Immune evasion strategy involving propionylation by the KSHV interferon regulatory factor 1 (vIRF1)

Post-translational modifications (PTMs) are essential for host antiviral immune response and viral immune evasion. Among a set of novel acylations, lysine propionylation (Kpr) has been detected in both histone and non-histone proteins. However, whether protein propionylation occurs in any viral proteins and whether such modifications regulate viral immune evasion remain elusive. Here, we show that Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded viral interferon regulatory factor 1 (vIRF1) can be propionylated in lysine residues, which is required for effective inhibition of IFN-β production and antiviral signaling. Mechanistically, vIRF1 promotes its own propionylation by blocking SIRT6’s interaction with ubiquitin-specific peptidase 10 (USP10) leading to its degradation via a ubiquitin-proteasome pathway. Furthermore, vIRF1 propionylation is required for its function to block IRF3-CBP/p300 recruitment and repress the STING DNA sensing pathway. A SIRT6-specific activator, UBCS039, rescues propionylated vIRF1-mediated repression of IFN-β signaling. These results reveal a novel mechanism of viral evasion of innate immunity through propionylation of a viral protein. The findings suggest that enzymes involved in viral propionylation could be potential targets for preventing viral infections.


Introduction
Post-translational modifications (PTMs) were discovered over 50 years ago, as a regulatory mechanism in which a protein is covalently linked to new functional groups, such as phosphate, methyl, and acetyl groups [1].The modifications of positively charged amino acids, especially lysine, serve to regulate the activity, stability, and folding of proteins involved in a variety of cellular processes, such as metabolism, cell cycle, signal transduction and immune response [2][3][4][5][6].Over the past decade, acetylation, ubiquitination and methylation are the best characterized PTMs on lysine residues.Diverse types of lysine acylation have been elucidated according to the differences in hydrocarbon chain length, hydrophobicity and charge, including propionylation (Kpr), butyrylation (Kbu), crotonylation (Kcr), hydroxyisobutyrylation (Khib), β-hydroxybutyrylation (Kbhb), malonylation (Kmal), succinylation (Ksu), and lactylation (Kla) [7].The canonical lysine acetylation is characterized by a dynamic and reversible process modulated by specific enzymes, either "writers" or "erasers" that add or remove the acetyl group.However, it is not yet clear whether the atypical lysine PTMs in proteins are catalyzed by enzymes, and what are the "writers" and "erasers"?Moreover, the functional roles of these novel lysine PTMs have been minimally characterized.
To initiate infection, viruses consume host cellular resources to replicate, which may also involve cellular lysine PTMs.The PTMs of host proteins activate innate immunity to control viral replication; meanwhile, the PTMs of viral proteins endow them the ability to evade innate immunity through regulating the compartmentalization, trafficking, and physical interaction of key molecules involved in immunological processes.For instance, viral interferon regulatory factor 1 (vIRF1) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with ubiquitin-specific protease 7 (USP7) to repress the p53 enzymatic activities, thus inhibiting p53-mediated antiviral responses [8].However, whether viruses, such as KSHV, can evade host immune response through novel lysine PTMs remains unknown.
KSHV, also known as human herpesvirus 8 (HHV-8), is a large double stranded DNA (dsDNA) virus initially detected in a Kaposi's sarcoma (KS) lesion from an AIDS patient in 1994 [9].In addition to KS, KSHV is also associated with primary effusion lymphoma (PEL), a subset of multicentric Castleman's disease (MCD), and KSHV-associated inflammatory cytokine syndrome (KICS) [10,11,12].Like other herpesviruses, KSHV establishes a persistent infection within the host through two distinct phases, including a latent phase and a productive lytic replication phase.During the latent phase, the viral genome is maintained as episome expressing only a minimal number of viral genes, which enables the virus to persist and evade host immunity.During the lytic phase, most viral genes are expressed and the virus utilizes multiple immunomodulatory strategies to sustain replication, including expression of viral homologs of cellular interferon (IFN) regulatory factors (vIRFs).
Containing an N-terminal DNA-binding domain (DBD) and a C-terminal IRF interaction domain (IAD), vIRF1 is the most studied of the four KSHV vIRFs [13].It shares 26.6% and 26.2% of protein homologs with human IRF3 and IRF7, respectively [13].However, unlike its cellular homologs, vIRF1 cannot bind to DNA directly, but still exhibits inhibitory effects on

