BRLF1 suppresses RNA Pol III‐mediated RIG‐I inflammasome activation in the early EBV lytic lifecycle
2020; Springer Nature; Volume: 22; Issue: 1 Linguagem: Inglês
10.15252/embr.202050714
ISSN1469-3178
AutoresXubing Long, Jing Yang, Xiaolin Zhang, Ziwei Yang, Yang Li, Fan Wang, Xiaojuan Li, Ersheng Kuang,
Tópico(s)Kawasaki Disease and Coronary Complications
ResumoArticle23 November 2020free access Source DataTransparent process BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle Xubing Long Xubing Long orcid.org/0000-0003-3563-0697 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Jing Yang Jing Yang orcid.org/0000-0002-7208-1474 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Xiaolin Zhang Xiaolin Zhang Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Ziwei Yang Ziwei Yang Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Yang Li Yang Li Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Fan Wang Fan Wang Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Xiaojuan Li Corresponding Author Xiaojuan Li [email protected] Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Ersheng Kuang Corresponding Author Ersheng Kuang [email protected] orcid.org/0000-0002-4976-3311 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Ministry of Education, Guangzhou, Guangdong, China Search for more papers by this author Xubing Long Xubing Long orcid.org/0000-0003-3563-0697 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Jing Yang Jing Yang orcid.org/0000-0002-7208-1474 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Xiaolin Zhang Xiaolin Zhang Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Ziwei Yang Ziwei Yang Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Yang Li Yang Li Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Fan Wang Fan Wang Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Xiaojuan Li Corresponding Author Xiaojuan Li [email protected] Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Search for more papers by this author Ersheng Kuang Corresponding Author Ersheng Kuang [email protected] orcid.org/0000-0002-4976-3311 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Ministry of Education, Guangzhou, Guangdong, China Search for more papers by this author Author Information Xubing Long1, Jing Yang1, Xiaolin Zhang1, Ziwei Yang1, Yang Li1, Fan Wang1, Xiaojuan Li *,1 and Ersheng Kuang *,1,2 1Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China 2Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Ministry of Education, Guangzhou, Guangdong, China *Corresponding author. Tel: +86 20 87335346; E-mail: [email protected] *Corresponding author. Tel: +86 20 87335346; E-mail: [email protected] EMBO Reports (2021)22:e50714https://doi.org/10.15252/embr.202050714 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Latent infection with herpesviruses constitutively activates inflammasomes, while lytic replication suppresses their activation through distinct mechanisms. However, how Epstein–Barr virus (EBV) lytic replication inhibits the activation of inflammasomes remains unknown. Here, we reveal that the EBV immediate-early protein BRLF1 inhibits inflammasome activation, and BRLF1 deficiency significantly increases the activation of inflammasomes and pyroptosis during early lytic lifecycle. BRLF1 interacts with RNA polymerase III subunits to suppress immunostimulatory small RNA transcription, RIG-I inflammasome activation, and antiviral responses. Consequently, BRLF1-deficient EBV primary infection induces robust T-cell and NK cell activation and killing through IL-1β and IL-18. A BRLF1-derived peptide that inhibits inflammasome activation is sufficient to suppress T-cell and NK cell responses during BRLF1-deficient EBV primary infection in lymphocytes. These results reveal a novel mechanism involved in the evasion of inflammasome activation and antiviral responses during EBV early lytic infection and provide a promising approach for the manipulation of inflammasomes against infection of