(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence

['Guangwen Wang', 'State Key Laboratory Of Veterinary Biotechnology', 'Harbin Veterinary Research Institute', 'Chinese Academy Of Agricultural Sciences', 'Harbin', 'The People S Republic Of China', 'Yuhui Zhao', 'Yuan Zhou', 'Li Jiang', 'Libin Liang']

Date: 2022-05

Host defense systems employ posttranslational modifications to protect against invading pathogens. Here, we found that protein inhibitor of activated STAT 1 (PIAS1) interacts with the nucleoprotein (NP), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) of influenza A virus (IAV). Lentiviral-mediated stable overexpression of PIAS1 dramatically suppressed the replication of IAV, whereas siRNA knockdown or CRISPR/Cas9 knockout of PIAS1 expression significantly increased virus growth. The expression of PIAS1 was significantly induced upon IAV infection in both cell culture and mice, and PIAS1 was involved in the overall increase in cellular SUMOylation induced by IAV infection. We found that PIAS1 inhibited the activity of the viral RNP complex, whereas the C351S or W372A mutant of PIAS1, which lacks the SUMO E3 ligase activity, lost the ability to suppress the activity of the viral RNP complex. Notably, the SUMO E3 ligase activity of PIAS1 catalyzed robust SUMOylation of PB2, but had no role in PB1 SUMOylation and a minimal role in NP SUMOylation. Moreover, PIAS1-mediated SUMOylation remarkably reduced the stability of IAV PB2. When tested in vivo, we found that the downregulation of Pias1 expression in mice enhanced the growth and virulence of IAV. Together, our findings define PIAS1 as a restriction factor for the replication and pathogenesis of IAV.

SUMOylation appears to be an important posttranslational modification mechanism of proteins, including viral proteins. In the present study, we found that the SUMO E3 ligase PIAS1 interacts with the PB2, PB1, and NP proteins of the RNP complex of IAV. PIAS1 expression was found to suppress the viral RNP complex activity. Mechanistically, the SUMO E3 ligase activity of PIAS1 led to robust SUMOylation of IAV PB2, but had no or a minimal effect on the SUMOylation of PB1 and NP, respectively, and PIAS1-mediated SUMOylation significantly decreased the stability of PB2. The expression of PIAS1 was markedly induced upon IAV infection in cell culture and mice, indicating that PIAS1 is actively involved and biologically important in the inhibition of IAV replication. Of note, the role of Pias1 in restricting the replication and virulence of IAV was directly verified in Pias1 +/- mice. Our findings thus identify a SUMO E3 ligase that interacts with and SUMOylates IAV PB2, thereby leading to reduced virus replication and virulence in vitro and in vivo.

Funding: This work was supported by the National Natural Science Foundation of China (NSFC) [31521005(HC), 31672582(LJ), 31902260(GW)], the National Key Research and Development Program of China [2016YFD0500205(CL)], the Heilongjiang Provincial Science and Technology Department [JQ2019C005(CL)], and the Central Public-Interest Scientific Institution Basal Research Fund [Y2017JC35(GT)]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In the present study, we found that PIAS1 interacts with three viral RNP components of IAV: NP, PB1, and PB2. SiRNA knockdown or CRISPR/Cas9 knockout of PIAS1 expression dramatically increased the replication titer of a range of IAVs, whereas overexpression of PIAS1 inhibited IAV propagation. PIAS1 expression was markedly induced in IAV-infected cells or mice, and the presence of PIAS1 was important for the overall increase in cellular SUMOylation induced by IAV infection. We further demonstrated that PIAS1 expression suppressed the viral RNP activity, which was dependent on the SUMO E3 ligase activity of PIAS1. The SUMO E3 ligase activity of PIAS1 had no effect on the stability of NP and PB1, but led to apparent degradation of PB2. Moreover, downregulation of Pias1 expression in Pias1 +/- mice resulted in enhanced virus replication and virulence. Our findings thus established that PIAS1 is a restriction factor for IAV infection and pathogenesis.

Protein inhibitor of activated STAT 1 (PIAS1) was initially identified as an inhibitor of the STAT1 signaling pathway [ 28 ], and functions as a negative regulator of innate immunity [ 29 , 30 ]. However, many studies have now revealed that PIAS1 is a multi-functional protein that is essential in a variety of biological processes, such as DNA repair [ 31 ], apoptosis [ 32 – 34 ], tissue development [ 35 , 36 ], differentiation [ 37 – 41 ], and tumorigenesis [ 42 – 44 ]. PIAS1 is involved in the posttranslational SUMO modification of numerous target proteins, including many vital transcription factors, such as p53 and Myc [ 43 , 45 , 46 ]. PIAS1 generally relies on its SUMO E3 ligase activity during these different biological processes [ 47 ]; however its interaction with various binding partners could also lead to a change in the localization of a target protein or affect the interaction of the target proteins with other proteins in a SUMOylation-independent manner [ 48 , 49 ]. PIAS1 has also been reported to be involved in the infection course of different viruses, such as Ebola virus (EBOV), Epstein-Barr virus (EBV), Herpes simplex virus 1 (HSV-1), and Vesicular stomatitis virus (VSV) [ 29 , 50 – 53 ].