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IFN-mediated signaling by interacting with cellular proteins [14,15].It is generally agreed that vIRF1 interacts with IRF3 to disrupt the formation of IRF3-CBP/p300 complexes, resulting in the suppression of the early response of IFN to virus infection [16].The DNA sensing via the cGMP-AMP synthase (cGAS) and stimulator of IFN-dependent genes (STING) pathway is a powerful innate immune response to DNA viruses.vIRF1 represses the cGAS-STING pathway at multiple nodes to inhibit IFN-β production [17].However, whether the novel lysine PTMs are involved in vIRF1-related host IFN response has not been verified.
In this study, we showed that vIRF1 underwent lysine propionylation (Kpr) at Lys406 and Lys442 to downregulate IFN-β production in response to virus infection.Among the sirtuins family responsible for removing acyl-lysine modifications, SIRT6 played a counteractive role in vIRF1 propionylation, and its association with ubiquitin-specific peptidase 10 (USP10) could be inhibited by vIRF1 by targeting it for degradation.Mechanically, SIRT6-mediated vIRF1 propionylation inhibited IFN-related innate immunity by blocking IRF3-CBP/p300 recruitment, as well as the STING DNA sensing pathway.Here we propose a novel mechanism by which a viral homolog of cellular IRF hijacks the cellular lysine propionylation system to facilitate immune evasion.This study provides insights into the immune evasion strategies of KSHV.

KSHV vIRF1 is propionylated at Lys406 and Lys442 to repress antiviral response
To investigate the post-translational modifications (PTMs) of viral protein, immunoprecipitation (IP) assay was performed to analyze lysine acetylation (Kac), propionylation (Kpr), butyrylation (Kbu), crotonylation (Kcr), hydroxyisobutyrylation (Khib), β-hydroxybutyrylation (Kbhb), lactylation (Kla), malonylation (Kmal) and succinylation (Ksu) on KSHV vIRF1 with a various of pan-acyl-lysine antibodies.We found that vIRF1 was modified by both acetylation and propionylation but not other acyl-lysine modifications in HEK293T cells (Fig 1A).Since KS tumor cells express endothelial cell markers, the same experiments were performed in endothelial cell lines EA.hy926 and TIVE.We observed both acetylation and propionylation of vIRF1 in these cells (Fig 1B), implying that the PTMs pattern of vIRF1 is independent of cell type.To examine whether the above findings could be confirmed in the context of viral infection, we induced a KSHV infected cell line iSLK-RGB with doxycycline to trigger the expression of KSHV lytic genes, including vIRF1.Immunoprecipitation assay (IP) demonstrated the presence of both propionylation and acetylation modifications on endogenous vIRF1 after KSHV reactivation (Fig 1C).
To determine which lysine residue mediates vIRF1 acetylation and propionylation, we transduced vIRF1-expressing plasmid into HEK293T cells, and then isolated vIRF1 by immunoprecipitation for LC-MS/MS analysis to map sites of lysine acetylation and propionylation.Consistent with the results of IP analysis (Fig 1A -1C), we identified 12 lysine propionylation sites and 2 acetylation sites in vIRF1 (Fig 1D).Noticeably, most predicted propionyl-lysines of vIRF1 were located in the functional domains.
To confirm the results from mass spectrometry, all lysine residues within the N-terminal DNA-binding domain (DBD) and the C-terminal IRF interaction domain (IAD) of vIRF1 were mutated to arginine, named as MD1 and MD2, respectively, to mimic its positive charge state in vivo (Fig 1E).Mutations in the IAD (MD2), rather than the DBD (MD1), completely abolished vIRF1 propionylation; however, mutations at lysine in both domains did not affect the acetylation level of vIRF1 (Fig 1F).Previous studies have shown that the primary and most important function of vIRF1 is blocking IFN-β production in the antiviral response [16,17].To illustrate whether KSHV evasion of immune response depends on vIRF1 propionylation, HEK293T cells expressing wild type (WT), DBD mutant (MD1) or IAD mutant (MD2) of vIRF1 were infected with herpes simplex virus type 1 (HSV-1) or treated with a synthetic analog of viral double stranded RNA (Poly I:C).RT-qPCR showed that ectopic expression of vIRF1 inhibited the mRNA level of IFN-β.However, lysine mutations in the IAD (MD2), but not the DBD (MD1) abolished the vIRF1 inhibitory effect (Fig 1G and 1H).Similarly, vIRF1 overexpression suppressed the IFN-β promoter activity during HSV-1 or vesicular stomatitis virus (VSV) infection in a luciferase reporter assay, and this inhibitory effect was abolished following mutations of lysine to arginine in the IAD (MD2), but not the DBD (MD1) (Fig 1I and 1J).Consistently, mutations of lysine in IAD resulted in a higher level of IFN-β protein upon the infection by HSV-1 or VSV (Fig 1K and 1L).
Previous studies have shown that ubiquitin-specific peptidase 10 (USP10) interacts with SIRT6 and suppresses its ubiquitination and degradation [21,22].We therefore investigated whether vIRF1 mediated SIRT6 degradation was involved with USP10.USP10 overexpression increased SIRT6 protein expression (S5  and 4O).Collectively, these results suggest that by blocking the interaction between USP10 and SIRT6, vIRF1 promotes SIRT6 degradation via an ubiquitin-proteasome pathway resulting in self-propionylation and inhibition of IFN-β production.

