The Class IV human deacetylase, HDAC11, exhibits anti‐influenza A virus properties via its involvement in host innate antiviral response
2018; Wiley; Volume: 21; Issue: 4 Linguagem: Inglês
10.1111/cmi.12989
ISSN1462-5822
AutoresAshley Nutsford, Henry Galvin, Farjana Ahmed, Matloob Husain,
Tópico(s)Immune Cell Function and Interaction
ResumoCellular MicrobiologyVolume 21, Issue 4 e12989 RESEARCH ARTICLEFree Access The Class IV human deacetylase, HDAC11, exhibits anti-influenza A virus properties via its involvement in host innate antiviral response Ashley N. Nutsford, Department of Microbiology and Immunology, University of Otago, Dunedin, New ZealandPresent Address: Ashley N. Nutsford, School of Biological Sciences, University of Auckland, Auckland, New Zealand.Search for more papers by this authorHenry D. Galvin, Department of Microbiology and Immunology, University of Otago, Dunedin, New ZealandSearch for more papers by this authorFarjana Ahmed, Department of Microbiology and Immunology, University of Otago, Dunedin, New ZealandSearch for more papers by this authorMatloob Husain, Corresponding Author matloob.husain@otago.ac.nz orcid.org/0000-0002-4413-6220 Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand Correspondence Dr. Matloob Husain, Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand. Email: matloob.husain@otago.ac.nzSearch for more papers by this author Ashley N. Nutsford, Department of Microbiology and Immunology, University of Otago, Dunedin, New ZealandPresent Address: Ashley N. Nutsford, School of Biological Sciences, University of Auckland, Auckland, New Zealand.Search for more papers by this authorHenry D. Galvin, Department of Microbiology and Immunology, University of Otago, Dunedin, New ZealandSearch for more papers by this authorFarjana Ahmed, Department of Microbiology and Immunology, University of Otago, Dunedin, New ZealandSearch for more papers by this authorMatloob Husain, Corresponding Author matloob.husain@otago.ac.nz orcid.org/0000-0002-4413-6220 Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand Correspondence Dr. Matloob Husain, Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand. Email: matloob.husain@otago.ac.nzSearch for more papers by this author First published: 04 December 2018 https://doi.org/10.1111/cmi.12989Citations: 11 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Histone deacetylase 11 (HDAC11) is most recently discovered deacetylase. Here, we demonstrate that human HDAC11 exhibits anti-influenza A virus (IAV) properties. We found that knockdown of HDAC11 expression augments IAV growth kinetics in human lung epithelial cells A549 by up to 1 log. One of the ways HDAC11 exerts its anti-IAV function is by being a part of IAV-induced host antiviral response. We found that the kinetics of both IAV- and interferon-induced innate antiviral response is significantly delayed in HDAC11-depleted cells. Further, in the absence of HDAC11 expression, there was a significant decrease in the expression of interferon-stimulated genes—IFITM3, ISG15, and viperin—previously implicated in anti-IAV function. One of the ways IAV antagonises HDAC11 is by downregulating its expression in host cells. We found that there was up to 93% reduction in HDAC11 transcript levels in A549 cells in response to IAV infection. HDAC11 is the smallest HDAC with majority of its polypeptide assigned to catalytic domain. Evolutionarily, it seems to be the least evolved and most closely related to common ancestral HDAC gene(s). Furthermore, HDAC11 has also been described as a deacylase. Therefore, our findings present exciting prospects for further investigations into significance of HDAC11 in virus infections. 