SUMOylation, the process of conjugating a small ubiquitin-like modifier (SUMO) to target proteins, has been recognized as an important posttranslational modification mechanism. SUMOylation is primarily involved in processes that occur in the nucleus [ 24 , 25 ], and leads to the modification of the localization, activity, stability, and function of target proteins [ 26 ]. In mammals, there are four members of the SUMO protein family: SUMO1, SUMO2, SUMO3, and SUMO4 [ 26 , 27 ]. SUMO1 is a 101-amino-acid protein of approximately 11.6 kDa. SUMO2 and SUMO3 differ from each other by only three N-terminal residues, and together they share only approximately 47% homology with SUMO1. SUMO1, SUMO2, and SUMO3 can covalently attach to the acceptor lysines on target substrates. SUMO4 is very similar to SUMO2/3. However, SUMO4 seems to be blocked in its maturation and is probably non-conjugated to substrates under physiological conditions. The SUMOylation cascade involves the SUMO E1 activating enzyme SAE1/SAE2, SUMO E2 conjugase Ubc9, and a limited number of SUMO E3 ligases.

The genome of IAV comprises eight single-stranded negative-sense RNA segments, encoding ten essential proteins as well as up to eight accessary proteins [ 6 ]. However, compared with its host, the genome and proteome of IAV are too simple to complete virus replication unaided. Therefore, IAV must rely on its host’s cellular machinery and factors to support its life cycle and become pathogenic [ 7 – 11 ]. In turn, the host encodes restriction factors (e.g., IFITM3, LSD1, MOV10, PKP2, PLSCR1, TRIM25, TRIM32, TRIM35, and TUFM) [ 12 – 20 ] to suppress the replication and virulence of IAVs. The transcription and replication of the IAV genome take place in the nucleus of virus-infected cells [ 21 , 22 ], catalyzed by the viral ribonucleoprotein (RNP) complex, which is composed of polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), polymerase acidic protein (PA), and nucleoprotein (NP) [ 23 ]. To fulfill the function of transcribing and replicating the viral genome, the newly synthesized viral polymerases and NP must assemble into a complete complex with the viral RNAs. The central role of the vRNP complex in the virus life cycle makes it a key target of the host defense system.

Influenza A virus (IAV) is a widespread zoonotic pathogen that can infect various host species, including birds, lower mammals, and humans [ 1 , 2 ]. Although global efforts have been made to respond to, prevent, and control the threat posed by IAVs, seasonal influenza epidemics and occasional pandemics continue to challenge public health. In addition, increasing numbers of subtypes of avian influenza viruses, such as H5N1 and H7N9, have acquired the ability to cross the species barrier to infect and kill humans [ 3 – 5 ], raising concerns of the evolution of new pandemic viruses. It is therefore imperative that we understand the mechanisms of viral pathogenesis and host defense in order to develop better countermeasures.