Propionylated vIRF1 represses IFN antiviral response by blocking IRF3-CBP/p300 recruitment and the STING DNA sensing pathway
A previous study has shown that vIRF1 interferes with IFN response and IRF3-mediated transactivation by competing for CBP/p300 co-activator recruitment [16].Therefore, we determined whether SIRT6-mediated vIRF1 propionylation is essential for IRF3-CBP/p300 complex formation in the antiviral activities of IFN.Mutation of either Lys406 or Lys442 immunoprecipitation.The immuno-isolated proteins were analyzed by Western blot using anti-propionyllysine (Kpr) antibody.(C).HEK293T cells were transduced with lentiviral vIRF1 (vIRF1-Flag) or its control (pHAGE), and then subjected to anti-Flag immunoprecipitation.The precipitated proteins were analyzed by Western blot using anti-SIRT6 antibody.(D).EA.hy926 cells transduced with lentiviral vIRF1-Flag and SIRT6-Myc (vIRF1-Flag + SIRT6-Myc) were employed to detect the expression of vIRF1 and SIRT6 by immunofluorescence staining.(E).EA.hy926 cells transduced with lentiviral vIRF1-Flag and SIRT6-Myc (vIRF1-Flag + SIRT6-Myc) were further treated with MG132 (5 μM) for 24 h, and then were employed to examine the colocalization of vIRF1 and SIRT6 by immunofluorescence staining.(F).HEK293T cells transduced with lentiviral vIRF1 (vIRF1-Flag) or its control (pHAGE) were infected with lentiviral SIRT6 (SIRT6-Myc) or its control (pCDH), and then subjected to anti-Flag immunoprecipitation.The immuno-isolated proteins were analyzed by Western blot using anti-propionyllysine (Kpr) and anti-acetyllysine (Kac) antibodies, respectively.(G).iSLK-RGB cells transduced with lentiviral SIRT6 (SIRT6-Myc) or its control (pCDH) and induced with Doxycycline (Doxy) for 48 h were subjected to the anti-vIRF1 immunoprecipitation, and the precipitated proteins were examined by anti-propionyllysine (Kpr) and anti-acetyllysine (Kac) antibodies, respectively.(H-I).vIRF1-expressing HEK293T cells were transduced with lentiviral SIRT6 (SIRT6) or its control (pCDH), and further infected with HSV-1 for 16 h (H) or transfected with Poly I:C for 8 h (I) before IFN-β mRNA levels were measured by RT-qPCR.***, P < 0.001 by Student's t test.(J-K).vIRF1-expressing HEK293T cells were transduced with lentiviral SIRT6 (SIRT6) or its control (pCDH), and further co-transfected with IFN-β promoter luciferase reporter plasmid (IFN-β pro) or the control (pGL3-basic) for 24 h.The indicated cells were further infected with HSV-1 (J) or VSV (K) for 16  abolished the interaction between vIRF1 and IRF3 (Fig 5A and 5B).To further examine whether vIRF1 propionylation at Lys406 and Lys442 is linked to vIRF1 inhibition of the formation of IRF3-CBP/p300 complexes, Myc-tagged IRF3 and HA-tagged p300 or CBP were transiently co-transfected into HEK293T cells with the wild type or mutant vIRF1.As expected, wild type vIRF1 interacted with CBP/p300 co-activators to block its association with IRF3; however, mutation of any lysine in IAD (Fig 5C and 5E), particularly at Lys406 or Lys442, abolished the vIRF1 inhibitory effect (Fig 5D and 5F).Next, we examined the role of SIRT6mediated vIRF1 propionylation in this process.Overexpression of SIRT6 significantly increased the amount of IRF3 immunoprecipitated p300 or CBP (Fig 5G and 5H).
In addition to blocking IRF-3 recruitment of CBP/p300 co-activator, vIRF1 can also inhibit IFN-β activation through alternative mechanisms, such as by interacting with STING to suppress the phosphorylation of IRF3 and TBK1 in response to KSHV reactivation [17].Thus, we examined the effect of vIRF1 propionylation on vIRF1 and STING interaction.Compared with wild type vIRF1, mutation of Lys406 or Lys442 alone, or all lysines in IAD, abolished vIRF1 binding to STING (Fig 6A and 6B).To determine the role of vIRF1 propionylation in the inhibition of STING-dependent IFN-β response, Western blot was performed to evaluate the phosphorylation and activation of TBK1 and IRF3 in EA.hy926 cells overexpressing wild type and mutant vIRF1.While wild type vIRF1 inhibited the phosphorylation of TBK1 and IRF3, mutation of Lys406 or Lys442 alone, or all lysines in IAD reversed this effect (Fig 6C -6F).Moreover, overexpression of SIRT6 reversed vIRF1 inhibition of phosphorylation of IRF3 and TBK1 in response to exogenous DNA or HSV-1 infection (Fig 6G and 6H).
Together these results indicate that vIRF1 propionylation inhibits the antiviral response of IFN by blocking IRF3-CBP/p300 recruitment and the STING DNA sensing pathway.

