PIAS1 Potentiates the Anti-Viral Activity of SAMHD1 Through Sumoylation
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PIAS1 Potentiates the Anti-viral Activity of SAMHD1 through SUMOylation Farjana Saiada Virginia Commonwealth University Kun Zhang Virginia Commonwealth University Renfeng Li ( [email protected] ) Virginia Commonwealth University https://orcid.org/0000-0002-9226-4470 Research Keywords: SAMHD1, PIAS1, restriction factor, Epstein-Barr virus, cytomegalovirus, SUMOylation, herpesvirus, deoxynucleotide triphosphohydrolase, phosphorylation Posted Date: March 17th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-296406/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Cell & Bioscience on July 8th, 2021. See the published version at https://doi.org/10.1186/s13578-021-00636-y. 1 PIAS1 potentiates the anti-viral activity of SAMHD1 through 2 SUMOylation 3 4 5 6 Farjana Saiada1, Kun Zhang1, and Renfeng Li1,2,3,4 7 8 1Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth 9 University, Richmond, Virginia 23298, USA 10 2Department of Microbiology and Immunology, School of Medicine, Virginia Commonwealth 11 University, Richmond, Virginia 23298, USA 12 3Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298, USA 13 14 4Corresponding author: [email protected] (RL) 15 1 16 Abstract 17 Background: Sterile alpha motif and HD domain 1 (SAMHD1) is a host restriction factor that 18 suppresses the infection of a variety of RNA and DNA viruses, including herpesviruses. The 19 anti-viral activity of SAMHD1 is finely tuned by post-translational modifications, including 20 phosphorylation, acetylation and ubiquitination. Our recent studies also demonstrated that the E3 21 SUMO ligase PIAS1 functions as an Epstein-Barr virus (EBV) restriction factor. However, 22 whether SAMHD1 is regulated by PIAS1 to restrict viral infection remains unknown. 23 Results: In this study, we showed that PIAS1 interacts with SAMHD1 and promotes its 24 SUMOylation. We identified three lysine residues (K469, K595 and K622) located on the 25 surface of SAMHD1 as the major SUMOylation sites. We demonstrated that phosphorylation of 26 SAMHD1 slightly promotes its SUMOylation by PIAS1 and SUMOylated SAMHD1 can still be 27 phosphorylated by viral protein kinases. We further demonstrated that SUMOylation-deficient 28 SAMHD1 loses its anti-EBV activity. 29 Conclusion: Our study reveals that PIAS1-mediated SUMOylation of SAMHD1 enhances its 30 anti-viral activity. 31 32 Keywords 33 SAMHD1; PIAS1; restriction factor; Epstein-Barr virus; cytomegalovirus; SUMOylation; 34 herpesvirus; deoxynucleotide triphosphohydrolase; phosphorylation 35 36 Background 37 Restriction factors serve as the first line of defense against viral infection. One recently 38 discovered restriction factor is the Sterile alpha motif and HD domain 1 (SAMHD1) protein, 39 which hydrolyzes deoxyribonucleoside triphosphates (dNTPs) to reduce the cellular dNTP pool 40 required for viral infection and propagation. In addition to limiting the infection of human 41 immunodeficiency virus-1 (HIV-1) (1), SAMHD1 has also been shown to restrict the infection 42 of herpesviruses consisting of Epstein-Barr virus (EBV), human/mouse cytomegalovirus 43 (HCMV/MCMV) and human simplex virus 1 (HSV-1) (2-7), vaccinia virus (2), human T cell 44 leukemia virus type 1 (8), hepatitis B virus (9) and human papillomavirus 16 (10). 45 46 The anti-viral activity of SAMHD1 is counterbalanced by a group of viral proteins, including 47 HIV-2/SIV virion-associated Vpx accessory proteins and the conserved herpesvirus protein 48 kinases. Vpx proteins bind to SAMHD1 and E3 ubiquitin ligase complex to promote SAMHD1 49 ubiquitination and proteasome-dependent degradation (11-13). Phosphorylation of SAMHD1 by 50 the conserved herpesvirus protein kinases (4-7) and cellular kinases CDK1 and CDK2 51 diminishes its anti-viral activity (3, 14-16). Although earlier studies suggested that SAMHD1’s 52 deoxynucleotide triphosphohydrolase (dNTPase) activity is not regulated by phosphorylation, 53 recent studies revealed that phosphorylation suppress the dNTPase activity to elevate the dNTP 54 level for optimal viral DNA synthesis (6, 17-22). SAMHD1 phosphorylation has also been 55 implicated in its re-localization to cytoplasm during viral infection (16). SAMHD1 is acetylated 56 by the acetyltransferase ARD1 to enhance its dNTPase activity (23). 57 2 58 Protein SUMOylation is mediated by a series of enzymes that catalyze the transfer of small 59 ubiquitin-related modifier (SUMO) to a protein substrate. These enzymes include an E1 60 activating enzyme complex SAE1/UBA2, an E2 conjugating enzyme UBC9 and an E3 protein 61 ligase that controls substrate specificity (24). Protein SUMOylation plays important roles in 62 diverse biological processes, including gene transcription, DNA replication, and viral infection 63 (24, 25). With the advances in mass spectrometry, recent studies identified thousands of 64 SUMOylation sites within the human proteome (26-28). 