (21). suggest that mainly the cytoplasmic isoforms of LANA interact To provide further evidence that cytoplasmic isoforms of with cGAS. LANA can negatively modulate cGAS activity, we generated a stable HuAR2T cell line expressing LANAΔ161 by lentiviral The Cytoplasmic Isoforms of LANA Antagonize the Function of cGAS. transduction. HuAR2T cells stably transduced with eGFP served To determine whether the cytoplasmic isoforms of LANA an- as control. As shown in HeLa MZ cells, LANAΔ161 was local- tagonize cGAS function, we first analyzed the interaction of ized in the cytoplasm of transduced HuAR2T cells (Fig. 4F). We next induced the cGAS-STING-IRF3 signaling pathway by ISD transfection and found that LANAΔ161 inhibited IRF3 and IP TBK1 phosphorylation compared with HuAR2T cells stably A IgG α-cGAS expressing eGFP (Fig. 4F). Together, these results indicate that a input control M.W. (kD) cytoplasmic isoform of LANA interacts with cGAS and antago- -250 nizes the ability of cGAS to initiate an innate immune response α-LANA -150 ← β ← resulting in IFN- production. -100 After the induction of the cGAS-STING-IRF3 signaling path- -75 α-cGAS way by transfection of ISD in HeLa MZ cells stably expressing LANAΔ161 or GFP, we also quantified the level of 2′3′cGAMP produced in these cells by RP-HPLC/MS. We did not detect ′ ′ Δ B Input IP 2 3 cGAMP from HeLa MZ cells expressing LANA 161 after CE NE CE NE ISD transfection, compared with a low amount of 2′3′cGAMP -250 IgG α-LANA IgG α-LANA detected in the cells expressing GFP at 48 h after ISD transfection, -150 control control α-LANA -250 which is consistent with the observation of reduced IRF3 phos- -100 Δ α-LANA -150 phorylation in HeLa MZ cells expressing LANA 161 (Fig. S4). α-cGAS -50 -100 These results confirm that the cytoplasmic isoform of LANA an- α-Calnexin -100 tagonizes cGAS function and inhibits the subsequent 2′3′cGAMP * -75 α-cGAS and type I interferon production. α-LaminA/C - IgG heavy To address the biological significance of this observation, we chain took advantage of the observation that cGAS can restrict HSV-1 Fig. 3. cGAS interacts with the cytoplasmic isoforms of LANA. (A) BCBL-1 replication (42, 43). We infected the LANA FL and LANAΔ161 cells were lysed, and endogenous cGAS was immunoprecipitated with a expressing HeLa MZ cell lines with HSV-1 at an MOI (multi- cGAS-specific antibody or IgG as negative control. Input lysates and immu- plicity of infection) of 0.1 and measured the levels of newly noprecipitates were analyzed by immunoblotting with LANA and cGAS- produced viral progeny in the cell culture supernatant by plaque specific antibodies. Arrows indicate the shorter isoform of LANA. (B) BCBL-1 assay on Vero cells. As shown in Fig. 4G, in HeLa MZ cells cells were lysed, and nuclear (NE) and cytoplasmic (CE) extracts were pre- expressing LANAΔ161, we found markedly (∼10-fold) enhanced pared. NE and CE were analyzed by immunoblotting (Left). CE and NE were Δ immunoprecipitated with a LANA-specific antibody or IgG as control and HSV-1 replication, in line with the ability of LANA 161 to in- analyzed by immunoblotting with antibodies to LANA and cGAS (Right). hibit IRF3 and TBK1 phosphorylation, whereas LANA FL

*cGAS coprecipitates with the cytoplasmic isoforms of LANA in the cyto- did so only moderately (∼4-fold). Together, our findings MICROBIOLOGY plasmic extract. suggest that a cytoplasmic isoform of LANA can antagonize

Zhang et al. PNAS | Published online January 25, 2016 | E1037 Downloaded by guest on September 28, 2021 A B LANA DAPI 1 1162 LANA△161 LANA-FL NTD Internal repeat CTD ↑ ↑ NLS NLS M161 1162 LANA△161 LANA DAPI NTD Internal repeat CTD ↑ NLS LANA-FL

C CE NE LANA FL △161 FL △161 CE cGAS ++++ M.W. (kD) LANA-FL+cGAS △161+cGAS -250 IgG α-LANA IgG α-LANA α-LANA -150 control control 250 -100 - α-cGAS α-LANA -150 α-calnexin -100 α-laminA/C α-cGAS - IgG heavy chain D ISD 2h 4h E LANA -FL△161 -FL△161

