Visualising the Role of Histone Deacetylase 6 in Influenza Virus Assembly and Budding
Influenza virus causes seasonal disease that continues to place a huge burden on global health and economy. Due to rapid mutations in the virus, universal vaccine development remains futile and the efficacy of seasonal vaccines are unstable. Furthermore, resistance to antiviral drugs such as M2 ion channel and neuraminidase inhibitors calls for development of alternative treatment strategies. Influenza virus requires host cell machinery in order to complete its life cycle. With this in mind, the use of host-directed therapy could be an alternative to current virus-directed strategies. Recently, a class of host proteins, histone deacetylases (HDACs) have been shown to possess antiviral properties. In particular, a class II HDAC, HDAC6 is mainly localised to the cytoplasm and inhibits trafficking of influenza virus within the cell. In this study, we investigated the mechanism by which HDAC6 could be inhibiting viral trafficking and how this affects assembly and budding. HDAC6 deacetylase activity regulates cytoskeleton such as microtubules, which are important for trafficking of viral proteins. Indeed, inhibition of HDAC6 deacetylase activity, which leads to increased acetylation and structural changes in microtubules, resulted in increase of viral release. (unfinished-waiting on EM results to complete this section)
Future research aims to investigate whether HDAC6 affects virion integrity during assembly and budding.
Influenza virus is an enveloped member of Orthomyxoviridae (Bouvier and Palese, 2008). The viral particle is between 80-120nm in size and contains a segmented, negative sense, single-stranded RNA genome encased in a capsid (Taubenberger and Kash, 2010; Bouvier and Palese, 2008; Vajda, et al, 2016). The virion may be spherical or filamentous and the three major components includes the envelope, M1 matrix protein, and viral ribonucleoprotein (vRNP) core (Watanabe, et al, 2010). The envelope is a lipid bilayer composed of glycoproteins haemagglutinin (HA), neuraminidase (NA), and M2 ion channel. The M1 matrix encloses the nuclear export protein (NEP) and vRNP. The vRNP incorporates eight viral RNA segments that associates with nucleoprotein (NP) and polymerase complex. The polymerase complex constitutes polymerase acidic protein (PA), and polymerase basic proteins PB1 and PB2 (At the centre: influenza A virus Ribonucleoproteins-review original). There are currently four influenza virus types identified based on their antigenicity. These types are A, B, C, and recently discovered D, with types A and B contributing to seasonal epidemics (World Health Organisation, 2018). Furthermore, influenza B is divided into lineages and strains, which include the Yamagata and Victoria lineages originally identified in the 1980s (Fang, et al, 2015; Ni, F., Kondrashkina and Wang, 2013). The more prevalent influenza A virus (IAV) is subtyped according to combinations of HA and NA, with currently 18 HA and 11 NA subtypes identified (The evolution of seasonal influenza viruses-review original).
IAV subtypes H1N1 and H3N2 are the current circulating strains in humans, causing epidemics and global pandemics (World Health Organisation, 2018). HA and NA variants result from amino acid changes at binding sites due to frequent point mutations (antigenic drift) and gene reassortment (antigenic shift), the latter being the cause of pandemics (Petrova and Russell, 2018; Boivin, et al, 2010). Gene reassortment occurs when different strains mix within a host, leading to novel strains in which seasonal vaccines cannot protect against (Parrish, et al, 2015; Bouvier and Palese, 2008). In addition, the lack of proofreading by viral RNA-dependent RNA polymerase (RdRp) results in constant accumulation of mutations in the viral genome. This further contributes to the evolution of the virus, allowing it to effectively evade the immune evasion and develop drug resistance (Boivin, et al, 2010).