SIRT6 activator UBCS039 promotes vIRF1 depropionylation to assist antiviral innate immunity
We further explored SIRT6-mediated vIRF1 propionylation as potential target for inhibiting KSHV evasion of host immunity.UBCS039 is a selective activator of SIRT6.Treatment with UBCS039 in both iSLK-RGB and THP-1 cells not only enhanced SIRT6 expression but also depropionylated vIRF1 (Fig 7A and 7B).Furthermore, UBCS039 effectively increased the level of IFN-β transcript in response to virus infection (Fig 7C and 7D).UBCS039 activation of SIRT6 increased IRF3 recruitment of CBP/p300 co-activator that was blocked by propionylated vIRF1 (Fig 7E and 7F).Meanwhile, UBCS039 restored the levels of phosphorylated TBK1 and IRF3 suppressed by propionylated vIRF1 (Fig 7G).These results collectively indicate that HEK293T cells transduced by lentiviral vIRF1 (vIRF1-Flag) or its control (pHAGE) were treated with CHX (10 μg/mL) for 0, 8, 16, 24 h.The protein expression levels of SIRT6 in the indicated cells were detected by Western blot to monitor its stability.(E).Results were quantified in (D).***, P < 0.001 by Student's t test.(F).HEK293T cells transduced by lentiviral vIRF1 (vIRF1-Flag) or its control (pHAGE) were treated with MG132 (MG132; 5 μM) or its control (DMSO) for 24 h.The expression levels of SIRT6 in the indicated cells were examined by Western blot to verify its degradation pathway.(G).Results were quantified in (F).***, P < 0.001 by Student's t test.(H).HEK293T cells treated as in (F) were transfected with the HA-Ub and SIRT6-Myc constructs, and then subjected to anti-Myc immunoprecipitation for detection of SIRT6 ubiquitination.(I).HEK293T cells with HA-Ub and SIRT6-Myc transfection were treated with MG132 (5 μM) for 24 h, and then infected with KSHV (KSHV) or its control (PBS) for another 24 h.The indicated cells were subjected to anti-Myc immunoprecipitation for detection of SIRT6 ubiquitination.(J).HEK293T cells transduced by lentiviral vIRF1 (vIRF1-Flag) or its control (pHAGE) were transfected with the SIRT6 plasmid (SIRT6-Myc) or its control (pCDH), and treated with MG132 (5 μM) for 24 h.The indicated cells were then subjected to anti-Myc immunoprecipitation and analyzed by Western blot using anti-USP10 antibody.(K).HEK293T cells infected with KSHV (KSHV) or its control (PBS) were transfected with the SIRT6 plasmid (SIRT6-Myc) or its control (pCDH), and treated with MG132 (5 μM) for 24 h.The indicated cells were then subjected to anti-Myc immunoprecipitation and analyzed by Western blot using anti-USP10 antibody.(L).HEK293T cells with co-transfection of HA-Ub and SIRT6-Myc plasmids were transfected with USP10 plasmid (USP10-Flag) or its control (pCDH), and then treated with MG132 (5 μM) for 24 h.The indicated cells were subjected to anti-Myc immunoprecipitation for detection of SIRT6 ubiquitination.(M).HEK293T cells transduced by lentiviral vIRF1 (vIRF1-Flag) or its control (pHAGE) were transfected with the USP10 plasmid (USP10-HA) or its control (pCDH).Cells were subjected to anti-Flag immunoprecipitation and analyzed by Western blot using anti-propionyllysine (Kpr) antibodies.(N-O).HEK293T cells treated as in (M) were infected with HSV-1 (N) or VSV (O) for 16 h before IFN-β mRNA levels were measured by RT-qPCR.***, P < 0.001 by Student's t test.