**

α-LANA **

reporter activity reporter β α-pIRF3 α-pTBK1

α-actin Relative IFN- Relative HeLa MZ control LANA-FL LANA△161

F LANA DAPI HUAR2T LANA△161

G *** 105 ISD - 4h 8h ** GFP △161 GFP △161 GFP△161

4

α-LANA 10 pfu/ml

α-pIRF3 103 α-pTBK1 HeLa MZ control LANA-FL LANA△161

α-GFP α-actin

Fig. 4. A cytoplasmic isoform of LANA antagonizes cGAS-dependent IFN-β signaling and antiviral activity. (A) Schematic diagram of full-length LANA (LANA-FL) and the cytoplasmic LANA mutant LANAΔ161, which starts at the internal methionine 161. (B) Representative images of HeLa MZ-LANA FL and HeLa MZ LANAΔ161 stable cell lines. Cells were fixed and probed with a LANA antibody and stained with Hoechst to visualize nuclei. (C) HEK293T cells were cotransfected with cGAS and LANA-FL or LANAΔ161. Cells were fractionated into cytoplasmic and nuclear fractions, and input samples were analyzed by immunoblotting analysis (Left). The cytoplasmic extracts were immunoprecipitated with a LANA-specific antibody or IgG as control and analyzed by immunoblotting with anti- bodies to LANA and cGAS (Right). (D) HeLa-MZ cells stably expressing LANA-FL or LANAΔ161 were stimulated with ISD or transfection reagent as the control. Lysates were generated 2 and 4 h later and analyzed by immunoblotting. (E)IFN-β promoter luciferase reporter assay in HeLa MZ cells stably expressing LANA FL or LANAΔ161, transfected with an IFN-β promoter reporter construct. The value of the relative reporter activity of parental control is normalized and set to 1. (F) Cytoplasmic localization of LANAΔ161 in HuAR2T cells transduced with a lentiviral vector for LANAΔ161. Cells were fixed, and LANA was visualized with a LANA antibody; nuclei were stained with DAPI (Upper). Cells were stimulated by ISD transfection or transfection reagent as control, and lysates were prepared 4 and 8 h later for immunoblotting analysis. (G) HeLa MZ cells stably expressing LANA-FL or LANAΔ161 were infected with HSV-1 at an MOI of 0.1. Supernatants were collected 36 h after infection, and viral titers were measured by plaque assay on Vero cells. **P < 0.01; ***P < 0.001; Student t test.

the cGAS-mediated innate immune response and thereby LANA, shown here to antagonize cGAS, could increase following promote productive herpesviral replication. activation of the lytic replication and thereby promote the pro- gression of the lytic replication cycle. Expression of Cytoplasmic LANA Isoforms Is Increased on Lytic Reactivation As shown in Fig. 5B, induction of the lytic replication cycle by of KSHV. Because LANA is known to be required for the estab- TPA in BCBL-1 cells resulted in increased expression of lower- lishment and maintenance of KSHV latency (47, 48, 53), a role for molecular-weight forms of LANA in the cytoplasm of fraction- the cytoplasmic forms of LANA to promote productive (lytic) ated cells, which were detected by an antibody to the internal herpesviral replication seems at first counterintuitive. We therefore repeat region of LANA (anti-CR2-3), but not with an anti- examined whether expression of the cytoplasmic isoforms of body to an epitope in the N-terminal domain (NTD) of LANA

E1038 | www.pnas.org/cgi/doi/10.1073/pnas.1516812113 Zhang et al. Downloaded by guest on September 28, 2021 (anti-NTD) (Fig. 5 A and B). Similarly, induction of the lytic viral lytic replication by an inhibition of the cGAS-dependent PNAS PLUS replication cycle in a persistently KSHV-infected BJAB B-cell signaling pathway in LANAΔ161 expressing cells (Fig. 5D). line, BJAB.rKSHV.219 (54, 55), with an antibody to surface IgM was also accompanied by the increased expression of short iso- Discussion forms of LANA in the cytoplasmic fraction (Fig. 5C). As ob- As one of the major viral proteins expressed in KSHV latency, served for BCBL-1 cells, these inducible cytoplasmic isoforms of the function of LANA in the nucleus of KSHV-infected cells has LANA were only detected with the anti–CR2-3 LANA antibody, been well studied. In contrast, the function of the recently dis- but not with the anti-NTD LANA antibody (Fig. 5A). This result covered cytoplasmic isoforms of LANA, generated by non- suggests that the N-terminal end of full-length LANA (LANA canonical translation initiation within the LANA NTD (35, 56) FL), which contains a nuclear localization signal, is lacking in the or possibly by caspase cleavage (24), has not been explored to inducible cytoplasmic forms of LANA. date. In this study, we report that LANA, especially its cyto- To investigate whether the cytoplasmic isoforms of LANA plasmic isoforms, antagonizes the function of cGAS, hereby might facilitate lytic replication by antagonizing cGAS, we trans- promoting lytic replication after lytic cycle induction. duced HuAR2T.rKSHV.219 cells with LANAΔ161 or eGFP- Following our initial observation that LANA interacts with expressing lentiviruses and activated KSHV lytic replication with cGAS, we show that this interaction occurs mainly in the cytoplasm RTA and sodium butyrate. We observed enhanced levels of the of KSHV-infected cells and that cGAS immune-precipitated from KSHV early protein K-bZIP in HuAR2T.rKSHV.219 expressing the cytoplasm is associated with lower-molecular-weight forms of LANAΔ161 after reactivation, compared with the GFP-expressing LANA (Fig. 3). We found that overexpression of one of the cy- control HuAR2T.rKSHV.219 cell line (Fig. 5D). These results toplasmic isoforms of LANA, lacking amino acids 1–160, reduced suggest that the cytoplasmic isoforms of LANA might enhance IRF3 and TBK1 phosphorylation levels during ISD stimulation in