65 66 In contrast to ubiquitination, there are only a few E3 ligases for SUMOylation. The protein 67 inhibitor of STAT (PIAS) family proteins are E3 SUMO ligases with RING domains responsible 68 for the transfer of SUMO proteins from UBC9 to the protein substrates. The PIAS family 69 proteins consist of PIAS1, PIASx/PIAS2, PIAS3 and PIASy/PIAS4 (29). PIAS1 restricts HSV-1 70 infection when ICP0 of HSV-1 is deleted (30). Our group recently identified PIAS1 as an EBV 71 restriction factor, which is cleaved by caspases upon lytic induction (31). PIAS1 is the SUMO 72 E3 ligase for a group of cellular and viral proteins, including p53 (32-35) and EBV immediate- 73 early protein RTA (36). 74 75 In this study, we demonstrated that PIAS family members PIAS1, PIAS2, PIAS3 and PIAS4 76 interact with SAMHD1 and PIAS1 has the capability to trigger SAMHD1 SUMOylation both in 77 vitro and in intact cells. We identified K469, K595 and K622 as the major SUMOylation sites on 78 SAMHD1. We showed that PIAS1 promotes the anti-EBV activity of SAMHD1 in a 79 SUMOylation dependent manner, which expands our understanding of SAMHD1 regulation by 80 post-translational modifications. 81 82 83 Results 84 85 PIAS family proteins interact with SAMHD1 86 87 Our previous studies have demonstrated that both PIAS1 and SAMHD1 restrict EBV lytic 88 replication (6, 31). PIAS family is consist of 4 members with different substrate specificity 89 (Figure 1A). To determine whether PIAS1 and other members of the PIAS family interact with 90 SAMHD1, we co-transfected SAMHD1 with individual PIAS construct into 293T cells and 91 performed co-immunoprecipitation (Co-IP) experiments. We found that SAMHD1 is strongly 92 Co-IPed by PIAS1, PIAS2, PIAS4 (Figure 1B, lanes 1, 2 and 4). The weaker Co-IP signal of 93 SAMHD1 by PIAS3 is possibly due to PIAS3 protein expression level (Figure 1B, lanes 3). 94 Interestingly, we also observed an extra band when SAMHD1 is Co-IPed by PIAS1 (Figure 1B, 95 lanes 1), suggesting a post-translational modification on SAMHD1. 96 97 PIAS1 promotes the SUMOylation of SAMHD1 98 Because PIAS1 could serve as an E3 SUMO ligase, we reasoned that PIAS1 could trigger the 99 SUMOylation of SAMHD1. To test this possibility, we performed in vitro SUMOylation assay 100 with recombinant proteins (Figure 2A). We first performed Western Blot (WB) using anti- 101 SUMO2/3 antibody (Figure 2B). We found that SAMHD1 is SUMOylated when incubated with 102 E1, E2 and wild-type (WT) SUMO2 (Figure 2B, lane 3) but not with SUMO2 mutant (Figure 103 2B, lane 2). We observed that PIAS1 further promotes SAMHD1 poly-SUMOylation with WT 3 104 SUMO2 but not SUMO2 mutant (Figure 2B, lane 4 vs 2). As a positive control, we also found 105 that PIAS1 triggers the SUMOylation of p53 with WT SUMO2 but not SUMO2 mutant (Figure 106 2B, lane 6 vs 5). To further confirm these results, we used anti-SAMHD1 antibody for WB and 107 found that PIAS1 indeed promotes the SUMOylation of both SAMHD1 and p53 (Figure 2C, 108 lanes 4 and 6). 109 110 PIAS1 synergizes with SAMHD1 to restrict EBV replication 111 To determine the regions within PIAS1 responsible for binding to SAMHD1, we co-transfected 112 SAMHD1 with our previously created PIAS1 truncation mutants into 293T cells (Figure 3A and 113 3B). We found that all the PIAS1 truncation mutants (aa 1-433, 101-433, 1-415 and 409-651) all 114 bind to SAMHD1 but only full-length PIAS1 can trigger SAMHD1 modification (Figure 3B, 115 lanes 1-5). PIAS1 (aa 1-205) showed less binding to SAMHD1 possibly due to the reduced 116 expression level (Figure 3B, lane 6). 117 118 To demonstrated whether PIAS1 and SAMHD1 association plays a role in EBV lytic replication, 119 we transfected 293 (EBV+) cells with vectors expressing ZTA (a trigger for EBV lytic 120 reactivation), SAMHD1 and different PIAS1 truncation mutants. As expected, transfection of 121 SAMHD1 only reduced EBV lytic replication (Figure 3C, lane 2 vs 3). Interestingly, we found 122 that co-transfection of WT PIAS1 and SAMHD1 greatly suppresses EBV DNA replication 123 (Figure 3C, lane 3 vs 4). However, PIAS1 truncation mutants or E3 ligase deficient mutant 124 (C351S) failed to synergize with SAMHD1 in blocking viral replication (Figure 3C, lanes 4 vs 125 5-10). Together, these results suggested that PIAS1-mediated SAMHD1 SUMOylation plays a 126 role in limiting EBV replication. 127 128 PIAS1 SUMOylates SAMHD1 at K469, K595, and K622 129 To identify the SUMOylation sites on SAMHD1, we searched the mass spectrometry database 130 on protein SUMOylation and found that three sites (K469, K595, K622) within SAMHD1 131 protein are SUMOylated in Hela cells (26-28). These sites are located within the typical KxE/D 132 motif that is required for SUMOylation (Figure 4A). 133 134 To determine whether these are the SAMHD1 SUMOylation sites, we mutated these sites 135 individually and in combination to generate four SAMHD1 mutants, namely K469R, K595R, 136 K622R and RRR (K469R/K595R/K622R).