Results

PIAS1 interacts with multiple proteins in the RNP complex of IAV We utilized the same yeast-two-hybrid (Y2H) approach, which has been previously described [19], to identify potential interacting partners of IAV NP. A high frequency clone encoding PIAS1 was obtained as a potential NP-interactor in the screen. The interaction of PIAS1 with NP was confirmed by co-transforming the bait plasmid pGBDT7-NP of A/Anhui/2/2005 (AH05, H5N1) virus and the prey plasmid pGADT7-PIAS1 into the Y2HGold yeast strain and growing the transformant on plates with special medium (S1 Fig). To determine whether NP interacts with PIAS1 in mammalian cells, HEK293T cells were transfected with V5-tagged NP of A/WSN/33 (WSN, H1N1) virus and Myc-tagged PIAS1 constructs, either alone or together. Cell lysates were subject to immunoprecipitation with a mouse anti-V5 monoclonal antibody (mAb), followed by western blotting with a rabbit anti-V5 or anti-Myc polyclonal antibody (pAb). Myc-tagged PIAS1 was co-immunoprecipitated with V5-tagged WSNNP only when they were co-expressed (Fig 1A). Conversely, V5-tagged WSNNP was also co-immunoprecipitated with Myc-tagged PIAS1 when the co-IP experiment was performed with a mouse anti-Myc mAb (Fig 1B). Together, these results indicate that PIAS1 interacts with IAV NP in transiently transfected mammalian cells. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. PIAS1 interacts with multiple proteins in the RNP complex of IAV. (A, B) Interaction of V5-WSNNP and Myc-PIAS1 in HEK293T cells by using a co-IP assay. HEK293T cells were individually transfected or co-transfected with plasmids expressing V5-WSNNP and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Myc mAb (B), and subjected to western blotting with a rabbit anti-V5 pAb and a rabbit anti-Myc pAb for the detection of WSNNP and PIAS1, respectively. (C) Interaction of GST-WSNNP and His-PIAS1 by using a GST pull-down assay. His-tagged PIAS1 was expressed in E. coli BL21 (DE3) and purified by using Ni Sepharose Excel resin, and the GST or GST-NP protein was expressed in HEK293T cells and purified by using Glutathione Sepharose 4 Fast Flow. An equal amount of purified PIAS1 was mixed with the Glutathione Sepharose 4 Fast Flow samples that bind GST or GST-NP. After rocking and washing, the mixed samples were separated by SDS-PAGE and stained with Coomassie blue. (D) Interaction of IAV NP and PIAS1 in virus-infected cells. HEK293T cells were transfected for 24 h to express Myc-PIAS1, and were then infected with WSN (H1N1) virus (MOI = 5). At 30 h p.i., cell lysates were immunoprecipitated with a mouse anti-NP mAb, followed by western blotting with a rabbit anti-NP pAb and a rabbit anti-Myc pAb. (E) Co-localization of IAV NP and PIAS1 in A549 cells infected with WSN (H1N1) virus. A549 cells were infected with WSN (H1N1) virus (MOI = 5). At 2, 4, 6, and 8 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb and a rabbit anti-PIAS1 pAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H+L) (red) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green). The nuclei were stained with DAPI. (F-H) Co-IP assay to examine the interactions between Myc-PIAS1 and PB2, PB1, and PA of WSN (H1N1) virus in HEK293T cells. HEK293T cells were individually transfected or co-transfected with plasmids expressing WSNPB2, WSNPB1, WSNPA, and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-Myc mAb and were subjected to western blotting with a rabbit anti-PB2 pAb (F), a rabbit anti-PB1 pAb (G), a rabbit anti-PA pAb (H), and a rabbit anti-Myc pAb (F-H) for the detection of PB2, PB1, PA, and PIAS1, respectively. https://doi.org/10.1371/journal.ppat.1010446.g001 Next, we performed a GST pull-down experiment to determine whether NP and PIAS1 directly interact with each other. HEK293T cells were transfected individually with constructs expressing GST or GST-WSNNP. At 48 h post-transfection, the expressed GST or GST-WSNNP in the cell lysates were purified with Glutathione Sepharose 4 Fast Flow. His-tagged PIAS1 was expressed in E. coli BL21 (DE3) and purified by using Ni Sepharose Excel resin. An equal amount of purified PIAS1 was mixed with the Glutathione Sepharose 4 Fast Flow samples that bind GST or GST-NP. After rocking and washing, the mixed samples were separated by SDS-PAGE and stained with Coomassie blue. As shown in Fig 1C, purified PIAS1 was only pulled down by GST-WSNNP, and not GST alone, indicating that IAV NP directly binds to PIAS1 in vitro. To further explore the interaction between NP and PIAS1 during IAV infection, HEK293T cells were transfected for 24 h to express Myc-PIAS1, and then subjected to infection with WSN (H1N1) virus at an MOI of 5. At 30 h post-infection (p.i.), the cell lysates were immunoprecipitated with a mouse anti-NP mAb, and the presence of NP and PIAS1 in the immunoprecipitates was revealed by western blotting. We found that IAV NP efficiently interacted with PIAS1 during virus replication (Fig 1D). To reveal the localization of NP and PIAS1 during virus infection, we performed immunostaining and confocal microscopy analysis. A549 cells infected with WSN (H1N1) virus at an MOI of 5 were fixed at 2, 4, 6, or 8 h p.i., followed by immunostaining with anti-NP and anti-PIAS1 antibodies. Confocal microscopy showed that PIAS1 was distributed in both the cytoplasm and nucleus (Fig 1E). Most of the NP was localized in the cytoplasm at 2 h p.i., accumulated in the nucleus at 4 h p.i., was exported to the cytoplasm at 6 h p.i., and gathered around the plasma membrane at 8 h p.i. Of note, PIAS1 partially colocalized with NP during the shuttle of NP between the cytoplasm and nucleus at different stages of virus infection. The above results indicate that PIAS1 interacts with IAV NP in both transfected and infected cells. Given that the RNP complex is a compact functional unit for the replication of IAV [54,55], we next attempted to investigate whether PIAS1 also interacts with other protein components of the RNP complex. To that end, we performed co-IP experiments in transiently transfected HEK293T cells and found that PB2 and PB1, but not PA, were also co-immunoprecipitated with Myc-tagged PIAS1 (Fig 1F–1H). Consistent with these data, we found that PIAS1 was also co-immunoprecipitated with PB2 and PB1, but not PA, during IAV infection (S2 Fig). We further investigated whether PIAS1 interacted with other internal (M1) or surface proteins (HA, NA and M2) of IAV by co-IP experiments, and found that PIAS1 did not interact with these structural components of IAV (S3 Fig). These data indicate that three components of the IAV RNP complex, that is, PB2, PB1, and NP, efficiently interact with cellular PIAS1.