Discussion
Similar to phosphorylation, methylation and ubiquitination play critical roles in various biological processes, including immune defense against viruses.New advancements in mass spectrometry and biochemistry technologies have led to the discovery of many types of lysine acylation.Among them, lysine propionylation, known as covalent binding of a propionyl group (CH 3 -CH 2 -CO-) to lysine residues in proteins, was initially reported in histones, and later characterized as a transcriptionally active PTM in an in vitro system [23,24].In 2009, Cheng et al. identified the first three non-histone protein substrates of lysine propionylation in eukaryotic cells, including p53, p300, and CBP [25].However, whether propionylation exists in viral proteins is yet to be explored.Our study is the first to show that propionylation occurs in a viral protein, and this modification functionally regulates viral immune evasion.
Like other lysine acylations, propionylation is post-translational, reversible, and enzymatically regulated by the same set of acetyltransferases and deacylases.In eukaryotic cells, p300 and CBP, two previously known histone acetyltransferases (HATs), can catalyze propionyl transfer both in vitro and in vivo [23,25], and propionylation of CoA synthetase (propionyl-CoA synthetase) of Salmonella is catalyzed by the acetyltransferase Pat [26].Interestingly, lysine deacylase SIRT1 has depropionylase activity in eukaryotic cells [25], and sirtuin-like deacetylase CobB regulates the propionylation of CoA synthetase in Salmonella [26], suggesting that sirtuins might function to remove propionyl groups.Therefore, we focused on the sirtuin family to screen the depropionylases for vIRF1.Noticeably, SIRT6 was shown to interact with vIRF1.A previous study showed the association between SIRT6 and KSHV DNA, which inhibited viral reactivation [27].Here, we found that SIRT6 decreased the propionylation level of vIRF1, suggesting the potential enzymatic activity of SIRT6 in lysine propionylation in vivo.Therefore, vIRF1 may promote the degradation of depropionylase SIRT6 to prevent its inhibition of KSHV reactivation and immune escape.
As a highly conserved NAD + -dependent protein deacetylase, SIRT6 facilitates the removal of acyl groups from the ε-amino group of lysines [28].Both activators and inhibitors have HEK293T cells with IRF3 overexpression (IRF3-Myc) were co-transfected with the wild type vIRF1 (vIRF1-WT-Flag), IAD-mutated vIRF1 (vIRF1-MD2-Flag) or its control (pHAGE), along with the p300 plasmid (p300-HA) or its control (pcDNA3.1).Cells were subjected to the anti-HA immunoprecipitation and analyzed by Western blot using anti-Myc antibody.The relative intensities of the bands were quantified and normalized to HAtagged p300.The relative values of proteins in the p300-HA plus pHAGE group were set as "1" for comparison and labeled under the protein bands.(D).HEK293T cells with IRF3 overexpression (IRF3-Myc) were co-transfected with the wild type vIRF1 (vIRF1-WT-Flag), the mutant vIRF1 (vIRF1-K406R-Flag, vIRF1-K442R-Flag, vIRF1-MD2-Flag) or its control (pHAGE), along with the p300 plasmid (p300-HA) or its control (pcDNA3.1).Cells were subjected to the anti-HA immunoprecipitation and analyzed by Western blot using anti-Myc antibody.The relative intensities of the bands were quantified as in (C).(E).HEK293T cells with IRF3 overexpression (IRF3-Myc) were co-transfected with the wild type vIRF1 (vIRF1-WT-Flag), IAD-mutated vIRF1 (vIRF1-MD2-Flag) or its control (pHAGE), along with the CBP plasmid (CBP-HA) or its control (pcDNA3.1).Cells were subjected to the anti-HA immunoprecipitation and analyzed by Western blot using anti-Myc antibody.The relative intensities of the bands were quantified and normalized to HAtagged CBP.The relative values of proteins in the CBP-HA plus pHAGE group were set as "1" for comparison and labeled under the protein bands.(F).HEK293T cells with IRF3 overexpression (IRF3-Myc) were co-transfected with the wild type vIRF1 (vIRF1-WT-Flag), the mutant vIRF1 (vIRF1-K406R-Flag, vIRF1-K442R-Flag, vIRF1-MD2-Flag) or its control (pHAGE), along with the CBP plasmid (CBP-HA) or its control (pcDNA3.1).Cells were subjected to the anti-HA immunoprecipitation and analyzed by Western blot using anti-Myc antibody.The relative intensities of the bands were quantified as in (E).(G).HEK293T cells with IRF3 overexpression (IRF3-Myc) were co-transfected with the vIRF1 plasmid (vIRF1-WT-Flag) or its control (pHAGE), the p300 plasmid (p300-HA) or its control (pcDNA3.1),along with the SIRT6 plasmid (SIRT6-Myc) or its control (pCDH).Cells were subjected to the anti-HA immunoprecipitation and analyzed by Western blot using anti-Myc antibody.The relative intensities of the bands were quantified and normalized to HAtagged p300.The relative values of proteins in the p300-HA + pHAGE + pCDH group were set as "1" for comparison and labeled under the protein bands.(H).HEK293T cells with IRF3 overexpression (IRF3-Myc) were co-transfected with the vIRF1 plasmid (vIRF1-WT-Flag) or its control (pHAGE), the CBP plasmid (CBP-HA) or its control (pcDNA3.1),along with the SIRT6 plasmid (SIRT6-Myc) or its control (pCDH).Cells were subjected to the anti-HA immunoprecipitation and analyzed by Western blot using anti-Myc antibody.The relative intensities of the bands were quantified and normalized to HAtagged CBP.The relative values of proteins in the CBP-HA + pHAGE + pCDH group were set as "1" for comparison and labeled under the protein bands.https://doi.org/10.1371/journal.ppat.1011324.g005been developed for SIRT6 given its multiple roles of in human diseases.UBCS039, a newly synthesized pyrrolo[1,2-a] quinoxaline derivative, is the first synthetic activator and specific for SIRT6 deacetylase activity [29], which has been reported to trigger SIRT6-dependent autophagy in different types of human cancer cell lines [30], and to activate SIRT6 to shift the macrophages from M1 to M2 [31].Interestingly, UBCS039 has been used to uncover the function of SIRT6 in the a mouse model of thioacetamide (TAA)-induced acute liver failure (ALF) [32].Our study has shown that UBCS039 can effectively inhibit vIRF1 propionylation and impair vIRF1 suppression of IFN-β response.
Viral infection triggers multifaceted antiviral responses in the host, with the IFN system as the most potent.Viruses have co-evolved strategies to undermine host innate responses.KSHV evades innate immunity through encoding the viral homologues of IFN regulatory factors [14,15].KSHV vIRFs have low amino acid homology with cellular IRFs and distinct spatial conformation.Similar to cellular IRFs, vIRF1 contains an N-terminal DBD and a C-terminal IAD [33].However, since its DBD lacks three of five conserved tryptophan residues that are essential for DNA binding, vIRF1 loses its DNA-binding ability [14].Instead, vIRF1 counters host innate immunity via direct interaction with cellular proteins [16,17,[33][34][35].Previous reports have shown that the N-terminal of vIRF1 is mainly responsible for its association with the constitutively active form of IRF3 [16], while multiple domains of vIRF1 and STING interact with each other [17].In our study, mutations of all lysine residues in the IAD or a mutation at a critical propionylation site can dramatically block vIRF1-mediated recruitment of CBP/ p300 coactivator by IRF3, as well as the binding of vIRF1 to STING, thereby relieving vIRF1 inhibition of the interferon signaling pathway.Two reasons may explain the difference between our results and those of previous studies.First, vIRF1 binding sites to IRF3 were identified based on the constitutively active form of IRF3 [IRF3(5D)], rather than the wide type form, by replacing the serine and threonine residues in the C-terminal domain of IRF3 with the phosphomimetic aspartic acid [36].Thus, it cannot be ruled out that there is association between vIRF1 C-terminal domain and wild type IRF3.Second, the biological activities of proteins may be due to their three-dimensional (3D) structures and folding; therefore, the modification of amino acid residues may endow proteins with different structures and functions [37,38].Similarly, the mutational perturbation of propionylated lysine residues in vIRF1 IAD domain may influence its structural properties, such as neighboring residues, secondary structure and surrounding hydrophobicity, which in turn affects subsequent formation of proteinprotein complexes based on the residues at binding sites.
In the present study, although we characterized a novel acylation modification in a viral protein by combining mass spectrometry and lysine mutation analysis, the results obtained by the two methods were not exactly the same.For instance, Lys442 in vIRF1 has been fully proved to exhibit the functional role in vIRF1 propionylation and immune escape by lysine mutation analysis.However, we did not identify the propionylation Lys442 polypeptide in the mass spectrometry analysis.We speculated that the peptide was not protected from trypsin digestion, which resulted in its destruction.Moreover, besides propionylation, we also found an increased vIRF1 acetylation level.However, the lysine-to-arginine mutation in the DBD or IAD had no effect on vIRF1 acetylation.Using mass spectrometry, we only detected 2 potential acetylation sites (Fig 1D), in which the Lys2 located outside the functional domains showed a EA.hy926 cells with the wild type vIRF1 (vIRF1-WT-Flag), the mutant vIRF1 (vIRF1-K406R-Flag, vIRF1-K442R-Flag, vIRF1-MD2-Flag), or its control (pHAGE) overexpression were transfected with ISD90 (E) or infected with HSV-1 (F).The phosphorylated TBK1 (p-TBK1), TBK1 (TBK1), phosphorylated IRF3 (p-IRF3) and IRF3 (IRF3) were examined by Western blot.(G-H).EA.hy926 cells transduced with lentiviral vIRF1 (vIRF1-Flag) or its control (pHAGE) were infected with lentiviral SIRT6 (SIRT6-Myc) or its control (pCDH), and further transfected with ISD90 (G) or infected with HSV-1 (H).The phosphorylated TBK1 (p-TBK1), TBK1 (TBK1), phosphorylated IRF3 (p-IRF3) and IRF3 (IRF3) were measured by Western blot.https://doi.org/10.1371/journal.ppat.1011324.g006