As an obligate intracellular parasite, influenza virus requires host machinery to complete its life cycle (Lakadamyali, Rust, and Zhuang, 2004). Firstly, influenza virus attaches to host receptors via HA spike proteins (Cellular Networks Involved in the Influenza Virus Life Cycle). The HA0 precursor is composed of HA1, which recognises its cell surface receptor, and HA2 which is required for membrane fusion (Influenza A: Understanding the Viral Life Cycle-review original). The binding properties of HA globular domain to its receptor, either α-2,6 or α-2,3 sialic acid linkages determines the tissue tropism of the virus (reference). Sialic α-2,6 is the predominant receptor for human influenza virus and is localised to the cells of the upper respiratory tract, whereas α-2,3 linkages are found on the surface of the lower respiratory tract (The biology of influenza viruses-review original). (Consider adding the importance of these two different sialic acid receptors in regards to the difference in humans, birds and pigs)
Recognition of HA to its receptor induces clathrin-mediated endocytosis and subsequent entry of the virus into an endosomal compartment. The low endosomal pH of 5 to 6 causes a conformational change in HA0, which exposes the HA2 fusion peptide. HA2 can then insert into the endosomal membrane and induce fusion with the viral envelope. In addition, the acidic environment opens up the proton pumping M2 ion channel, causing acidification of the viral core. This results in the release of vRNP from M1 matrix and subsequent entry into the cytoplasm (REFERENCES).
The vRNP needs to be transported to the nucleus in order to replicate the viral genome. Nuclear localisation signals (NLSs) present in NP, M1, nonstructural protein 1 (NS1) and polymerase complex bind to nuclear import machinery (α and β importins) which direct vRNP to the nucleus (Contributions of Two Nuclear Localization Signals of Influenza A Virus Nucleoprotein to Viral Replication; Influenza A: Understanding the Viral Life Cycle). Influenza proteins are synthesised by RdRp and despite having a minimal genome, influenza virus is able to synthesise at least 17 proteins due to strategies such as alternative start codons, ribosomal frameshifting, alternative splicing, and leaky ribosomal scanning (Hussain et al, 2017-review, see original).
Once the viral genome and proteins are synthesised, components are trafficked to the plasma membrane. HA and NA initiate virion assembly by inserting into membrane lipid rafts causing curvature (Rossman, and Lamb, 2011). M1 is recruited to the membrane by HA and NA cytoplasmic domains followed by vRNP and NP. M2 stabilises the budding site ensuring components are assembled correctly before membrane scission occurs. Finally, NA is required to cleave membrane sialic acid, resulting in release of the virion and acquisition of the lipid envelope (Rossman, and Lamb, 2011).
(will use this to expand on targeting life cycle?)
3.2 Epidemiology of Influenza Virus.
Influenza virus infection causes acute respiratory febrile disease, commonly known as “flu”. Types A and B cause seasonal epidemics that result in over 500,000 deaths per year (Petrova and Russell, 2018; Nayak and Barman, 2004). The virus is spread through aerosol, respiratory droplets and although all demographics are affected, most at risk groups include young children, elderly, pregnant women and immunocompromised patients (Moghadami, 2017; Lagace-Wiens et al, 2010). Type C typically causes mild, sporadic incidents and only type A has caused global pandemics, such as the 1918 Spanish H1N1 flu that killed over 50 million people (Petrova and Russell, 2018). Influenza is a successful zoonotic pathogen with a wide host range including dogs, horses, pigs, and birds (Parrish, et al, 2015). The potential for sustained transmission from avian and swine viruses to humans poses great concern due to high mortality rates from pneumonia or respiratory failure (Taubenberger and Kash, 2010). The rate of avian zoonosis is infrequent as avian influenza recognises α-2,3 linkage receptors that are lowly abundant in the lungs, and therefore relatively inaccessible to the virus (Perhaps mention H5N1 and its high mortality rate in humans. This is a good segway into why swine acting as a mixing vessel is so important) (The biology of influenza viruses- review original). Swine however play a significant role as mixing agents when co-infected with both human and avian viruses, leading to gene reassortment (Bouvier and Palese, 2008). This brings about novel viruses with increased transmissibility and pathogenicity, with the potential to cause pandemics (Dlugolenski, et al, 2015).