oncogenic herpesviruses. Synopsis The Epstein-Barr virus immediate-early protein BRLF1 interacts with RNA polymerase III subunits and inhibits RIG-I-inflammasome activation. Primary infection with BRLF1-deficient EBV induces T cell and NK cell activation and virus killing. BRLF1 inhibits inflammasome activation and pyroptosis during the EBV lytic lifecycle. BRLF1 interacts with RNA-POL III to suppress immunogenic small RNA transcription and 5ʹ-ppp-RNA production. BRLF1 inhibits RNA polymerase III-mediated RIG-I inflammasome activation and antiviral responses. BRLF1-deficient EBV activates T cells and NK cells through inflammasome-dependent IL-1β and IL-18 secretion. Introduction Epstein–Barr virus (EBV) is a lymphotropic oncogenic herpesvirus and acts as a causative agent of several lymphoproliferative diseases and malignancies in humans (Hislop, 2015; Taylor et al, 2015). It primarily infects B cells and epithelial cells and also infects T and NK cells at low frequencies with a default latent lifecycle. In the presence of certain stimuli, latent EBV infection is reactivated to lytic replication, which leads to the expression of whole panel of viral genes and eventually produces infectious progeny virion particles (McKenzie & El-Guindy, 2015). During EBV latent infection, the latent gene products activate multiple signaling pathways and induce the expression of a series of inflammatory factors to immortalize infected cells and produce the inflammation and senescence-associated secretory phenotype (SASP); however, these phenomena are altered during the switch from the latent to the lytic lifecycle by immediate-early proteins, such as BZLF1, to promote the optional lytic replication and attenuate SASP and the paracrine senescence of uninfected cells (Li et al, 2015; Long et al, 2016). Alternatively, the inflammasomes are constitutively activated through recognition by IFI16 of the viral DNA genome and recognition by RIG-I of viral small RNA EBERs (Samanta et al, 2006; Ansari et al, 2013). However, whether and how inflammasomes are regulated during the EBV lytic lifecycle remain unknown. Pattern recognition receptors (PRRs) sense endogenous or pathogenic DNA, RNA, or other products and intracellular damage signaling to induce immune responses and inflammasome activation (Brennan & Bowie, 2010; Hayward et al, 2018). Several PRRs have been characterized in organisms as simple as yeast and as complex as mammals, and inflammasome-related PRRs include the DNA sensors AIM2 and IFI16, the RNA sensor RIG-I and NOD-like receptors (NLRs) that recognize microbial or stress-related molecules. Inflammasomes are multiple protein complexes that function in caspase-1 activation and the cleavage of IL-1β, IL-18, and other pro-inflammatory factors. After their formation and activation are triggered by PRRs, inflammasome-dependent cytokines are processed and released to the extracellular compartment to induce sterile and inflammatory responses; therefore, inflammasomes play important roles in immune responses, inflammation, and the pathogenesis of infectious and inflammatory diseases and many malignant tumors (Patel et al, 2017; Karki & Kanneganti, 2019). Infection with herpesviruses is recognized by multiple sensors (Brennan & Bowie, 2010); IFI16 and cGAS recognize viral DNAs (Kerur et al, 2011; Ansari et al, 2013; Diner et al, 2016) and RIG-I recognizes viral small RNAs or specific RNA fragments (Zhao et al, 2018). In addition, RNA polymerase III also acts as a DNA sensor to activate RIG-I signaling through synthesizing 5ʹ-pppRNA (Ablasser et al, 2009; Chiu et al, 2009), and it is also responsible for the transcription of viral small RNAs, such as HSV-1 LAT and EBV EBERs. Moreover, 5ʹ-pppRNA induces inflammasome activation through RIG-I, which can bind to ASC and activate caspase-1-dependent inflammasomes (Poeck et al, 2010). Consequently, inflammasomes are constitutively activated during the latent infection of herpesviruses through IFI16, AIM2, or