1 INTRODUCTION Influenza virus is an ever-evolving human pathogen, which presents a constant threat to global public and animal health. Influenza virus impacts the global human population mainly in two ways: by causing regular seasonal epidemics alternating in northern and southern hemispheres and intermittent unpredictable pandemics (Webster & Govorkova, 2014). Both these events result in significant morbidity and mortality as well as productivity and economic losses worldwide (Iuliano et al., 2017; Ortiz et al., 2014; Petrie et al., 2016). In addition, zoonotic outbreaks of newly emerged avian influenza A virus (IAV) in humans, which have lately become more frequent, result in an unusually high mortality rate (~35–50%; Husain, 2014), as well as pose the threat of the emergence of a highly virulent pandemic IAV. These influenza virus events are aided by the constant circulation and rapid evolution of influenza viruses in various clinical and reservoir hosts globally (Xue, Moncla, Bedford, & Bloom, 2018). The individual infections and co-infections of each host give rise to genetically diverse populations of influenza viruses due to de novo mutations and genetic reassortment. Some of these influenza variants transmit to other hosts and continue the cycle of evolution. Such evolution of influenza viruses has precluded the development of a universal influenza vaccine and makes the annually formulated influenza vaccines only variably effective (Kim, Webster, & Webby, 2018). Furthermore, this phenomenon also aides the rapid emergence of drug resistance and has caused half of the available anti-influenza virus drugs, that is, adamantanes, to become practically obsolete and the other half, that is, neuraminidase inhibitors, prone to be ineffective over time (Hussain, Galvin, Haw, Nutsford, & Husain, 2017). Therefore, there is an undeniable need to identify the missing links—both host and viral—that are critical for influenza virus multiplication and pathogenesis to develop alternative, effective, and long-lasting anti-influenza virus strategies. One such strategy could be to develop host-directed therapies, particularly the strengthening of host defences, against which influenza virus is potentially less likely to evolve rapidly. We have identified and characterised a role of multiple human histone deacetylases (HDACs) in IAV infection (Husain & Cheung, 2014; Nagesh & Husain, 2016; Nagesh, Hussain, Galvin, & Husain, 2017). The HDACs are a family of enzymes that were originally described to catalyse the deacetylation of acetylated histones (Seto & Yoshida, 2014). However, a variety of non-histone proteins, both cytoplasmic and nuclear, are now known to be HDAC substrates (Seto & Yoshida, 2014). HDACs work in equilibrium with histone acetyl transferases to control the acetylation level of proteins and influence diverse biological processes such as gene expression (Sterner & Berger, 2000), protein trafficking (Li & Yang, 2015), and the innate immune response (Zhou, He, Wang, & Ge, 2017). Consequently, the imbalance in protein acetylation due to aberrant function of HDACs or histone acetyl transferases contributes to multiple human diseases such as cancer (Liu, Li, Wu, & Cho, 2017), neurodegeneration (Cook, Stankowski, Carlomagno, Stetler, & Petrucelli, 2014), and infection (Jeng, Ali, & Ott, 2015; Song & Walley, 2016). So far, 18 HDACs have been identified in human HDAC family and classified into four classes (Seto & Yoshida, 2014). Class I composes of HDAC 1, 2, 3, and 8, and Class II contains HDAC 4, 5, 6, 7, 9, and 10. The Class III HDACs are known as sirtuins (SIRT) with seven members, SIRT 1–7. Finally, Class IV possesses a lone member, HDAC11. We have found that members of both the Class I (HDAC 1 and 2) and Class II (HDAC6) possess anti-IAV properties (Husain & Cheung, 2014; Nagesh et al., 2017; Nagesh & Husain, 2016). Furthermore, all Class III HDACs (SIRT) have been found to exhibit anti-IAV properties too (Koyuncu et al., 2014). However, no such role has been described for HDAC11—the sole member of Class IV. HDAC11 is the smallest and the most recently discovered member of the HDAC family (Gao, Cueto, Asselbergs, & Atadja, 2002). HDAC11 shares very little sequence homology with Class I and II HDACs, as most of the HDAC11 polypeptide is predominantly composed of catalytic domain. However, human HDAC11 possesses all nine active sites, which are conserved in other eukaryotic HDACs and prokaryotic HDAC-like proteins (Gao et al., 2002). On the basis of this, we hypothesised that, like Class I and II HDACs, HDAC 1, 2, and 6, respectively, HDAC11 also possesses anti-IAV properties. Indeed, the data presented here support this hypothesis and demonstrate anti-IAV potential of human HDAC11. 