A α-NTD (CM 6.43.24) α-CR2-3 (CDLGDDLHLQPKRRKHVAD) repetitive EQEQE, EQEQ 1 1162 LANA-FL N-term CR1 CR2 CR3 C-term ↑ ↑ NLS NLS

B BCBL-1 C BJAB BJAB.rKSHV CE NE

TPA -+-+ M.W. (kD) CE NE CE NE - 250 anti-IgM - +-+-+-+ α-LANA ← - 150 -250 ← (α-CR2-3) α-LANA ← - -150 100 (α-CR2-3) ← -250 * -100 -150 α-LANA α-LANA -250 (α-NTD) * -100 (α-NTD) -150 α-K-bZIP -75 α-cGAS α-actin α-actin α-calnexin α-K-bZIP α-lamin A/C α-calnexin

α-lamin A/C

D HUAR2T.rKSHV GFP △161 GFP △161

RTA+SB: -- ++

-250

α-LANA -150

-100

α-K-bZIP

α-actin

Fig. 5. Increased expression of cytoplasmic LANA isoforms following lytic reactivation of KSHV and enhancement of lytic replication by a cytoplasmic LANA isoform. (A) Schematic representation of LANA showing epitopes detected by the anti-LANA antibodies anti-CR2-3 and anti-NTD (CM 6.43.24). (B and C) Immunoblotting showing the expression of LANA isoforms in the cytoplasmic (CE) and nuclear (NE) extracts after lytic cycle induction by TPA (24 h) in BCBL-1 cells (B) and after lytic cycle induction by anti-IgM antibody (24 h) in BAJB.rKSHV.219 and the control cell line BJAB (C). LANA isoforms were detected by an antibody to the CR2-3 domains in the internal repeat region of LANA (Top) and an antibody to an amino-terminal epitope (second panel from the top). Arrows, cytoplasmic LANA bands increasing after lytic reactivation; asterisk, full-length, nuclear LANA isoform. (D) HuAR2T.rKSHV.219 cells transduced with a

lentivirus expressing LANAΔ161 or GFP were treated with baculovirus expressing RTA and Na-butyrate to induce the lytic cycle or left untreated. Lysates were MICROBIOLOGY analyzed 36 h later by immunoblotting.