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KSHV vIRF1 propionylation inhibits IFN-β signaling higher mass spectrometry score than that of Lys195.It may explain why the lysine mutation in vIRF1 functional domains alone did not influence its overall acetylation level.As the first newly discovered lysine acylation, propionylation is also structurally and functionally similar to acetylation, but the propionyl group is slightly larger than the acetyl group and may be functionally different.Several studies have demonstrated that the lysine acetylation and propionylation pathways share many substrates and regulatory enzymes.Indeed, we and others have discovered that several lysine-acetylation-regulating enzymes, such as SIRT6, SIRT1, p300 and CBP, also act on lysine propionylation [23,25].How such modification achieves specificity in regulating different patterns of acylation modifiers remains unknown.The distinction of acetylation and propionylation at a particular lysine residue regulated by the same enzyme is also unclear.Finally, what is the functional difference between lysine acetylation and propionylation at the same substrate?As a relatively new identified modification, the understanding of propionylation is still limited by technologies and methods.Recent works have demonstrated that, acetylated and propionylated lysines, despite their similar structures, may lead to different protein-protein interactions and functions [20,39].
The innate immune response is the first line of defense against viral infections [40].Viral DNAs and RNAs are sensed by cGAS and mitochondrial antiviral-signaling protein (MAVS) in the cytosol, respectively, and then signal via TBK1 to activate IRF3 [41,42].Activated IRF3 enters the nucleus and binds to CBP/p300 co-activator, thereby triggering the expression of pro-inflammatory cytokines and type I IFNs.Our study has identified a novel mechanism of viral evasion of innate immunity through propionylating a viral protein.KSHV vIRF1 disrupts SIRT6 interaction with USP10 causing its degradation, hence stabilizing vIRF1 propionylation at K406 and K442, which is necessary for inhibiting IFN-β production by blocking IRF3-CBP/ p300 recruitment and repressing the STING DNA sensing pathway (Fig 7H).Finally, we have found that an activator of SIRT6 UBCS039 is effective in inhibiting vIRF1 propionylation and enhancement of IFN-β signaling, which could potentially be explored for inhibiting KSHV infection and treatment of KSHV-related diseases.