Annual vaccination is currently the best anti-influenza strategy available (reference). The major challenges with influenza include the development of drug resistance and absence of a universal vaccine. Drug resistance emerges during treatment with antivirals (Dobrovolny and Beauchemin, 2017). The two main classes of antiviral drugs include adamantanes, which are M2 ion channel inhibitors and neuraminidase inhibitors (Hurt, 2014). Adamantanes such as amantadine and rimantadine were amongst the first clinically approved antivirals, which began in the 1960s (Hurt, 2014). These drugs bind the M2 ion channel pore by steric or allosteric hindrance, preventing proton conductance and viral core acidification, leading to inhibition of vRNP release into the cytoplasm (Drug resistance in influenza A virus: the epidemiology and management-review original).Mutations in this channel can alter structural properties including pore size, hydrophobicity, and helix assembly, leading to adamantane resistance (Drug resistance in influenza A virus: the epidemiology and management-review original). M2 ion channel S31N mutation is the predominant cause for development of adamantane resistance in H1N1, H3N2, and avian strains (Drug resistance in influenza A virus: the epidemiology and management-review original). In some geographic locations such as Asia, the S31N mutation has reached greater than 90% of IAV subtypes, rendering adamantanes virtually obsolete (Clinical Implications of Antiviral Resistance in Influenza-review original). Neuraminidase inhibitors prevent NA cleavage of host sialic acid, stopping release and spread of influenza (Drug resistance in influenza A virus: the epidemiology and management-review original). Neuraminidase inhibitors block cleavage activity by competing with the interaction between viral NA and host cell sialic acid (Drug resistance in influenza A virus: the epidemiology and management-review original). Neuraminidase inhibitors including zanamivir and oseltamivir are effective as prophylactic treatments but they need to be taken within 48 hours of symptom onset to prevent the spread of influenza (Drug resistance in influenza A virus: the epidemiology and management-review original). The development of H3N2 resistance to oseltamivir is mainly due to R29K and E119V mutations while H1N1 resistance is due to H275Y mutations, which originated in Europe (Clinical Implications of Antiviral Resistance in Influenza-review original). Seasonal vaccines are selected by computational predictions of circulating strains (Steinbruck et al, 2014). The effectiveness of the IAV vaccine varies and is impeded by antigenic drift and antigenic shift. Alternative strategies are needed and targeting host factors could be a great alternative to virus-directed treatment since host interactions are important for influenza replication cycle (Lou, Sun and Rao, 2014). Host-directed antiviral therapy is possible but improving our understanding of the host-virus interactions are critical in developing such treatments (REFERENCE).
Previously our lab and others have shown that some members of a family of enzymes known as histone deacetylases (HDACs) play a role during influenza infection. Histone deacetylases (HDACs) are a family of host enzymes presently under investigation for their role during influenza infection. Currently, 18 HDACs have been identified and are categorized into four classes based on sequence similarity and homology to their related yeast counterparts (Seto and Yoshida, 2014). Class I HDACs (HDAC1, 2, 3, and 8) are Rpd3-like proteins and are ubiquitously expressed (Haberland, et al, 2009-review original). Class II HDACs are Hda1-like proteins and are further categorised into class IIa (HDAC4, 5, 7, and 9) and class IIb (HDAC6 and 10). Class IIb HDACs are unique in that they possess a second putative catalytic domain (Seto and Yoshida, 2014). Class III HDACs are Sir2-like proteins (SIRT1, 2, 3, 4, 5, 6 and 7) and unlike the zinc-dependent deacetylase activity of class I, II and IV HDACs, class III HDACs are NAD+-dependent (Seto and Yoshida, 2014). HDAC11 is the only class IV HDAC and has sequence similarities to classes I and II.
HDACs catalyse the acetyl group removal of histones and several non-histone proteins (Seto and Yoshida, 2014). HDACs therefore play an important role in post-translational modifications that regulate cellular function including gene regulation, cell signalling, motility, and protein degradation (Li, Shin and Kwon, 2013). With their wide role in human biology, it is not surprising that mutations in HDACs correlate with a multitude of diseases. Current investigation on HDACs as potential therapeutic targets for many diseases including cancer, neurodegenerative diseases and potentially viral infections are underway (Eckschlager, 2017). Whereas class I HDACs mainly reside in the nucleus, class II HDACs are able to shuttle between the nucleus and cytoplasm (Marmorstein, 2001). In particular, HDAC6 is a class IIb HDAC that contains two catalytic deacetylase domains (DD1 and DD2) (Li, Shin and Kwon, 2013). These catalytic domains are involved in deacetylation of cell substrates including cortactin, heat shock protein 90 (Hsp90), and tubulin (Zhang et al., 2007; Kovacs et al., 2005; Hubbert et al. 2002). Due to the nuclear export signal (NES) and cytoplasmic retention signal (serine-glutamic acid repeat (SE14) domain), HDAC6 mainly localizes in the cytoplasm where it regulates cellular functions (Class II histone deacetylases: versatile regulators–review original). HDAC6 contains a C-terminal BUZ domain, a ubiquitin-binding zinc finger that is able to bind DNA as well as mono and polyubiquitinated proteins to regulate gene transcription and aggresome formation, respectively (Li, Shin and Kwon, 2013; Ouyang et al., 2012). Herein, the evidence that HDAC6 antagonises influenza virus during infection is discussed.