RIG-I-dependent pathways (Lupfer et al, 2015). These sensors activate both antiviral immune responses and inflammatory responses to inhibit viral lytic replication and establish latent infection. For successful lytic replication, herpesviruses employ diverse strategies to escape from antiviral responses through the inhibition of interferon-related responses and inflammatory responses and the attenuation of the activation and maturation of lymphocytes (Coscoy, 2007; Means et al, 2007; Biolatti et al, 2018). Similarly, they suppress the activation of inflammasomes and inflammasome-related responses; for example, HSV-1 VP22 and KSHV ORF63 inhibit AIM2 and NLRP1/3 inflammasome activation, respectively, to promote HSV-1 and KSHV lytic replication (Gregory et al, 2011; Maruzuru et al, 2018). Similar to infection with other herpesviruses, EBV latent infection induces inflammasome activation through the recognition of viral DNAs by IFI16 and the recognition of viral small RNA EBERs by RIG-I (Chen et al, 2012; Ansari et al, 2013; Torii et al, 2017). Although an EBV miRNA can inhibit NLRP3 expression and consequent NLRP3 inflammasome activation (Haneklaus et al, 2012), the mechanism underlying the inhibition of inflammasome activation and inflammasome-dependent responses during the EBV lytic lifecycle remains unknown. Here, we reveal that the EBV immediate-early protein BRLF1 inhibits RIG-I inflammasome activation and antiviral responses via RNA polymerase III during the early stages of primary infection and reactivation and then evades antiviral responses of T and NK cells through inflammasome-dependent factors. Results BRLF1 suppresses the activation of inflammasomes during the early stages of herpesvirus infection To investigate how EBV lytic replication inhibits the activation of inflammasomes, the systemic screening of an EBV lytic ORF-expressing library was performed using a Gaussia luciferase-based reporter of inflammasome activation, in which pro-IL-1β with a 31 aa N-terminal truncation was fused with Gaussia luciferase (Fig EV1A). It is well-known that HSV-1 infection activates several kinds of inflammasomes (Johnson et al, 2013; Coulon et al, 2019); as such it was used as activator of inflammasomes in our studies. Following inflammasome activation stimulated by HSV-1 infection (MOI = 1), we found that the lytic genes BHRF1 and BoRF2 significantly activate inflammasomes, whereas the lytic genes BRLF1 and BCRF2 significantly inhibit inflammasomes, and the immediate-early gene BRLF1 exhibited the most significant inhibition (Fig EV1B). To confirm that BRLF1 inhibits inflammasome activation, BRLF1 was introduced into THP-1 cells and Ramos cells using lentivirus-based transduction, after which inflammasomes were activated by HSV-1 infection. The levels of cleaved caspase-1 (p20), mature IL-1β, and mature IL-18 were increased by HSV infection in both cell lysates and supernatants, and all three were inhibited by BRLF1 expression in both cell lines (Figs 1A and EV1C). The secretion of IL-1β and IL-18 was analyzed by enzyme-linked immunosorbent assay (ELISA), both were similarly decreased by BRLF1 expression (Fig 1B and C). Click here to expand this figure. Figure EV1. EBV ORF-expressing library screening reveals BRLF1 as an inhibitor of inflammasomes The diagram showing the Gaussia luciferase-based reporter of inflammasome activation. Pro-IL-1β with a 31 aa N-terminal truncation was fused with Gaussia luciferase and expressed with the CMV promoter and was named pro-IL-1β-DN reporter. The pro-IL-1β-DN reporter was transfected into A549 cells with empty vector or EBV lytic ORF-expressing plasmids. Cells were infected with HSV-1 (MOI = 1) for 12 h, and then, the supernatants were collected and measured using a reagent for Renilla luciferase activity. The values are shown as the mean ± standard deviation of triplicate analyses from three independent experiments. *P < 0.01. Tukey's multiple comparison test. Ramos cells were transduced with BRLF1-expressing or empty lentivirus for 24 h and then were left uninfected or infected with HSV-1 (MOI = 1). Twelve hours after HSV-1 infection, the levels of pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18, and mature IL-18 in supernatants and whole-cell lysates were detected by Western blotting as indicated. The release of mature IL-1β and IL-18 from Ramos cells was measured by ELISA. Results are presented as the mean ± SD, n = 3 biological replicates, *P < 0.05, **P < 0.01, Tukey's multiple comparison test. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. BRLF1 inhibited inflammasome activation induced by herpesviruses A. THP-1 cells were transiently infected with BRLF1-expressing or empty control lentivirus. Twenty-four hours later, the control or BRLF1-expressing THP-1 cells were primed with 40 ng/ml TPA overnight and then were either uninfected or infected with HSV-1 (MOI = 1) in serum-free medium for 12 h. The supernatants and whole-cell lysates were collected and analyzed by Western blotting to detect pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18, and mature IL-18 as indicated. B, C. The release of mature IL-1β (B) and IL-18 (C) from THP-1 cells was measured by enzyme-linked immunosorbent assay (ELISA) following HSV-1 infection. Results are presented as the mean ± SD, n = 3 biological replicates, *P < 0.05, **P < 0.01, Tukey's multiple comparison test. D–F. Ramos cells were infected with wild-type EBV-WT- or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI = 100). After being transduced with empty control or BZLF1-expressing lentivirus, the supernatants and cell pellets of EBV-WT- or EBV-ΔBRLF1-harboring Ramos cells were collected after 12 h in serum-free culture, and then, the levels of EBNA1, BRLF1, BZLF1, pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18, mature IL-18, GSDMD, and cleaved N-GSDMD in the supernatants and whole-cell lysates were analyzed as indicated (D). The release of mature IL-1β (E) and IL-18 (F) from Ramos cells was measured by enzyme-linked immunosorbent assay (ELISA). Results are presented as the mean ± SD, n = 3 biological replicates, *P < 0.05, **P < 0.01, Tukey's multiple comparison test. G. After being transduced with control or BZLF1-expressing lentivirus for 48 h, EBV-WT- and EBV-ΔBRLF1-infected Ramos cells were stained with PI and measured using fluorescent flow cytometry. Representative images of pyroptotic cells are shown, and the percentages of pyroptotic cells were calculated in duplicate. Source data are available online for this figure. Source Data for Figure 1 [embr202050714-sup-0008-SDataFig1.pdf] Download figure Download PowerPoint To further investigate whether BRLF1 inhibits inflammasomes during the EBV lytic lifecycle, primary EBV infection was established in Ramos cells with wild-type EBV-WT- or BRLF1-deficient EBV-ΔBRLF1 virus. After the cells were transduced with control or BZLF1-expressing lentivirus, the whole-cell lysates and supernatants were analyzed for inflammasome activation. In the BZLF1-transduced cells, the levels of cleaved caspase-1 (p20), mature IL-1β, and mature IL-18 were increased in both cell lysates and supernatants from EBV-ΔBRLF1-infected cells compared with those from EBV-WT cells (Fig 1D), and the secretion of both IL-1β and IL-18 was similarly increased in the EBV-ΔBRLF1-infected cells compared with the EBV-WT-infected cells (Fig 1E and F). The cleavage of the pyroptotic modulator GSDMD by caspase-1 was elevated in EBV-ΔBRLF1-infected cells as well (Fig 1D). Studies have shown that the cleaved GSDMD isoform forms permeable pores in plasma membrane in cells undergoing pyroptosis, and staining with propidium iodide (PI) in living cells can detect cell pyroptosis (Wree et al, 2014; Gu et al, 2019). EBV-ΔBRLF1-infected cells exhibited an increased percentage of pyroptosis compared with wild-type cells during the lytic stage via PI staining and flow cytometry analysis (Fig 1G). All these results confirm that BRLF1 inhibits