Cell culture and reagents
HEK293T cells and telomerase-immortalized human umbilical vein endothelial (TIVE) cells were maintained as previously described [43,44].The human umbilical vein endothelial cell line EA.hy926 (#CRL-2922) and the human acute monocytic leukemia cell line THP-1

Lentivirus, virus and infection
The packaging plasmid psPAX2 and the envelope plasmid pMD2.G were co-transfected with the lentivirus plasmids into HEK293T cells as previously described [46].Lentivirus was collected to infect cells, and the infection efficiency was monitored by fluorescence microscopic examination.Herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV), both of which were kindly provided by Drs.Junjie Zhang and Ke Lan from Wuhan University, respectively, were collected from supernatants of infected Vero cells at the appearance of cytopathic effect (CPE).The viral titers in the supernatants were determined by standard plaque assay.HSV-1 and VSV were diluted in DMEM at multiplicity of infection (MOI) of 10 PFU/ mL and collected at the indicated times post infection.
Production of KSHV was performed according to the previous study [47].Briefly, the stable iSLK-BAC16 cells were induced with doxycycline (1 μg/mL) for 48 h, and then the maintaining medium was replaced for another 2 or 3 days.KSHV particles were pelleted of the cell PLOS PATHOGENS supernatant through 20% sucrose cushion at 24 000 rpm for 3 h (4˚C), and re-suspended in a desired volume.