Zhang et al. PNAS | Published online January 25, 2016 | E1039 Downloaded by guest on September 28, 2021 HeLa MZ and HuAR2T cells and inhibited IFN-β promoter ac- clude the leakage of viral or host DNA into the cytosol due to cell tivity in an IFN-β luciferase reporter assay (Fig. 4 and Fig. S4). We death during KSHV reactivation, which may be recognized by also show that expression of this cytoplasmic isoform of LANA cGAS and thereby activate this innate response. DNA fragmen- enhances HSV-1 replication and KSHV reactivation (Figs. 4G and tation and cell death by apoptosis were observed during KSHV 5D). Together these results suggest that, during lytic replication, reactivation in PEL- and KSHV-infected -immortal- the cytoplasmic isoforms of LANA antagonize the activity of cGAS ized human umbilical vein endothelial cells (TIVEs) (67–69). in the cGAS-STING-IRF3 pathway and reduce the phosphoryla- Fragmented DNA due to DNA damage might accumulate in the tion of IRF3 and TBK1 and thereby production of type I IFNs, cytoplasm and initiate DNA sensing (70). The mitochondrial which, in turn, further enhances viral lytic replication. Indeed, in DNA (mtDNA) instability induced by herpesviruses can also in- keeping with this interpretation of our finding, we observed that duce the cGAS-STING-IRF3–mediated antiviral innate immune the expression of several cytoplasmic isoforms of LANA increases response (71). Interestingly, the RIG-MAVS signaling pathway on lytic cycle induction by TPA in BCBL-1 cells and by anti-IgM was also activated during KSHV reactivation, and dsRNA was in BJAB.rKSHV.219 cells. This result suggests that the translation detected during viral reactivation in latently KSHV-infected iSKL of LANA mRNA is regulated differently during latency and lytic cells (72). Activation of the cGAS-STING-IRF3 signaling pathway reactivation, with the start codon in position 1 of ORF73 being may therefore be part of a larger surveillance network that re- used during latency and internal initiation codons coming into play sponds to viral nucleic acids in the cytoplasm of infected cells and during the lytic cycle. It has been shown that expression of LANA against which KSHV may have evolved countermeasures. Our can be directed from two different promoters that generate dif- observation that the cytoplasmic isoforms of LANA may serve as a ferent 5′ UTRs of LANA mRNA. Although the constitutive (la- viral antagonist of cellular innate immunity extends the role of tent) promoter generates a longer 5′ UTR that occurs in a spliced LANA into the lytic replication cycle and could provide an ex- or unspliced form, the lytic LANA promoter, localized in the in- planation both for the existence of a lytic LANA promoter and the tron within the LANA 5′ UTR, directs the expression of an recently described initiation of alternative forms of LANA at in- unspliced mRNA with a shorter 5′ UTR (4, 57, 58). One could ternal start codes in its N-terminal domain. envisage that the shorter 5′ UTR generated during lytic reac- tivation could favor, as a result of a different RNA structure fold, Materials and Methods the use of internal start codons in the LANA NTD and thereby the Cell Culture and Reagents. HEK293T, Vero, and HeLa MZ cells were maintained generation of LANA variants lacking the N-terminal NLS (4, 5), in DMEM with 10% (vol/vol) FCS, 50 IU/mL penicillin, and 50 mg/mL strepto- which localize to the cytoplasm. This hypothesis will have to be mycin. BJAB and BCBL-1 cells were maintained in RPMI 1640 supplemented addressed experimentally in the future. Our conclusion that, during with 20% (vol/vol) FCS, 50 IU/mL penicillin, and 50 mg/mL streptomycin. HuAR2T. KSHV lytic reactivation, the cytoplasmic isoforms of LANA an- rKSHV.219 is an endothelial cell line (HuAR2T) conditionally immortalized by human telomerase RT and SV40 large T antigen and stably infected with tagonize cGAS activity and inhibit the subsequent production of β rKSHV.219 (51). HuAR2T.rKSHV.219 cells were maintained in EGM-2MV medium IFN- , thereby further facilitating lytic replication, highlights the (Lonza) supplemented with 10% (vol/vol) FCS, 50 IU/mL penicillin, and 50 mg/mL role of the IFN system in regulating herpesviral latency and the streptomycin, in the presence of 1 μg/mL doxycycline and 4.2 μg/mL puromycin. role of cGAS as a cellular determinant of KSHV latency. 2′3′-cGAMP (tlrl-cga23-s), 3′3-cGAMP (tlrl-cga-s), c-di-UMP (tlrl-cdu), ISD We observed robust viral reactivation on knockdown of cGAS (tlrl-isdn), and pUNO1-hCGAS (MB21D1) were purchased from Invivogen or STING in HuAR2T.rKSHV.219, as shown by enhanced ex- and human IFN β1a (11415-1) from PBL Assay Science. pression of the early lytic viral K-bZIP and viral progeny production (Fig. 2). Pretreatment with 2′3′cGAMP or IFN-β be- Immunoblotting. The following antibodies were used to detect proteins after fore reactivation was able to repress or completely inhibit viral separation by SDS/PAGE and transfer to nitrocellulose membranes: anti-LANA lytic replication. These results suggest that activation of the cGAS- CR2-3 (13-210-100; Advanced Biotechnologies), anti-LANA NTC (CM 6.43.24; STING-IRF3 pathway and the resulting IFN-β production restrict a kind gift from Y. Chang and P. Moore, University of Pittsburgh, Pittsburgh), KSHV reactivation and therefore play an important role in the anti-cGAS (HPA031700-100UL; Sigma), anti-TMEM173/STING (LS-C108557; LSBio), anti–phospho-IRF3 (4947S; Cell Signaling Technology), anti–phospho- maintenance of viral latency from reactivation. There are several TBK1/NAK (Ser172) (5483S; Cell Signaling Technology), anti-T7 Tag (69522-3; lines of evidence supporting a role for type I IFNs in supporting Novagen); anti–K-bZIP (sc-69797; Santa Cruz), anti-calnexin (C-20) (sc-6465; persistent viral infections. MHV-68 replicates more efficiently Santa Cruz), anti–β-actin (A5441; Sigma), and anti-lamin A/C (2032S; Cell from macrophages of IFN-γ receptor-deficient mice and from Signaling Technology). As a negative control in immunoprecipitation ex- splenocytes of IFN-α/β receptor-deficient mice (59–61). IFN-α periments, rat IgG2c isotype control [SB68b] (GTX35063; Genetex) and also promoted the establishment of HSV-1 latency and pseu- normal rabbit IgG (sc-2027; Santa Cruz) were used. dorabies virus (PRV) in vitro in neurons of the trigeminal ganglion (62). IFN-β could block MCMV reactivation in mice latently In Vitro Translation and GST Pulldown Assay. GFP-cGAS cloned into the infected with MCMV (63). Chronic IFN-I signaling was also pIRESneo3 vector containing a T7 promoter (kindly provided by A. Ablasser, reported to be associated with LCMV persistence (64–66). After École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland) was transcribed and translated in vitro using the TNT rabbit reticulocyte lysate the viruses successfully establish viral latency, the IFN-I signaling system (Promega). GST-fused N-LANA or GST alone were produced in may contribute to virus persistence. We therefore hypothesize that Escherichia coli Rosetta and bound to glutathione beads. Equivalent IFN-α/β may not only control γ-herpesviruses, but also play an amounts of GST proteins were incubated with 20 μL in vitro-translated important role in the maintenance of viral latency. cGAS, and the reaction volume was brought up to 300 μLbyTBSTlysis If, as our findings suggest, the role of cytoplasmic LANA iso- buffer containing protease inhibitors. Proteins were incubated overnight forms is to antagonize cGAS and thereby promote lytic re- at 4 °C on a rotating wheel. The beads were washed six times with TBST plication, this raises the question of how cGAS is activated in lysis buffer. Bound proteins were eluted with 10 μL5× SDS sample buffer, KSHV-infected cells. We did not observe spontaneous reac- boiled 5 min, and analyzed by Western blot. tivation in HuAR2T.rKSHV.219 in the absence of cGAS without lytic replication induction (Fig. 2), suggesting that lytic replication Construction of Lentiviral Vectors. The full-length LANA-FL construct was amplified by PCR from BAC36 with the following primers: LANA Avrll for- is necessary for cGAS activation. During the viral lytic replication ward, TAT CCT AGG GCG CCC CCG GGA ATG CGC; LANA NotI reverse, TAT cycle, newly synthesized viral DNA is incorporated into capsids, GCG GCC GCT TAT GTC ATT TCC TGT GGA GAG TC. which are then transported from the nucleus into the cytoplasm. It The LANA-NC construct (LANA lacking the internal repeat, aa 329–931) is conceivable that viral DNA leaking from imperfectly assembled was obtained by PCR from pcDNA3.1-LANA-NC, which was originally cloned capsids might be recognized by cGAS and trigger the activation of from BAC36 LANAΔ329–931 (47) with the following primers: LANA Avrll the cGAS-STING-IRF3 signaling pathway. Other possibilities in- forward, TAT CCT AGG GCG CCC CCG GGA ATG CGC; ACL NotI reverse, ATT