the activation of inflammasomes during both HSV-1 and EBV lytic infection. To map the key BRLF1 region that is responsible for the inhibition of inflammasome activation, a series of BRLF1 mutants were constructed. Several key functional domains present in the BRLF1 protein, including the dimerization domain (1–232 aa), the DNA-binding domain (1–280 aa), the nuclear localization sequence (NLS) (410–413 aa), and the transcriptional activation domain (TA domain) (415–605 aa), were subject to mutation (Appendix Fig S1A). The results of the mutagenesis indicated that neither the dimerization domain nor the DNA-binding domain of BRLF1 exhibited an inhibitory effect. However, the NLS and the TA domain region in BRLF1 were required for inhibition. The requirement of NLS indicates that BRLF1 nuclear localization is important for its inhibition, which plays an important role in BRLF1 function by either direct DNA binding or other indirect mechanisms mediated by cellular components or transcriptional factors (Hsu et al, 2005). We then constructed a series of mutants with deletions and mutations in the TA domain to map the specific functional sites (Appendix Fig S1A–C). Deletion of the NLS or the 577–578 region or the L578A mutation abolished the inhibitory effects (Appendix Fig S1C and Fig EV2C). Interestingly, fusion of an 11 aa-fragment from the BRLF1 572–582 region with the NLS was sufficient to inhibit inflammasome activation induced by HSV-1 infection (Appendix Fig S1D and Fig EV2C). Therefore, a TAT-driven cell-permeable peptide was chemically synthesized, which was named TAT-N572 (Fig EV2B). This peptide was able to inhibit the activation of caspase-1 and the cleavage of pro-IL-1β in both HSV-1-infected THP-1 cells and EBV-positive p3HR-1 lymphoma cells in a dose-dependent manner (Fig EV2D and E). These results suggested that a short region in BRLF1 and its functional peptide were sufficient for inhibiting inflammasome activation induced by herpesviruses. Click here to expand this figure. Figure EV2. Mapping of the domain of BRLF1 involved in the inhibition of inflammasomes A. The diagram of BRLF1 functional domains and sites. B. The sequences of the TAT-fused Flag and BRLF1 NLS-fused 572–582 peptides, named TAT-Flag and TAT-N572, respectively. C. Flag-tagged BRLF1 wild type, ΔNLS, Δ577-579, L578A, and NLS-fused 572–582 constructs were transfected into THP-1 cells, which were then infected with HSV-1 (MOI = 1). Twelve hours post-infection, the levels of cleaved caspase-1 (p20) and mature IL-1β in the supernatants were detected. D, E. TAT-fused cell-permeable Flag peptide or BRLF1 N572 peptide were added to p3HR-1 EBV-positive cells for 12 h (D) or added to THP-1 cells that were immediately infected with HSV-1 (MOI = 1) for 12 h (E). The levels of pro-caspase-1, cleaved caspase-1, pro-IL-1β, and mature IL-1β in the whole-cell lysates were detected. Source data are available online for this figure. Download figure Download PowerPoint BRLF1 interacts with RNA polymerase III subunits to downregulate small RNA transcription and 5ʹ-pppRNA production To reveal the mechanism underlying the inhibition of inflammasome activation by BRLF1, GST-tagged BRLF1 or a control vector was transfected into A549 cells, which was followed by HSV-1 stimulation, and then, the BRLF1-binding proteins were isolated by affinity purification with GST-agarose beads (Fig 2A) and sequenced by mass spectrometry. Several proteins, including hsp90, S100A7, RAC1, POLR3F, and POLR3G, were shown to bind to BRLF1; however, only POLR3F and POLR3G knockdown attenuated the inhibition of inflammasome activation in the presence of BRLF1 expression (Fig EV3A). When POLR3F and POLR3G expression were depleted by shRNAs in EBV-ΔBRLF1-infected HNE1 cells and HSV-1-infected A549 cells, BRLF1 no longer exhibited the inhibition of the cleavage of caspase-1 or IL-1β (Fig 2B), indicating that RNA polymerase III subunits play an essential role in the BRLF1-mediated inhibition of inflammasome