Co-immunoprecipitation (Co-IP) and mass spectrometry (MS)
Co-IP assay was performed as previously described [43].Briefly, the IP lysis were centrifuged and incubated with 10 μL of anti-tagged immunomagnetic beads for overnight at 4˚C.The beads were washed and eluted to collect the immunoprecipitated proteins for Western blot or MS analysis.For Western blot, the IPKine HRP Goat anti-mouse or anti-rabbit IgG LCS (Abbkine Scientific Co., Ltd, China) reacted with kappa light chains on IgG were utilized to avoid the detection of the heavy chains of IgG.The MS analysis was performed by Applied Protein Technology (Zhejiang, China).

Western blot and antibodies
Western blot was performed as previously described [48].Specific commercial primary antibodies used in Western blot was shown in S2 Table .Anti-vIRF1 rabbit polyclonal antibody was generated by immunization of rabbits (Abclonal, China).Generally, the recombinant vIRF1 protein (1-223 aa) was obtained though molecular cloning and prokaryotic expression system.Then the New Zealand white rabbits were immunized with the recombinant proteins and adjuvant for at least 4 times.After serum titer detection, the antibodies were collected by affinity or Protein A/G purification.The antibodies were validated by Western blot before use.

RT-qPCR
Total RNA was isolated with TRIzol (Life Technologies, NY, USA), and then reverse transcribed into cDNA by HiScript III RT SuperMix (Vazyme Biotech Co., Ltd.).ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.) on StepOnePlus Real-Time PCR System (Applied Biosystems) was used for RT-qPCR analysis according to the manufacturer's instructions, and the results were further analyzed using the 2 -ΔΔCt method and standardized with GAPDH.The sequences of RT-qPCR primers were listed in S3 Table.

Luciferase reporter assay
The IFN-β promoter reporter plasmid or the pGL3-basic construct was transfected into HEK293T cells, as well as the Renilla vector pRL-TK (Promega) for the transfection efficiency normalization.Relative luciferase activity was measured by the dual-luciferase reporter assay system obtained from Promega (Beijing) Biotech Co., Ltd.

Enzyme-linked immunosorbent assay (ELISA)
The human IFN-β ELISA Kit (RK01630; ABclonal Technology Co.,Ltd., China) was used to detect the IFN-β level in cell culture supernatants.The absorbance was measured using a Microplate plate reader (BioTek, USA) set to 450 nm.

Immunofluorescence assay (IFA)
vIRF1-Flag and SIRT6-Myc were transfected into HEK293T cells for 24 h to detect their colocalization, and the indicated cells were seeded on 12 mm diameter round glass coverslips in 24-well plates for overnight.Then, the cells were fixed with cold acetone for 15 min, permeabilized with 0.2% Triton X-100 for 15 min, blocked with 1% bovine serum albumin for 1 h, and incubated with the indicated primary antibodies and the corresponding secondary antibodies conjugated with Alexa Fluor fluorescent dyes (S2 Table ).4,6 diamidino-2-phenylindole (DAPI; Beyotime, China) were further incubated for 10 min, and images were observed with a confocal microscopy (Carl Zeiss, Freistaat Thu ¨ringen, Germany).

Statistical analysis
Numerical data are expressed as the mean±SD.Two group comparisons were analyzed using two-sided Student's t-test.Differences with a P value of 0.05 or less were considered statistically significant.All the experiments were repeated at least for three times, unless otherwise stated.

Fig 1 .
Fig 1. vIRF1 is propionylated by lysine to repress IFN-β production.(A).HEK293T cells were transduced with lentiviral vIRF1 (vIRF1-Flag) or control lentivirus (pHAGE), and then subjected to anti-Flag immunoprecipitation Fig 1E).Consistent with the results of mass spectrometry (Fig 1D and S1 Fig), mutation of Lys406 in the IAD but not other predicted lysines in vIRF1, to arginine (K406R) decreased the level of vIRF1 propionylation in HEK293T cells (Fig 2A).Surprisingly, mutation of Lys442 to arginine (K442R) also reduced vIRF1 propionylation (Fig 2A) albeit Lys442 propionylation was not observed in the mass spectrum (Figs 1D and