Several studies have investigated the antiviral role of HDAC6 during infection. Researchers suggest that HDAC6 regulates the immune system, which is important for host defence against invading pathogens. As part of the the innate immune system, retinoic acid-inducible gene I (RIG-I) is important for intracellular sensing of RNA viruses including influenza (Rudnicka and Yamauchi, 2016). HDAC6 deacetylation of RIG-I leads to downstream signalling and ultimately an antiviral response through expression of interferon-β and other immune regulators (Choi et al., 2016). Phosphorylation and subsequent activation of HDAC6 by protein kinase C alpha during Sendai virus infection leads to deacetylation of β-catenin, and therefore activation of the interferon regulatory transcription factor, IRF3 (Zhu, Coyne, and Sarkar, 2011). The reduced protection against human immunodeficiency virus (HIV) in HDAC6 knockout mice further illustrates its activity as a modulator of the immune response (Valenzuela-Fernández et al., 2005). In addition, HDAC6 deacetylation of microtubules is important for stabilisation of the immune synapse where T-cells and antigen presenting cells interact to promote the adaptive immune response (Serrador, 2004). It is possible that deacetylation of substrates which regulate other components of the cytoskeleton are also involved in immune synapse stability.
The cytoskeleton includes components such as actin and microtubules that are important for cell motility, structure and protein transport and are involved in viral entry and propagation during infection (Ward, 2011). When viruses attach host cells, they can induce actin polymerisation at the cell membrane. This results in membrane ruffling which facilitates viral endocytosis. Microtubules are composed of α and β tubulin monomers, which are important for membrane fusion in viruses such as influenza or HIV (Lakadamyali, Rust, and Zhuang, 2004). HDAC6 regulation of actin filaments and tubulin therefore affects viral entry. The regulation of actin by HDAC6 occurs through deacetylation of the F-actin binding protein, cortactin. This stabilises F-actin binding ability of cortactin, and therefore regulates actin dynamics and possibly affects viral entry (Zhang et al., 2010). Studies have shown that HDAC6 deacetylation of tubulin reduces HIV infection in T lymphocytes, which also requires membrane fusion for host entry (Malinowsky, Luksza and Dittmar, 2008; Valenzuela-Fernández et al., 2005). Overexpression of HDAC6 during HIV infection leads to inhibition of membrane fusion and syncytia formation due to reduced acetylated tubulin (Valenzuela-Fernández et al., 2005).
Upon gaining entrance into host cells, uncoating of the viral capsid is required to release its ribonucleoprotein (RNP) into the cytoplasm where it propagates towards the nucleus for replication (Li et al., 2014). Viruses can achieve this by exploiting various processes within the host cell including those regulated by ubiquitin. Viruses may subvert ubiquitin to degrade unwanted cell proteins or aid its own replication (Banerjee et al., 2014). Through its BUZ domain, HDAC6 binds unanchored ubiquitin chains. HDAC6 links ubiquitin to dynein complexes that are part of the aggresome-autophagy pathway, an important pathway for clearance of misfolded proteins (Lee et al., 2010). Banerjee et al. (2014) suggest that via its BUZ domain, HDAC6 recognises ubiquitin-bound influenza as well as M1 protein. Ubiquitin-bound virus resembles misfolded proteins, and recognition by HDAC6 BUZ domain results in transportation of the virus particle along microtubules towards the aggresome (Banerjee et al., 2014). During transportation, opposing mechanical forces induced by HDAC6 leads to virus capsid uncoating and release of the genome (Banerjee et al., 2014). In addition, the presence of lysine 184 ubiquitination site on viral nucleoprotein (NP) suggests ubiquitination may be involved in the early phases of influenza cycle (Liao et al., 2010). Liao et al. (2010) suggest that ubiquitination stabilizes NP and RNA interactions that increases the efficiency of replication. Due to the importance of influenza ubiquitination, the next step would be determining whether influenza manipulates HDAC6 ubiquitin-binding activity to facilitate its uncoating during early infection.