2 RESULTS 2.1 HDAC11 mRNA expression is downregulated in IAV-infected cells Viruses, including IAV, employ multiple strategies to antagonise antiviral host factors and consequently host innate antiviral response in order to efficiently multiply (Duggal & Emerman, 2012). One such strategy is the downregulation of the expression of antiviral host factors by various mechanisms. We have found that IAV downregulates the expression of HDAC1 and HDAC2, both at mRNA and polypeptide level in A549 cells (Nagesh et al., 2017; Nagesh & Husain, 2016). Therefore, to determine the antiviral potential of HDAC11, we first examined the expression of HDAC11 mRNA in A549 cells in response to IAV infection. The A549 cells were infected with influenza virus A/PR/8/34(H1N1) strain (henceforth referred to as PR8) at a multiplicity of infection (MOI) of 3.0. After 0 (the time point following the removal of virus inoculum), 6, 12, and 24 hr of infection, the cells and the culture medium were harvested separately. The cells were processed to measure the mRNA levels of HDAC11 and two reference genes—actin and 18S ribosomal RNA (18SRNA) by quantitative real-time PCR (qPCR). In addition, the mRNA level of interferon (IFN)-stimulated gene, viperin, was measured as a positive control for infection as well as qPCR. The culture medium was titrated on Madin–Darby canine kidney (MDCK) cells to determine the progeny virus titre at each time point. We found that PR8 infection reduced the HDAC11 mRNA level in A549 cells in a time-dependent manner, regardless of the reference gene used for normalisation (Figure 1a). When normalised to actin, compared with 0-hr post-infection, there was a significant 43.1% (P = 0.0001), 77.6% (P = 0.0001), and 87.2% (P = 0.0001) reduction in HDAC11 mRNA level after 6, 12, and 24 hr of infection, respectively (Figure 1a). Similarly, when normalised to 18SRNA, there was a significant 39.4% (P = 0.0002), 87.2% (P = 0.0001), and 90% (P = 0.0001) decrease in HDAC11 mRNA level after 6, 12, and 24 hr of infection, respectively (Figure 1a). Further, consistent with previous findings, where the expression of viperin increased in response to virus infection (Fitzgerald, 2011; Nagesh et al., 2017), there was a significant and time-dependent increase in viperin mRNA levels in response to PR8 infection (Figure 1b). Furthermore, the titre of PR8 progeny was also increased in the culture medium collected after each time point in a time-dependent manner (Figure 1c). Figure 1Open in figure viewerPowerPoint HDAC11 mRNA expression is downregulated in influenza A virus-infected cells. A549 cells were infected with PR8 at a multiplicity of infection (MOI) of 3.0. After 0, 6, 12, and 24 hr of infection, the infected and pairing uninfected cells were harvested and processed, and HDAC11, viperin, actin, and 18SRNA mRNA levels were detected by qPCR. First, the HDAC11 and viperin mRNA levels in uninfected and infected cells at each time points were normalised with corresponding either actin or 18SRNA mRNA levels. Then, the levels of HDAC11 and viperin mRNAs in infected cells were normalised with their levels in pairing uninfected cells. Finally, such normalised level of (a) HDAC11 or (b) viperin mRNA at 0 hr was considered 100% or onefold, respectively, to compare their level at subsequent time points. The virus titre in culture medium collected from infected cells above were measured by (c) microplaque assay. (d and e) A549 cells were infected with PR8 at an MOI of 1.0, 3.0, and 5.0. After 24 hr, the levels of HDAC11, actin, and 18SRNA mRNAs in uninfected and infected cells were detected by qPCR. Then, the HDAC11 mRNA levels in uninfected and infected cells were normalised with corresponding either actin or 18SRNA mRNA levels. (d) Finally, the normalised level of HDAC11 