PIAS1 suppresses the replication of IAV in vitro We demonstrated that PIAS1 interacts with PB2, PB1, and NP of the RNP complex of IAV. To determine the role of PIAS1 in the regulation of IAV replication, we first established a lentiviral vector-based A549 cell line stably overexpressing PIAS1 to examine the effect of PIAS1 upregulation on IAV replication. Quantitative reverse transcription PCR (RT-qPCR) and western blotting showed that PIAS1 overexpression produced a 7.5-fold increase in mRNA transcript abundance and a 5.49-fold increase in protein expression compared with the empty lentivirus-transduced control cell line (Fig 2A and 2B). The PIAS1-overexpressing or control A549 cells were infected with WSN (H1N1) virus at an MOI of 0.01, and the culture supernatant was collected at 24 and 48 h p.i. for titration of infectious virus by use of plaque assays. As shown in Fig 2C, stable overexpression of PIAS1 led to a 3.5- and 4-fold decrease in viral titers at 24 and 48 h p.i., respectively. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. PIAS1 restricts IAV replication in vitro. (A, B) Establishment of a lentiviral-mediated PIAS1-overexpressing A549 cell line. The stable expression of PIAS1 was confirmed by quantitative reverse transcription PCR (RT-qPCR) (A) and western blotting with a rabbit anti-PIAS1 pAb (B). ***, P < 0.001. (C) The PIAS1-overexpressing or control A549 cells were infected with WSN (H1N1) virus (MOI = 0.01). Supernatants were collected at the indicated timepoints, and virus titers were determined by means of plaque assays on MDCK cells. **, P < 0.01. (D, E) siRNA knockdown of PIAS1 in A549 cells. A549 cells were transfected with siRNA targeting PIAS1 or with scrambled siRNA for 48 h. Cell lysates were subjected to RT-qPCR (D) or western blotting with a rabbit anti-PIAS1 pAb (E) to confirm the downregulation of PIAS1. ***, P < 0.001. (F) Cell viability of siRNA-treated A549 cells as in (D, E) was determined by using a CellTiter-Glo assay. (G to J) Virus replication in siRNA-treated A549 cells as in (D, E). PIAS1 siRNA- or scrambled siRNA-transfected A549 cells were infected with WSN (H1N1) (MOI = 0.01) (G), AH05 (H5N1) (MOI = 0.1) (H), AH13 (H7N9) (MOI = 0.1) (I), or SH13 (H9N2) (MOI = 0.1) (J) virus. Supernatants were collected at the indicated timepoints, and virus titers were determined by means of plaque assays on MDCK cells. *, P < 0.05, ***, P < 0.001. (K) Knockout of PIAS1 in PIAS1_KO A549 cells was confirmed by western blotting with a rabbit anti-PIAS1 pAb. (L) Cell viability of PIAS1_KO A549 cells was determined by using the CellTiter-Glo assay. (M) Virus replication in PIAS1_KO A549 cells. PIAS1_KO or control A549 cells were infected with WSN (H1N1) (MOI = 0.01) virus. Supernatants were collected at the indicated timepoints, and virus titers were determined by means of plaque assays on MDCK cells. **, P < 0.01, ***, P < 0.001. https://doi.org/10.1371/journal.ppat.1010446.g002 Next, we examined the effect of PIAS1 downregulation on virus replication by using siRNA knockdown. A549 cells treated with specific siRNA targeting PIAS1 or scrambled siRNA were infected with WSN (H1N1) virus at an MOI of 0.01, and the culture supernatant was collected at 24 and 48 h p.i. for virus titration. PIAS1-specific siRNA treatment efficiently reduced the expression of PIAS1 compared with scrambled siRNA treatment (Fig 2D and 2E), and PIAS1 knockdown had no obvious effect on the viability of the siRNA-treated cells (Fig 2F). As shown in Fig 2G, PIAS1 knockdown led to a 10.3- and 11.3-fold increase in WSN (H1N1) virus titer at 24 and 48 h p.i., respectively. Similarly, siRNA knockdown of PIAS1 expression led to 11.9-/8.7-, 4.2-/5.7-, and 7.8-/8.1-fold increases in the growth titers of AH05 (H5N1), A/Anhui/1/2013 (AH13, H7N9), and A/chicken/Shanghai/SC197/2013 (SH13, H9N2) virus at 24/48 h p.i. (Fig 2H–2J), demonstrating that PIAS1 downregulation enhances the replication of a broad range of IAVs. We then generated a PIAS1 knockout (PIAS1_KO) A549 cell line by using the CRISPR/Cas9 system (Fig 2K). Knockout of PIAS1 expression had no major effect on cell viability (Fig 2L). The titers of WSN (H1N1) virus grown on PIAS1_KO A549 cells increased 4.5 and 16.8 fold compared with those of virus grown on control cells at 24 and 48 h p.i., respectively (Fig 2M). To investigate the effect of PIAS1 knockout on viral protein expression, we infected PIAS1_KO A549 cells with WSN (H1N1) virus at an MOI of 5. Confocal microscopy showed during the course of WSN (H1N1) infection, between 2 and 8 h p.i., the expression of viral NP, an indicator of virus replication, was dramatically enhanced in PIAS1_KO A549 cells compared with that in control cells (S4 Fig). These data further confirm that PIAS1 negatively modulates IAV replication. Collectively, these results indicate that PIAS1 functions as a host restriction factor against IAV replication.