activation. When GFP-tagged BRLF1 or BRLF1 L578A was coexpressed with GST-tagged POLR3F or POLR3G in HEK293T cells, immunoprecipitation assays showed that BRLF1 interacted with both POLR3F and POLR3G, while BRLF1 L578A did not interact with either, which is consistent with the loss of inflammasome inhibition (Fig 2C).Further, GFP-BRLF1 or GFP-BRLF1 L578A was transfected in A549 cells followed by HSV-1 infection or in HNE-1-EBV-ΔBRLF1-infected cells, after which cells were collected and lysed to detect the interaction of BRLF1 with endogenous sensors of inflammasomes. The immunoprecipitation assays showed that BRLF1 interacted with endogenous POLR3F but not with AIM2, IFI16, RIG-I, or NLRP3, while BRLF1 L578A did not bind to any (Fig 2D). Moreover, immunoprecipitation assays with cell extracts undergoing EBV lytic replication showed that virus-expressed BRLF1 interacts with endogenous POLR3F (Fig 2E). Predicting ligand–receptor interactions in depth provides a basis and direct understanding of the binding structure. We predicted the 3D structure of BRLF1, the BRLF1 TA domain, and the peptide NLS572-582 in I-TASSER (Yang et al, 2015); the results showed that four helixes of the BRLF1 TA domain were appropriately arranged in parallel within the predicted structure and that the peptide fragment in BRLF1 protruded from the BRLF1 molecule as a helix (Fig 2F, Datasets EV1–EV3). We further analyzed the docking modes of this peptide with RNA Polymerase III open complex (PDB ID 6F40) (Vorländer et al, 2018) based on the CDOCKER analysis. Interestingly, the NLS572-582 peptide or the peptide-containing helix was found to bind with POLR3 in the DNA-binding pocket, likely to interfere with DNA binding and influence the transcription function of POLR3 (Fig 2F, Datasets EV4 and EV5). Figure 2. BRLF1 interacted with RNA polymerase III subunits GST-tagged BRLF1-expressing plasmid or control vector was transfected into A549 cells. After HSV-1 infection (MOI = 1) for 12 h, the cells were collected, and the whole-cell extracts were subjected to immunoprecipitation with GST-affinity beads. The immunoprecipitated complexes were then separated by SDS-PAGE, and the image obtained from silver staining is shown. Scramble (sc) or shRNAs against POLR3F and POLR3G with empty control vector (ctr) or BRLF1-expressing plasmid were transfected into EBV-ΔBRLF1-harboring HNE1 cells and A549 cells, followed by HSV-1 infection. Thirty-six hours after the transfection of the HNE1 cells and 12 h after HSV-1 infection (MOI = 1) of the A549 cells, the cell pellets were collected and then the cleavage of caspase-1 and IL-1β were measured as indicated. The release of mature IL-1β and IL-18 from HNE-1 and A549 cells was measured by enzyme-linked immunosorbent assay (ELISA) after ultrafiltration and concentration with Amicon® Ultra-0.5 centrifugal filter devices. Results are presented as the mean ± SD, n = 3 biological replicates, *P < 0.05, Tukey's multiple comparison test. GFP-tagged BRLF1 wild type, BRLF1 L578A, or empty vector was transfected into 293T cells with GST-tagged POLR3F or POLR3G expressing plasmids for 48 h. Cells were collected, and immunoprecipitation with GST-affinity beads was performed, and then, whole-cell lysates and immunoprecipitated complexes were analyzed as indicated. GFP-tagged empty vector or GFP-BRLF1- or GFP-BRLF1 L578A-expressing plasmids were transfected into A549 cells, after which the cells were then infected with HSV-1, or transfected into HNE-1-EBV-ΔBRLF1 cells. The cells were then collected and lysed. Immunoprecipitation with GST-affinity beads was subsequently performed, and then, whole-cell lysates and immunoprecipitated complexes were analyzed as indicated. P3HR-1 cells were induced by TPA plus NaB into lytic infection, and the cells were then collected. Immunoprecipitation with anti-mouse IgG or BRLF1 antibody was performed, after which whole-cell lysates and immunoprecipitated complexes were analyzed as indicated. 