E1040 | www.pnas.org/cgi/doi/10.1073/pnas.1516812113 Zhang et al. Downloaded by guest on September 28, 2021 TGC GG CCG CTT ATG TCA TTT CCT GTG GAG. Both LANA-NC and LANA-FL 0.1% Pyronin Y, and 3.5% β-mercaptoethanol). Samples were separated by PNAS PLUS inserts were first cloned into the pGEM-T Easy Vector (pGEM-T Easy Vector SDS/PAGE, the gels were stained with Coomassie brilliant blue, and complete Systems; Promega), and subsequently excised with AvrII and NotI to allow lanes were excised for MS analysis. ligation into the doxycycline-inducible lentiviral vector pSERS-GFP. Lentiviral Each lane of the SDS/PAGE gel was sliced and fragmented into small 1-mm3 vectors generated were designated pSERS-GFP-LANA-NC and pSERS-GFP-LANA- pieces. These gel slices were destained two times with 200 μL 50% aceto- FL. LANAΔ161 was amplified by PCR from plasmid pcDNA3.1 (+)/Zeo/LANAΔ161 nitrile (ACN) and 50 mM ammonium bicarbonate (ABC) at 37 °C for 30 min (kindly provided by P. Moore) with the following primers: LANAΔ161forward, and then dehydrated with 100% ACN. Solvent was removed in a vacuum CTCGTAAAGTCGACACCATGCGTCCGCCACCCTCG; LANAΔ161reverse, GGAC- centrifuge, and 20 μL of 6 ng/μL sequencing grade Trypsin (Serva) in 10% TAATCCGGAGCTTATGTCATTTCCTGTGGAGAGTCCCC); this was inserted into ACN and 40 mM ABC was added. Gels were rehydrated in trypsin solution the lentiviral vector pSERS-eGFP, which was digested with NcoI and NotI, for 1 h on ice and then covered with 10% ACN and 40 mM ABC. Digestion according to the protocol of Gibson Assembly (Gibson Assembly Cloning Kit, was performed overnight at 37 °C. Digestion was stopped, and peptides #E5510S; NEB). The generated lentiviral vector was designated pSERS- were extracted by adding 100 μL 50% ACN and 0.1% trifluoroacetic acid Δ LANA 161. (TFA) and incubation at 37 °C for 1 h. This step was repeated twice, and extracts were combined and dried in a vacuum centrifuge. Dried peptide Lentivirus Production and Generation of Stable Cell Lines. The expression extracts were redissolved in 30 μL 2% ACN and 0.1% TFA by soft shaking for vector for the RD114 envelope protein, M57-DAW (lentiviral gag/pol), kindly 20 min and then centrifuged at 20,000 × g, and the supernatant was stored provided by J. Bohne (Hannover Medical School, Hannover, Germany) and as aliquots of 12.5 μLat−20 °C. lentiviral vectors pSERS-GFP, pSERS-GFP-LANA-NC, or pSERS-GFP-LANA-FL A sample aliquot was injected into a nano-flow ultra-HPLC system (RSLC; were cotransfected into HEK293-T cells using the calcium phosphate method. Dionex) equipped with a trapping column (5-μm C18 particle, 2-cm length, The supernatant containing virus particles was harvested every 12 h after 75-μm ID, PepMap; Dionex) and a separating column (2-μm C18 particle, transfection for 60 h and centrifuged at 10,000 rpm for 14 h at 4 °C using a 50-cm length, 75-μm ID, PepMap; Dionex). The outlet of the RSLC system was SW32 rotor in a Beckmann ultracentrifuge. The supernatant was sucked off directly connected to the nano-ESI source (Thermo Fisher Scientific) of the ∼ μ gently, and 200 L medium was left. The pellet was resuspended in this Orbitrap mass spectrometer. A voltage of 1.2 kV was applied at the ESI − medium, and aliquots were stored at 80 °C. source, and ionized peptides were analyzed in a LTQ-Orbitrap velos mass × 4 BCBL-1 cells (5 10 ) were seeded per well of a 24-well-plate and in- spectrometer. Overview scans were acquired at a resolution of 60,000 in a μ fected with 100 L concentrated pseudotyped lentiviral stocks pSERS-GFP, mass range of m/z 300–1,600 in the orbitrap. The top 10 most intensive ions pSERS-GFP-LANA-NC, or pSERS-GFP-LANA-FL by centrifuging for 30 min at of charge 2 or 3, a minimum intensity of 2,000, and an isolation width of × 450 g at 32 °C. The medium was changed after an incubation time of 5 h 2 Th (a unit of mass-to-charge ratio) were selected for CID (collision-induced μ at 37 °C and 5% CO2. Doxycycline (1 g/mL) was added to the cells 24 h dissociation) fragmentation with a normalized collision energy of 38.0, an after transduction. The transduced cells were expanded, grown in the activation time of 10 ms, and an activation Q (parameter for Orbitrap mass μ presence of 1 g/mL doxycycline, and sorted for GFP-positive cells by spectrometry) of 0.250 in the LTQ (linear trap quadrupole) part of the LTQ FACS. The resulting transduced cell lines were named BCBL-1 GFP, BCBL-1 Orbitrap velos mass spectrometer. The m/z values in a 10-ppm mass window of GFP-LANA-NC, and BCBL-1 GFP-LANA-FL and cultured in the presence of the selected ions were subsequently excluded from the fragmentation for 70 s. 1 μg/mL doxycycline. Raw data were processed with the Proteome discoverer software package HuAR2T (1×105) or HuAR2T.rKSHV.219 cells were seeded in 12-well version 1.2 (Thermo Fisher) for identification of proteins using Mascot and the plates 1 d before transduction. The cells were transduced with 200 μL human entries of the Uniprot/Swissprot