IAV infection induces the expression of PIAS1 in vitro and in vivo To investigate whether the dynamics of PIAS1 expression is affected during IAV infection, we infected A549 cells with WSN (H1N1) virus at an MOI of 0.1 and determined the expression of PIAS1 by western blotting at 0, 12, and 24 h p.i. We found that the level of PIAS1 expression gradually increased as the infection progressed (S5A Fig). To investigate whether the increase in PIAS1 expression upon IAV infection is regulated by type I interferon (IFN), A549 cells were treated with or without 25 pg/mL IFN-β for 24 h. We found that the level of PIAS1 expression was unchanged by IFN-β treatment compared with that of control cells (S5B Fig). By contrast, the expression of MX1 and IFITM3, two well-known interferon-stimulated genes, was significantly triggered by IFN-β treatment. These data indicate that the expression of PIAS1 in vitro is induced solely by IAV infection, and is not dependent on IFN-β treatment. To determine whether the increase in PIAS1 expression also occurred in vivo, 6-week-old C57BL/6J mice were infected with 105 PFU of WSN (H1N1) and AH13 (H7N9), 102 PFU of AH05 (H5N1), 106 PFU of SH13 (H9N2) virus, or were mock-infected with PBS. The lungs of the infected mice were collected on day 3 p.i., and lung homogenates were western blotted with an anti-PIAS1 pAb. We found that for the four IAV strains, the level of Pias1 increased by 6.0, 12.6, 6.1, and 15.1 fold, respectively, in the lungs of IAV-infected mice compared with mock-infected mice (S5C Fig). These results demonstrate that the expression of PIAS1 is actively induced upon IAV infection in vitro and in vivo.

PIAS1 is involved in gross SUMOylation during IAV infection IAV infection triggers an increase in the abundance of proteins modified by both SUMO1 and SUMO2/3 [56]. To explore whether PIAS1 is a contributor to the overall increase in SUMOylation during IAV infection, A549 cells treated with specific siRNA targeting PIAS1 or scrambled siRNA were infected with WSN (H1N1) virus at an MOI of 0.01, and then the overall SUMOylation level was assessed at 0, 12, and 24 h p.i. by using an anti-SUMO1 or SUMO2/3 pAb. We found that IAV infection triggered an increase in the abundance of proteins modified by both SUMO1 and SUMO2/3, and that knockdown of PIAS1 expression reduced the overall level of cellular SUMOylation during IAV infection (Fig 3A and 3B). These data indicate that PIAS1 is involved in the gross cellular SUMOylation induced by IAV infection. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. PIAS1 regulates the gross SUMOylation induced by IAV infection. A549 cells treated with specific siRNA targeting PIAS1 or scrambled siRNA were infected with WSN (H1N1) virus at an MOI of 0.01. The overall cellular SUMOylation level was assessed at 0, 12, and 24 h p.i. by western blotting with an anti-SUMO1 (A) or anti-SUMO2/3 (B) pAb. https://doi.org/10.1371/journal.ppat.1010446.g003