3D structure of BRLF1, the BRLF1 TA domain, and the BRLF1 NLS572-582 peptide were predicted using I-TASSER. Receptor-ligand interaction mode of the BRLF1 TA domain and the BRLF1 NLS572-582 peptide with RNA Polymerase III open complex (PDB ID 6F40) was conducted using CDOCKER. The yellow sequence in top figure indicates the TA domain in BRLF1, and the red sequence represents the NLS572-582 peptide. The yellow sequence in the middle figure represents the BRLF1 TA domain, and the red helix in middle and bottom figures represents the DNA double helix. Source data are available online for this figure. Source Data for Figure 2 [embr202050714-sup-0009-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Supplementary data for Fig 3 The pro-IL-1β-DN reporter was transfected into A549 cells along with BRLF1 or empty vector in the presence of scramble or shRNAs against Hsp90, S100A7, RAC1, POLR3F, POLR3G, or POLR3F plus POLR3G. Twelve hours after HSV-1 infection (MOI = 1), the cell media were collected, and the Gaussia luciferase activity in the supernatants was measured. Left results are presented as the mean ± SD, n = 3 biological replicates, *P < 0.05, Tukey's multiple comparison test. The depletion of HSP90, S100A7, RAC1, and POLR3G was detected via real-time PCR, and the relative mRNA levels are shown. Right results are presented as the mean ± SD, n = 3 biological replicates, *P < 0.05, Student's t-test. The depletion of POLR3F was detected by Western blotting analysis, and the results of which are shown. Supplementary data for Fig 3B. Mean mRNA expression normalized to the housekeeping gene GAPDH. Results are presented as the mean ± SD, n = 3 biological replicates. Supplementary data for Fig 3C. Mean mRNA expression normalized to the housekeeping gene GAPDH. Results are presented as the mean ± SD, n = 3 biological replicates. EBV-ΔBRLF1-harboring HNE1 cells were transfected with control vector or BRLF1 in the absence or presence of shRNAs against the RNA polymerase subunits POLR3G plus POLR3F for 48 h. The total RNAs were extracted and subjected to real-time PCR analysis. Results are presented as the mean ± SD, n = 3 biological replicates, *P < 0.05. **P < 0.01, Tukey's multiple comparison test. The depletion of POLR3F was detected by Western blotting analysis, and the results of which are shown. A549 cells were transfected with control vector or BRLF1 along with scramble or shRNAs against POLR3G plus POLR3F for 36 h. After the cells were infected with HSV-1 (MOI = 1) for 12 h, the total RNAs were extracted and subjected to real-time PCR analysis. The depletion of POLR3F by shRNA is shown as indicated. Results are presented as the mean ± SD, n = 3 biological replicates, *P < 0.05. **P < 0.01, Tukey's multiple comparison test. Source data are available online for this figure. Download figure Download PowerPoint RNA polymerase III is responsible for small RNA transcription; therefore, a RNA-seq analysis was performed in Ramos cells infected with EBV-WT or EBV-ΔBRLF1 virus at a high titer (MOI = 100). After classification of all kinds of RNAs, POLR3-dependent RNA transcripts were selected and quantitated. We found that BRLF1 expression during EBV-WT primary infection down-regulated many POLR3-dependent small RNA transcripts, while the BRLF1-deficient EBV primary infection barely exhibited the differences, compared with that in uninfected cells. When the level of RNA expression in EBV-WT and EBV-ΔBRLF1 primary infection was compared, many POLR3-dependent small RNAs were specifically down-regulated for more than 5- to 10-fold or greater by BRLF1 expression, including RN7SL1, 7SK, SNORD14E, SNORA53, SNORA3B, SNORA47, and U6 (Fig 3A). We also detected the level of cellular and viral small RNA transcription in EBV- and HSV-1-infected cells. In EBV-ΔBRLF1-infected HNE1 cells, BRLF1 expression inhibited both EBER1 and EBER2 expression regardless of the presence of latent or lytic infection, while the BRLF
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