mRNA in uninfected (UNI) sample was considered 100% to compare its level in infected samples (1.0, 3.0, and 5.0). (e) The virus titre in culture medium collected from infected cells above was measured by microplaque assay. (f) A549 cells were infected with uninfected allantoic fluid (AF), live (INF), or UV-irradiated (INFUV) PR8 at an MOI of 3.0. After 24 hr, the HDAC11 and actin mRNA levels were detected by qPCR, and normalised and presented as above. (g and h) A549 cells were infected with CA09 at an MOI of 1.0, 3.0, and 5.0. (g) After 24 hr, the levels of HDAC11 and actin mRNAs were detected by qPCR and normalised and presented as above. (h) The CA09 progeny virus titre in culture medium collected from infected cells above was measured by microplaque assay. Error bar represents means ± standard errors of the means of three biological replicates; asterisks indicate the significant differences in means To determine if IAV had a dose-dependent effect on HDAC11 mRNA expression, A549 cells were infected with PR8 at an MOI of 1.0, 3.0, and 5.0 for 24 hr, and then, HDAC11 mRNA levels were determined by qPCR as above. Indeed, when normalised to actin, compared with uninfected cells, there was a significant dose-dependent 77.9% (P = 0.0001), 88% (P = 0.0001), and 91.3% (P = 0.0001) decrease in HDAC11 mRNA level after infection with an MOI of 1.0, 3.0, and 5.0, respectively (Figure 1d). Likewise, when normalised to 18SRNA, the infection with an MOI of 1.0, 3.0, and 5.0 caused a significant 89.6% (P = 0.0001), 93.4% (P = 0.0001), and 93.8% (P = 0.0001) reduction in HDAC11 mRNA level, respectively (Figure 1d). The productive infection of A549 cells with PR8 was confirmed by detecting the presence of viral progeny in the culture medium (Figure 1e). To ascertain that a productive IAV infection was causing the reduction in HDAC11 mRNA levels, we infected the A549 cells with uninfected allantoic fluid and ultraviolet (UV)-irradiated PR8 inoculum along with live PR8 inoculum for 24 hr and performed the qPCR as above. As shown in Figure 1f, compared with uninfected cells, there was no significant change in HDAC11 mRNA levels in cells treated with uninfected allantoic fluid or UV-irradiated inoculum, but there was a consistent 84.7% (P = 0.0001) decrease in HDAC11 mRNA level in cells treated with live inoculum. Finally, we infected A549 cells with influenza A/California/07/2009(H1N1) strain (henceforth referred to as CA09) at an MOI of 1.0, 3.0, and 5.0 to determine if a recent IAV clinical isolate would have a similar effect on HDAC11 mRNA levels. Like PR8, the 2009 pandemic isolate CA09 also reduced the HDAC11 mRNA levels, albeit less profoundly compared with PR8, in A549 cells in a dose-dependent manner. Compared with uninfected cells, there was a significant 53.8% (P = 0.0001), 66% (P = 0.0001), and 66.9% (P = 0.0001) decrease in HDAC11 mRNA level after infection with CA09 at an MOI of 1.0, 3.0, and 5.0, respectively (Figure 1g). Again, the productive infection of A549 cells with CA09 was confirmed by measuring viral progeny titre in the culture medium (Figure 1h). 2.2 The knockdown of HDAC11 mRNA expression promotes IAV infection Above data demonstrated a profound downregulation in HDAC11 mRNA expression in response to IAV infection. Unfortunately, we could not conclusively determine the same for HDAC11 polypeptide by western blotting (WB) due to non-availability of a reliable HDAC11 antibody. Nevertheless, like HDAC 1 and 2 (Nagesh et al., 2017; Nagesh & Husain, 2016), above data strongly suggested an antiviral role for HDAC11. Therefore, we went ahead and analysed the IAV growth characteristics in HDAC11-depleted cells. We employed RNA interference to deplete the HDAC11 expression in A549 cells. For this, we obtained two predesigned small-interfering RNA oligonucleotides (siRNAs) targeting human HDAC11 and delivered their different quantities—10, 25, 50, and 100 nM along with 50 nM of non-targeting control siRNA to A549 cells. After 72 hr, the cells were processed, and the depletion of HDAC11 mRNA was assessed by qPCR as