database. Proteins were stated concentrated pseudotyped retrovirus stocks containing pSERSeGFP or identified if at least two peptides per protein were identified with a peptide pSERSLANAΔ161 by centrifuging at 450 × g for 30 min at 32 °C. After ion score greater than 30 and a false discovery rate of less than 0.05. centrifugation, 200 μL fresh EGM-2MV medium was added to each well, and 4 h later, the medium was changed and replaced with 1 mL EGM-2MV Coimmunoprecipitation. Antibody-conjugated beads were prepared as fol- medium. The transduced HuAR2T and HuAR2T.rKSHV.219 cells were lows: 20 μL anti-LANA antibody (13-210-100; Advanced Biotechnologies; named HuAR2T-eGFP, HuAR2T-LANAΔ161, HuAR2T.rKSHV.219-eGFP, and 1 mg/mL) or 40 μL IgG (GTX35063; Genetex; 0.5 mg/mL) was incubated on ice HuAR2T.rKSHV.219-LANAΔ161 and cultured in EGM-2MV medium with μ 1 μg/mL doxycycline. for 15 min with 45 L PBS containing 4% sucrose and 0.02% sodium azide. Eighty microliters of protein A beads (17-5280-01; GE Healthcare) were HeLa MZ cells transduced with pSERS-eGFP or pSERS-LANAΔ161 were washed three times in TBST lysis buffer and incubated with the anti-LANA generated in a similar manner as the HuAR2T cells and named HeLa MZ- antibody overnight at 4 °C on a rolling platform. Before use, the antibody- pSERS-eGFP and HeLa MZ-pSERS-LANAΔ161. conjugated beads were washed three times with 500 μL TBST lysis buffer. To generate stable HeLa MZ cell transfectants, 8 × 104 HeLa MZ cells were BCBL-1 cells were suspended in TBST lysis buffer containing protease in- seeded per well of six-well plate and transfected with 2 μg/well of pcDNA3.1/ hibitors (1.5 mM aprotinin, 10 mM leupeptin, 100 mM PMSF, 1 mM benza- Zeo+/LANA FL or pcDNA3.1/Zeo+/LANAΔ161 with Lipofectamine 2000 trans- fection reagent (11668-019; Invitrogen). The medium was changed 6 h after midine, and 1.46 mM pepstatin A) on ice and treated with 200 U/mL transfection, and 24 h later, the transfected cells were selected with 100 μg/mL benzonase. After centrifugation, the lysates were precleared with protein A Zeocin. The selected cell clones were grown in the presence of Zeocin and beads for 2 h at 4 °C. The lysates were divided into two equal parts and μ named HeLa MZ-LANA FL and HeLa MZ-LANAΔ161. incubated with 15 L anti-LANA conjugated beads or control antibody conjugated beads overnight at 4 °C on a rolling platform. The beads were pelleted and washed six times with TBST buffer, and bound proteins were LC-MS Sample Preparation, Data Processing, and Statistical Analysis. Cells eluted with 5× SDS loading buffer and separated by SDS/PAGE. (1.5 × 108) from each stable cell line (BCBL-1 GFP, BCBL-1 GFP-LANA-FL, BCBL-1 GFP-LANA-NC) were washed once in PBS and resuspended in buffer A Co-IP of cytoplasmic and nuclear extracts from BCBL-1 cells was performed (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl , 5 mM DTT, and protease as follows: after centrifugation for 5 min at 900 rpm (Eppendorf centrifuge 2 μ inhibitors) for 30 min on ice and homogenized with 15 strokes in a cell 5810 R, rotor A-4-81, Hamburg, Germany), 50 L pelleted BCBL-1 cells were douncer (73). Nuclei were pelleted by centrifugation for 10 min at 1,200 rpm fractionated according to the protocol of NE-PER Nuclear and Cytoplasmic μ (Eppendorf centrifuge 5417R, rotor F45-30-11, Hamburg, Germany) and 4 °C Extraction Reagents (78833; Thermo Scientific). For IP, 500 L cytoplasmic ex- μ μ and resuspended in 2 mL TBST lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, tract was mixed with cold 500 L lysis buffer, and 250 L nuclear extract was μ 1 mM EDTA, and 1% TritonX-100). The suspension of nuclei was kept cold mixed with 250 L lysis buffer. Both cytoplasmic extract and nuclear extract and vortexed vigorously. Samples were then centrifuged at 13,000 rpm were precleared with 40 μL protein A beads for 2 h at 4 °C on a rolling plat- (Eppendorf centrifuge 5417R, rotor F45-30-11, Hamburg, Germany) for form, each was divided into two equal parts, and each part was incubated 10 min and precleared by incubation with 50 μL protein A beads (Protein A with 15 μL anti-LANA antibody-conjugated beads or IgG control beads. Sepharose 4 Fast flow; GE Healthcare) or GFP-trap control beads (GFP-Trap A; Chromotek) for 1 h at 4 °C on a rolling platform. Beads were removed by Transfection of siRNA by Electroporation. HuAR2T.rKSHV.219 cells (1 × 105) centrifugation, and precleared samples were incubated with 50 μL beads to were electroporated with 100 pmol of siRNA using the Neon Transfection which an anti-GFP antibody had been conjugated (Living Colors full-length System according to the manufacturer’s instructions (pulse voltage: 1,350 V; GFP Polyclonal Antibody; Clontech) or 50 μL GFP-trap beads (GFP-Trap A; pulse width: 30 ms; pulse number 1; tip type, 10 μL), and seeded in a 12-well Chromotek) on a rolling platform at 4 °C overnight. The precipitations were plate. The siRNAs were obtained from Dharmacon and Thermo Scientific