PIAS1 inhibits viral RNA transcription and replication Having found that PIAS1 interacts with three components of the viral RNP complex and restricts IAV replication, we hypothesize that PIAS1 protein expression may affect the transcription and replication of the viral genome. To test this hypothesis, we first performed a minigenome assay in HEK293T cells that were treated with PIAS1-specific siRNA or scrambled siRNA for 12 h, followed by transfection with constructs expressing the viral RNP proteins (PB2, PB1, PA, NP) of WSN (H1N1) virus, a firefly luciferase reporter flanked with the packaging signals of the NS segment, and an internal Renilla luciferase control. The viral RNP activity was evaluated by measuring the luciferase activity at 36 h post-transfection. The results revealed a 2.6-fold increase in the viral RNP activity in PIAS1-specific siRNA- versus scrambled siRNA-treated cells (Fig 4A and 4B). To confirm these results, we generated a PIAS1_KO HEK293T cell line by using the CRISPR/Cas9 system (Fig 4C). HEK293T cells and PIAS1_KO HEK293T cells were transfected with the constructs for the minigenome assay, and the viral RNP activity was evaluated by measuring the luciferase activity at 36 h post-transfection. We found that the viral RNP activity increased 2.7-fold in PIAS1_KO versus wild-type HEK293T cells (Fig 4D). Together, these data indicate that the expression of endogenous PIAS1 inhibits the RNP activity of IAV. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. PIAS1 relies on its SUMO E3 ligase activity to suppress IAV transcription and replication. (A) siRNA knockdown of PIAS1 in HEK293T cells. HEK293T cells were transfected with PIAS1 siRNA or scrambled siRNA for 48 h. Knockdown of PIAS1 expression was confirmed by western blotting with a rabbit anti-PIAS1 pAb. (B) Minigenome assay in siRNA-treated HEK293T cells to determine the effect of endogenous PIAS1 on viral RNP activity. HEK293T cells treated with siRNA as in (A) were transfected with plasmids for the expression of four viral RNP proteins (WSNPB2, WSNPB1, WSNPA, and WSNNP), together with pHH21-SC09NS F-Luc and pRL-TK. Thirty-six hours later, a dual-luciferase assay was performed in which the relative firefly luciferase activity was normalized to the internal control, Renilla luciferase activity. ***, P < 0.001. (C) Establishment of a PIAS1_KO HEK293T cell line. The knockout of PIAS1 was confirmed by western blotting with a rabbit anti-PIAS1 pAb. (D) Minigenome assay in PIAS1_KO HEK293T cells to determine the effect of endogenous PIAS1 on viral RNP activity. The minigenome assay was performed in PIAS1_KO HEK293T cells as in (B). ***, P < 0.001. (E) RT-qPCR analysis to determine the effect of PIAS1 knockdown on the transcription and replication of viral RNAs. A549 cells were transfected with siRNA targeting PIAS1 or with scrambled siRNA for 48 h, followed by infection with WSN (H1N1) virus (MOI = 5). Total RNA was extracted at 6 h p.i. by using TRIzol reagent, and the levels of NP-specific vRNA, cRNA, and mRNA were analyzed by RT-qPCR and then normalized to GAPDH mRNA. The values shown are standardized to the corresponding RNA expression level in the scrambled siRNA-treated A549 cells (100%). **, P < 0.01, ***, P < 0.001. (F-I) Minigenome assay in HEK293T cells to examine the effect of exogenously expressed wild-type PIAS1 and PIAS1 mutants on viral RNP activity. HEK293T cells were transfected with plasmids for the expression of four viral RNP proteins and increasing amounts of Myc-PIAS1 or Myc-PIAS1 mutant, together with pHH21-SC09NS F-Luc and pRL-TK. At 36 h post-transfection, a dual-luciferase assay was performed in which the relative firefly luciferase activity was normalized to the Renilla luciferase activity. **, P < 0.01, ***, P < 0.001. (J-M) Minigenome assay in PIAS1_KO HEK293T cells to examine the effect of exogenously expressed wild-type PIAS1 and PIAS1 mutants on viral RNP activity. The minigenome assay was performed in PIAS1_KO HEK293T cells as in (F-I). *, P < 0.05, **, P < 0.01, ***, P < 0.001. https://doi.org/10.1371/journal.ppat.1010446.g004 To further explore which step, transcription or replication, is inhibited by the expression of PIAS1, A549 cells treated with PIAS1-specific siRNA or scrambled siRNA were infected with WSN (H1N1) virus at an MOI of 5, followed by RT-qPCR analysis to measure the levels of the three species of viral RNA at 6 h p.i. The levels of vRNA, cRNA, and mRNA increased by 2.2-, 5.9- and 3.6-fold, respectively, in PIAS1-knockdown cells compared with scrambled siRNA-treated cells (Fig 4E). This result indicates that both transcription and replication of the viral genome are inhibited by the expression of PIAS1 protein.