above. The siRNA-1, which targeted the 3′-untranslated region of HDAC11 mRNA, knocked down the HDAC11 mRNA level by about 88% at 10-nM concentration (Figure 2a). Whereas the same concentration of siRNA-2, which targeted the HDAC11 open reading frame, reduced the level of HDAC11 mRNA by about 66%—this value did not improve considerably upon increasing the siRNA concentration to 100 nM (Figure 2a). Furthermore, at 10-nM concentration, both HDAC11 siRNAs had no considerable effect on the viability of A549 cells (Figure 2b). Therefore, we selected 10-nM concentration for both HDAC11 siRNAs and control siRNA for subsequent experiments. Notwithstanding the comparatively low depletion efficiency of HDAC11 siRNA-2, we included it in further experiments because we believed that it would serve as an additional control if this siRNA had an effect on IAV growth characteristic that was proportional to its depletion efficiency. Figure 2Open in figure viewerPowerPoint The knockdown of HDAC11 mRNA expression promotes influenza A virus infection. (a) A549 cells were transfected with indicated concentrations of HDAC11 (HD11) siRNA-1 and siRNA-2, and 50-nM concentration of non-targeting control (CTRL) siRNA. After 72 hr, the levels of HDAC11 and actin mRNAs were detected by qPCR. Then, the HDAC11 mRNA levels in CTRL siRNA-transfected cells and HD11 siRNA-1- and siRNA-2-transfected cells were normalised with corresponding actin mRNA levels. Finally, the normalised level of HDAC11 mRNA in CTRL siRNA-transfected cells was considered 100% to compare its level in HDAC11 siRNA-1- and siRNA-2-transfected cells. (b) A549 cells were transfected with either no siRNA, transfection reagent RNAiMax only, or 10 nM of CTRL siRNA, HD11 siRNA-1, or HD11 siRNA-2. After 72 hr, the cell viability was determined by MTT assay. (c–f) A549 cells were transfected with 10 nM of CTRL siRNA, HD11 siRNA-1, or HD11 siRNA-2. (c) After 72 hr, one set of the cells was harvested, and the knockdown of HDAC11 mRNA was confirmed by qPCR as above. The other sets of the cells were infected with (d) PR8 or (e) CA09 at a multiplicity of infection of 1.0. After 24 hr, the culture medium was harvested and subsequently titrated by microplaque assay to quantify the released viral progeny, whereas the infected cells were harvested and processed, and viral NP and M mRNA levels were detected by qPCR and normalised as above. (f) Then, the normalised levels of each viral mRNA in CTRL siRNA-transfected cells were considered onefold to compare their levels in HD11 siRNA-1-transfected cells. (g) A549 cells, transfected with 10 nM of CTRL siRNA or HD11 siRNA-1 for 72 hr, were infected with PR8 at a multiplicity of infection of 0.1. After 12, 24, and 48 hr of infection, the culture medium was harvested and titrated by microplaque assay to quantify the released viral progeny. Error bar represents means ± standard errors of the means of three biological replicates or repeats (g); asterisks indicate the significant differences in means Next, A549 cells were transfected with either HDAC11 siRNA-1, siRNA-2, or control siRNA in duplicates for 72 hr using the optimised 10-nM concentration. Thereafter, one set of the cells was harvested, and the knockdown of HDAC11 mRNA was confirmed by qPCR—this was done before the infection to avoid the confusion whether knockdown in HDAC11 mRNA levels was caused by the siRNAs or the IAV (see Figure 1). The other set of the cells was infected with PR8 or CA09 at an MOI of 1.0. After 24 hr, the culture medium was harvested and subsequently titrated by plaque assay to quantify the released viral progeny, whereas the infected cells were harvested and processed to analyse the viral gene (NP and M) expression by qPCR. As expected, siRNA-1 and siRNA-2 knocked down the HDAC11 mRNA levels by 84% (P = 0.0001) and 50% (P = 0.0006), respectively (Figure 2c). Additionally, IAV grew to higher titres in HDAC11-depleted cells that was proportional to the magnitude of siRNA knockdown efficiency (Figure 2d,e). The cells transfected with siRNA-1 released a significant 9.5-fold (P = 