washed four times in 500 μL TBST lysis buffer, and bound proteins were (cGAS siRNA MQ-015607–01-0002, STING siRNA M-024333–00-0010, non- MICROBIOLOGY eluted with 5× SDS loading buffer (5 mM Tris, pH 6.8, 45% glycerol, 5% SDS, targeting control siRNA D-001206-14-50).

Zhang et al. PNAS | Published online January 25, 2016 | E1041 Downloaded by guest on September 28, 2021 + HSV-1 Infection and Plaque Assay. HSV-1(17 )Lox-pMCMVmCherry (kindly incubated with 50 U/mL benzonase for 45 min on ice. The upper aqueous provided by B. Sodeik, Hannover Medical School, Hannover, Germany) was layer was collected after adding 500 μL P:I:C (phenol:chloroform:isoamyl propagated using BHK-21 cells and purified as previously described (74). The alcohol: 25:24:1; Sigma), vortexed vigorously, and centrifuged for 5 min at titer was determined by plaque assays, and the genome to plaque-forming 13,000 rpm (Eppendorf Centrifuge 5415D, rotor FA-45-24-11, Hamburg, units (PFU) ratio was determined by real-time PCR (74). Germany), which was repeated once. The collected aqueous layer was added Δ HeLa MZ, HeLa MZ-LANA FL, or HeLa MZ-LANA 161 cells were seeded in a to 500 μL chloroform, vortexed vigorously, and centrifuged for 5 min at 96-well plate at a density of 0.5 × 104 cells per well. Before infection with 13,000 rpm (Eppendorf Centrifuge 5415D, rotor FA-45-24-11, Hamburg, HSV-1, the medium was changed, and the cells were incubated with 50 μL/well Germany). The upper aqueous layer was poured into an Amicon 3K filter of CO -independent medium (Life Technologies Gibco) supplemented with 2 column (Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-3 membrane; 0.1% BSA on ice for 20 min. Subsequently, HSV-1 suspension was added, and × the cells were incubated for 60 min on ice on a slow rocking platform to allow Millipore) and centrifuged for 2 min at 14,000 g. The pellets were resuspended in 20 μLH2O after centrifuge in a SpeedVac Concentrator. virus attachment. After washing three times with 50 μLCO2-independent medium (with 0.1% BSA), 100 μL fresh DMEM was added, and cells were in- The method for detection and quantification of 2′3′cGAMP has been