The SUMO E3 ligase activity of PIAS1 is essential for its inhibitory effect on viral RNP activity To identify the biological property of PIAS1 associated with its inhibitory effect on the RNP activity of IAV, we first assessed the effect of PIAS1 overexpression on viral RNP activity. HEK293T cells were transfected with the constructs for the minigenome assay, together with gradually increasing amounts of the Myc-PIAS1 expression construct. At 36 h post-transfection, the luciferase activity of the cell lysates was measured. We found that the overexpression of PIAS1 reduced the viral RNP activity in a dose-dependent manner (Fig 4F). Ser90 phosphorylation of PIAS1 is essential for PIAS1-mediated repression of inflammatory gene activation [30]. To assess whether the inhibitory effect of PIAS1 on the RNP complex activity of IAV is dependent on Ser90 phosphorylation, HEK293T cells were transfected with the minigenome constructs, along with gradually increasing amounts of the Myc-PIAS1 S90A construct. The S90A PIAS1 mutant inhibited the viral RNP activity to a similar degree as wild-type PIAS1 (Fig 4G), indicating that Ser90 phosphorylation is not essential for PIAS1 to inhibit the RNP complex activity of IAV. PIAS1 functions as a SUMO E3 ligase, whose SP-RING domain interacts with the SUMO E2 conjugase Ubc9 and is essential for SUMOylation reactions [36]. The SUMOylation mutations of C351S or W372A in PIAS1 abolish its SUMOylation activity [30,57]. To determine whether the SUMO E3 ligase activity is essential for PIAS1 to inhibit the RNP complex activity of IAV, we transfected HEK293T cells with the minigenome constructs, along with gradually increasing amounts of the Myc-PIAS1 C351S or W372A construct. We found that compared with wild-type PIAS1, both the C351S and W372A mutants lost the ability to inhibit the RNP complex activity of IAV (Fig 4H and 4I). Instead, the expression of the C351S or W372A mutant led to increases in the RNP activity of IAV. These findings indicate that PIAS1 inhibits the IAV RNP complex activity in a SUMOylation-dependent manner. To validate the role of the SUMO E3 ligase activity of PIAS1 in inhibiting the RNP activity of IAV, we performed the minigenome assay in PIAS1_KO HEK293T cells. We found that the complement of wild-type or the S90A mutant of PIAS1 in the PIAS1_KO cells significantly reduced the viral RNP complex activity compared with the vector control (Fig 4J and 4K). In contrast, the addition of the C351S or W372A mutant of PIAS1 resulted in no inhibitory effect on the viral RNP complex activity (Fig 4L and 4M). These results further confirm that the SUMO E3 ligase activity is essential for PIAS1 to suppress the RNP activity of IAV.