0.0062) more PR8 progeny, whereas the cells transfected with siRNA-2 released a 4.1-fold (statistically non-significant) more PR8 progeny than the cells transfected with control siRNA (Figure 2d). Similarly, there was a significant 6.2-fold (P = 0.0001) and 1.4-fold (P = 0.019) increase in the release of CA09 progeny from the siRNA-1- and siRNA-2-transfected cells, respectively, compared with the control siRNA-transfected cells (Figure 2e). Furthermore, there was a significant 1.9-fold (P = 0.0004) and 2.0-fold (P = 0.003) increase in the expression of PR8 NP and M mRNAs, respectively, in HDAC11 siRNA-1-transfected cells compared with the control siRNA-transfected cells (Figure 2f). Likewise, the level of CA09 NP and M mRNAs was also significantly increased by 1.8-fold (P = 0.013) and 2.3-fold (P = 0.0004), respectively, in HDAC11 siRNA-1-transfected cells compared with control siRNA-transfected cells (Figure 2f). Finally, we analysed IAV growth kinetics in the absence of HDAC11. For this, A549 cells, transfected with HDAC11 siRNA-1 for 72 hr, were infected with PR8 at an MOI of 0.1, and the release of viral progeny in the culture medium was quantified after 12, 24, and 48 hr of infection. Consistent with above data, IAV exhibited faster growth characteristics in HDAC11-depleted cells (Figure 2g). Compared with control siRNA-transfected cells, there was a significant 11.0-fold (P = <0.0001), 8.1-fold (P = <0.0001), and 8.4-fold (P = 1,200-fold increase in the level of HDAC11 mRNA in cells transfected with HDAC11 plasmid confirmed its overexpression (Figure 3b). Figure 3Open in figure viewerPowerPoint The overexpression of HDAC11 mRNA inhibits influenza A virus infection. A549 cells were transfected with empty plasmid pcDNA3 or HDAC11 plasmid for 48 hr. Cells were then infected with PR8 at a multiplicity of infection of 1.0, and after 24 hr, the culture medium and the cells were harvested separately. (a) The culture medium was titrated by microplaque assay to quantify the released viral progeny. (b) The cells were processed for qPCR to detect HDAC11 and actin mRNAs. The HDAC11 mRNA levels in pcDNA3-transfected cells and HDAC11 plasmid transfected cells were normalised with corresponding actin mRNA levels. Then, the normalised HDAC11 mRNA level in pcDNA3-transfected cells was considered onefold to compare its level in HDAC11 plasmid transfected cells. Error bar represents means ± standard errors of the means of three biological replicates; asterisks indicate the significant differences in means 2.4 HDAC11 is important for IAV-induced expression of IFNs and subsequent antiviral response The data presented above demonstrating the noticeably significant proliferation of IAV in HDAC11-depleted cells and a profound downregulation of HDAC11 transcript level in infected cells—a sign of viral antagonism of HDAC11—indicated that, like HDAC 1 and 2 (Nagesh et al., 2017; Nagesh & Husain, 2016), HDAC11 is part of the host defences. Therefore, we next investigated the role of HDAC11 in IAV-mediated induction of host IFN response. For this, A549 cells were transfected with control siRNA and HDAC11 siRNA-1 as above and subsequently infected with PR8 at an MOI of 1.0 for 6 hr. Then, the expression of IFNα1, IFNα receptor (IFNαR1), IFNβ1, and IFNγ as well as HDAC11 was analysed by qPCR. We found that there was a significant 52% (P = < 0.0001) and 35% (P = < 0.0001) decrease in the expression of IFNα and IFNγ, respectively, in HDAC11-depleted cells compared with the control cells (Figure 4a). However, there was no significant change in the expression of IFNαR1 and IFNβ1 (Figure 4a). On the basis of this observation, we next investigated the IAV-induced phosphorylation of transcription factor, signal transducer and activator of transcription 1 (STAT1)—one of the main inducers of innate antiviral cascade, and subsequent expression of IFN-stimulated genes (ISGs; Chen et al., 2018; Iwasaki & Pillai, 2014; Schneider, Chevillotte, & Rice, 2014) in HDAC11-depleted cells. To analyse the STAT1 phosphorylation, A549 cells, transfected with the control