cubated at 37 °C and 5% CO2. described in ref. 76. Calibrators or sample extracts separation by reversed- The plaque titration was performed as previously described (75). Briefly, phase chromatography (RP-HPLC) was performed using an HPLC system Vero cells were seeded in six-well plates for 16–20 h before virus inoculation. (Shimadzu). The mobile phases were 3/97 methanol/water (vol/vol) (A) and μ After washing the cells with PBS, 500 LCO2-independent medium (with 97/3 methanol/water (vol/vol) (B), both contain 50 mM ammonium acetate 0.1% BSA) was added to each well. Then, the supernatant containing HSV-1 and 0.1% acetic acid. The following gradient was applied: 0–5min,0–50% virions produced by HeLa MZ cells (24 and 36 h after infection) was added, B, and 5–8 min, 0% B, with a flow rate of 500 μL/min. 2′3′cGAMP was de- and cells were incubated on a slow rocking platform at room temperature tected and quantified by a tandem mass spectrometer (MS/MS) 5500QTRAP μ + for 60 min. After removal of the virus, 2 mL fresh DMEM containing 40 g/mL (AB Sciex), and the following mass transitions [M+H] were identified for IgG solution (Sigma-Aldrich) was added, and cells were incubated at 37 °C. 2′3′-cGAMP: m/z 675.0 → 136.2 (quantifier), m/z 675.0 → 152.1 (identifier) and Three days later, the medium was removed, and the cells were fixed with 1 mL/well for tenofovir: m/z 288.0 → 176.0 (quantifier), m/z 288.0 → 159.1 (identifier). of prechilled (−20 °C) methanol. After removal of the methanol and drying of the samples, the cells were stained with 0.1% crystal violet and 2% EtOH. The plaques were counted after removal of the staining solution. Statistical Analysis. Statistical analysis was performed with the GraphPad Prism 5 software. All datasets were analyzed using t tests (unpaired t test, < Luciferase Reporter Assay. HeLa MZ, HeLa MZ-LANA, or HeLa MZ-LANAΔ161 two-tailed). P 0.05 was considered statistically significant. cells (4 × 104) were seeded per well of a 12-well plate in triplicate and transfected with 0.5 μg IFN-β promoter reporter construct using 1.5 μLLipo- ACKNOWLEDGMENTS. We thank Dr. P. S. Moore for kindly providing fectamine 2000 without further DNA stimulation. Four hours after trans- expression plasmids and anti-LANA antibody, and Dr. K. M. Kaye and Dr. A. Ablasser for generously providing expression plasmids. We thank fection, the medium was changed. The cells were lysed in 100 μL1× reporter μ Dr. B. Sodeik (Hannover Medical School) for kindly providing HSV-1 virus lysis buffer (Promega) 24 h after transfection, and 20 L cell lysate was used for stock. We thank the Core Facilities Cell Sorting (Dr. M. Ballmaier) and luciferase activity measurement using a luminometer. Metabolomics (Dr. V. Kaever) of the Hannover Medical School for assistance. This study was supported by grants to T.F.S. and M.M.B. of the Deutsche 2′3′cGAMP Quantification by HPLC/MS. HeLa MZ cells (8 × 104) stably expressing Forschungsgemeinschaft (DFG) [Collaborative Research Centre SFB900 “Chronic ” LANA-FL or LANAΔ161 were seeded in six-well plates and transfected with Infections: Microbial Persistence and its Control, Project C1 (to T.F.S.) and 4 μg ISD complexed with 4 μL Lipofectamine 2000 (Invitrogen), following the Project B3 (to M.M.B.)]. G.Z. is a scholarship holder of China Scholarship Council (2011621026) and supported by the Infection Biology international manufacturer’s protocol. μ PhD program of Hannover Biomedical Research School. B.C. and M.M.B. The cells in each well were resuspended in 500 L X-100 buffer (1 mM NaCl, were funded by the Helmholtz Association through the Helmholtz Virtual 3 mM MgCl2, 1 mM EDTA, 1% Triton X-100, and 10 mM Trip, pH 7.4) after Institute “Viral Strategies of Immune Evasion” (VH VI-424). A.B. was supported one cold PBS wash and kept on ice for 20 min. The cells were spun down at by the Niedersachsen-Research Network on Neuroinfectiology of the Minis- 1,000 × g at 4 °C after regular vortexing. The supernatant was collected and try of Science and Culture of Lower Saxony, Germany.

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