PIAS1-mediated SUMOylation destabilizes IAV PB2 We showed that PIAS1 differentially promoted the SUMOylation of PB2 and NP, but was not involved in the SUMOylation of PB1. To ascertain the effect of PIAS1-mediated SUMOylation on the viral RNP proteins, we determined their stability upon SUMOylation. PB1 was included as a control even though it was not SUMOylated by PIAS1. First, we examined the effect of SUMO modification by SUMO1, SUMO2, or SUMO3 on the stability of PB2. The constructs expressing WSNPB2, Flag-tagged SUMO1, Ubc9-V5, along with or without Myc-PIAS1, were co-transfected into HEK293T cells. Thirty-six hours later, the cells were treated with cycloheximide (CHX) to inhibit protein synthesis, followed by western blotting analysis at the indicated timepoints. We found that the exogenous expression of SUMO1 and Ubc9 affected the stability of PB2, and the further overexpression of PIAS1 led to dramatically reduced stability of PB2 (Fig 7A), indicating that PIAS1-mediated SUMOylation of PB2 by SUMO1 led to degradation of PB2. Similarly, the stability of PB2 was significantly decreased over time when the Flag-tagged SUMO1 was replaced with SUMO2 or SUMO3 in the transfections (Fig 7B and 7C). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 7. IAV PB2, PB1, and NP exhibit diverse stability in the presence of exogenously expressed SUMOylation components. (A-I) Stability of PB2, PB1 and NP in the presence of exogenous SUMOylation components. HEK293T cells were transfected with plasmids for the expression of WSNPB2, Flag-SUMO1/2/3, Ubc9-V5, along with/without Myc-PIAS1 (A-C), or transfected with plasmids for the expression of WSNPB1 (D-F) or WSNNP (G-I) in combination with Flag-SUMO1/2/3, Ubc9-V5, and Myc-PIAS1. At 36 h post-transfection, the cells were treated with CHX. At the indicated timepoints, cell lysates were subjected to western blotting. Data are representative of three independent experiments (A-I). The band intensities of PB2, PB1, and NP, quantified by using ImageJ software, were normalized to GAPDH and are expressed as relative ratios compared with untreated cells at 0 h. https://doi.org/10.1371/journal.ppat.1010446.g007 We then evaluated the effect of PIAS1 expression on the stability of PB1. Consistent with the finding that PIAS1 did not catalyze the SUMOylation of PB1 by SUMO1, SUMO2, or SUMO3, the co-expression of SUMO1, SUMO2, or SUMO3, Ubc9, and PIAS1 produced no visible effect on the stability of PB1 (Fig 7D–7F). Similarly, the co-expression of SUMO1, SUMO2, or SUMO3, Ubc9, and PIAS1 did not affect the stability of NP (Fig 7G–7I), implying that PIAS1-mediated minimal SUMOylation of NP by SUMO1 does not cause NP degradation. To investigate whether SUMOylated PB2 catalyzed by PIAS1 was degraded through the ubiquitin-proteasome pathway, HEK293T cells were co-transfected to express WSNPB2 and exogenous SUMOylation components (Ubc9, PIAS1, and SUMO1, SUMO2, or SUMO3), and were then treated with CHX to inhibit protein synthesis and with the proteasome inhibitor MG132. We found that the co-expression of SUMO1/2/3, Ubc9 and PIAS1 led to clear degradation of PB2, whereas treatment with MG132 prevented PB2 degradation in the presence of overexpressed SUMOylation components (Fig 8A–8C). Consistent with the finding that PIAS1 is able to mediate robust SUMOylation of PB2 by SUMO1/2/3 in the absence of Ubc9 overexpression, the co-expression of only SUMO1/2/3 and PIAS1 led to obvious degradation of PB2, whereas treatment with MG132 subverted the degradation of PB2 (Fig 8D–8F). These results demonstrate that SUMOylated PB2 catalyzed by PIAS1 is degraded through the ubiquitin-proteasome pathway. Furthermore, we found that endogenous SUMO and Ubc9 were sufficient to support PIAS1-mediated degradation of PB2 (Fig 8G), and that the addition of MG132 completely blocked PB2 degradation, causing a similar degradation effect to that observed in the presence of exogenously expressed SUMO and Ubc9. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 8. PIAS1-mediated SUMOylation leads to degradation of PB2 through the ubiquitin-proteasome pathway. (A-C) Stability of PB2 in cells overexpressing SUMO1/2/3, Ubc9, and PIAS1 in the presence or absence of MG132. HEK293T cells were transfected with plasmids for the expression of WSNPB2, Ubc9-V5, Myc-PIAS1, and Flag-SUMO1 (A), Flag-SUMO2 (B), or Flag-SUMO3 (C). At 36 h post-transfection, the cells were treated with CHX or with CHX and MG132. At the indicated timepoints, cell lysates were subjected to western blotting. (D-F) Stability of PB2 in cells overexpressing SUMO1/2/3 and PIAS1 in the presence or absence of MG132. HEK293T cells were transfected with plasmids for the expression of WSNPB2, Myc-PIAS1, and Flag-SUMO1 (D), Flag-SUMO2 (E), or Flag-SUMO3 (F). At 36 h post-transfection, the cells were treated with CHX or with CHX and MG132. At the indicated timepoints, cell lysates were subjected to western blotting. (G) Stability of PB2 in cells overexpressing PIAS1 in the presence or absence of MG132. HEK293T cells were transfected with plasmids for the expression of WSNPB2 and Myc-PIAS1. At 36 h post-transfection, the cells were treated with CHX or with CHX and MG132. At the indicated timepoints, cell lysates were subjected to western blotting. (H, I) Effect of the PIAS1 mutant on the stability of PB2. HEK293T cells were transfected with plasmids for the expression of WSNPB2, Myc-PIAS1, and Myc-PIAS1 W372A (H) or Myc-PIAS1 S90A (I). At 36 h post-transfection, the cells were treated with CHX. At the indicated timepoints, cell lysates were subjected to western blotting. Data are representative of three independent experiments (A-I). The band intensities of PB2, quantified by using ImageJ software, were normalized to GAPDH and are expressed as relative ratios compared with untreated cells at 0 h at the bottom of each panel. https://doi.org/10.1371/journal.ppat.1010446.g008 To confirm that PIAS1-mediated SUMOylation was responsible for the reduced stability of PB2, we did a side-by-side comparison of the effect of wild-type PIAS1 versus the PIAS1 S90A or W372A mutant on the stability of PB2. We found that the presence of the SUMO-ligase defective PIAS1 W372A mutant prevented the reduction in stability of PB2 (Fig 8H). In contrast, the SUMO-ligase active PIAS1 S90A mutant dramatically impaired the stability of PB2, demonstrating that PIAS1 is dependent on its SUMO-ligase activity to reduce the stability of PB2 (Fig 8I). Collectively, these results demonstrate that PIAS1-mediated SUMOylation of PB2 by SUMO1, SUMO2, and SUMO3 dramatically decreases the stability of PB2, but PIAS1-mediated SUMOylation of NP by SUMO1 has no effect on the stability of NP.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010446

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.