siRNA or HDAC11 siRNA-1, were infected with PR8 as above. After 0, 6, 12, and 24 hr of infection, cells were harvested, total cell lysates were prepared, and the levels of phosphorylated STAT1 (pSTAT1) were analysed by WB. In addition, the levels of total STAT1 (tSTAT1), actin, and viral NP were detected as a loading control and infection marker, respectively. We found that there was a noticeable decrease in pSTAT1 level in the cells transfected with HDAC11 siRNA-1 than the cells transfected with control siRNA after 6 hr of infection (Figure 4b). To quantify this decrease in pSTAT1 level, the intensity of pSTAT1 and tSTAT1 bands was quantified using the Image Studio Lite software (Version 5.0, LI-COR). Then, the amount of pSTAT1 was normalised with the corresponding tSTAT1 amount. Finally, the normalised amount of pSTAT1 at each time point in control siRNA-transfected cells was considered 100% to compare its amount in HDAC11 siRNA-transfected cells. We found that there was a significant 44% (P = 0.0006) decrease in pSTAT1 level in HDAC11 siRNA-transfected cells after 6 hr of infection (Figure 4c). This indicated a slow or delayed induction of host anti-IAV response in the absence of HDAC11. To investigate this further, we compared the expression of ISGs: IFN-induced transmembrane (IFITM) protein 1, 2, and 3, IFN-stimulated gene 15 (ISG15), viperin, cholesterol-25-hydroxylase (CH25H), tripartite motif protein 22 (TRIM22), myxovirus-resistance protein-1 (MX-1), oligoadenylate synthetase 3 (OAS3), and the retinoic acid inducible gene-1 (RIG-I) effector mitochondrial antiviral signalling protein (MAVS) that have been implicated in virus infections including IAV (Chen et al., 2018; Iwasaki & Pillai, 2014; Schneider et al., 2014), in control and HDAC11-depleted cells in response to PR8 infection by qPCR. Consistent with the above findings, there was a significant 44% (P = 0.013), 44% (P = 0.007), 42% (P = 0.026), 45% (P = 0.03), 38% (P = 0.039), 39% (P = 0.019), 45% (P = 0.017), and 42% (P = 0.016) reduction in the expression of IFITM1, IFITM2, IFITM3, ISG15, viperin, CH25H, TRIM22, and MX-1 transcripts, respectively, in HDAC11-depleted cells compared with the control cells (Figure 4d). However, there was no significant change in the expression of MAVS and OAS3 in HDAC11-depleted cells (Figure 4d). Figure 4Open in figure viewerPowerPoint HDAC11 is important for influenza A virus-induced host innate antiviral response. (a) A549 cells, transfected with 10 nM of control siRNA (CTRL) or HDAC11 siRNA-1 (HD11) for 72 hr, were infected with PR8 at a multiplicity of infection (MOI) of 1.0. After 6 hr, the levels of IFNα1, IFNαR1 IFNβ1, IFNγ, HDAC11, and actin mRNAs were detected by qPCR. The levels of IFNα1, IFNαR1 IFNβ1, IFNγ, or HDAC11 mRNA in both samples were normalised with the levels of corresponding actin mRNA. Finally, the normalised level of each gene mRNA in CTRL siRNA-transfected cells was considered 100% to compare its level in HD11 siRNA-transfected cells. (b and c) A549 cells, transfected with 10 nM of either control siRNA (CT) or HD11 siRNA-1 for 72 hr, were infected with PR8 at an MOI of 1.0. (b) After 0, 6, 12, and 24 hr of infection, cells were harvested, total cell lysates were prepared, and pSTAT1 (91/84 kDa), tSTAT1 (91/84 kDa), actin (42 kDa), and viral NP (56 kDa) were detected by western blotting. The intensity of pSTAT1 and tSTAT1 bands was quantified using the Image Studio Lite software (LI-COR). The levels of pSTAT1 was then normalised with the corresponding tSTAT1 levels. (c) Finally, the normalised level of pSTAT1 at each time point in control siRNA-transfected cells (CT) was considered 100% to compare its amount in HDAC11 siRNA-transfected cells (6, 12, and 24 hr). (d) A549 cells, transfected with 10 nM of either CTRL siRNA or HD11 siRNA-1 for 72 hr, were infected with PR8 at an MOI of 1.0. After 6 hr, the mRNA levels of actin and indicated interferon stimulated genes were detected by qPCR and normalised and presented as above. Error bar represen
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