Previously it has been shown that influenza induces caspase-mediated cleavage of HDAC6 resulting in the removal of the BUZ domain. In addition, caspase inhibition prevents degradation of the full-length HDAC6 polypeptide (Husain and Harrod, 2009). The significance of the cleavage is unknown and its effect on HDAC6 deacetylase activity is yet to be determined. It is possible that BUZ domain cleavage limits HDAC6 regulation of Hsp90, which is important for influenza replication (Kovacs et al., 2005; Chase, et al., 2008). The cleavage occurs at the caspase cleavage DMAD motif, between the SE14 and BUZ domains. This results in exposure of the SE14 cytoplasmic retention domain and may enhance influenza replication through the promotion of apoptosis (Husain and Harrod 2009). HDAC6 overexpression decreases virus release indicating HDAC6 inhibits late infection (Husain and Cheung, 2014). Due to its antiviral role, it is plausible that influenza is regulating HDAC6. Indeed, a reduction of HDAC6 deacetylase activity occurs during infection but the regulation of HDAC6 expression requires further investigation (Husain and Cheung, 2014).
Following replication, influenza components including envelope, matrix protein, and vRNP are trafficked towards the plasma membrane where assembly and budding occur (Nayak and Barman, 2004). Like viral entry, this requires dynamic assembly and disassembly of actin filaments and microtubules, which undergo modulation by HDAC6 deacetylase activity. Inhibiting HDAC6 deacetylase activity with tubacin, an HDAC6 specific inhibitor, leads to increased levels of acetylated tubulin and subsequent increase in influenza virus release (Husain and Cheung, 2014). It is possible that tubulin acetylation, which occurs on lysine 40 residues, reduces mechanical stress experienced by microtubules, allowing for the repair of lattice cracks by acetyltransferases before breakage occurs (Janke and Montagnac, 2017). This could improve mechanical resistance to the viscous cytoplasm and therefore enable influenza virus to traffic efficiently along microtubule tracks. Interestingly, both the acetylated and total tubulin levels appear to increase in infected cells, suggesting influenza could be regulating the total tubulin levels during infection (Husain and Harrod, 2011). Although evidence suggests that influenza may downregulate HDAC6 during the assembly phase of infection, this needs further research. As previously mentioned cortactin promotes viral entry, but may also restrict late stages of infection during assembly and budding. This could explain the influenza induced caspase-mediated cleavage of cortactin during the late stages of infection (Chen and Husain, 2016). The mechanism of HDAC6 deacetylation of cortactin leading to regulation of viral entry and egress needs further exploration, possibly by structural and microscopy analysis. Studies also suggest that via its ubiquitin-binding domain HDAC6 is able to recruit cortactin for F-actin assemble which is essential for autophagosome formation involved in removal of pathogens (Lee et al., 2010). Such observations reveal the wide role of HDAC6 in human biology through its ubiquitin-binding and deacetylase activity. This highlights the ability of HDAC6 to affect various stages of the virus cycle.
Much of the evidence points towards an antiviral role of HDAC6 during infection. Furthermore, in vivo studies by Wang et al. (2015) illustrate that HDAC6 overexpression in mice increases survival during infection by highly pathogenic influenza A H5N1 virus. However, there is a correlation between HDAC6 and cancer, and although mice appear to develop normally, it is uncertain whether overexpression could eventually lead to tumor formation (Eckschlager, 2017). In addition, the administration of HDAC6 targeting drugs could present a different outcome compared to HDAC6 overexpressing transgenic mice. As a potential target for antiviral therapy, more in vivo studies are required to assess the effect of manipulating HDAC6 in normal cell function. Although our lab has previously shown that HDAC6 restricts viral trafficking and release, it is uncertain what the effect of HDAC6 has on the stages of assemble and budding.
The present study aims to elucidate this restriction by visualising various stages of influenza A virus assembly and budding in HDAC6-deficient cells by confocal and electron microscopy as well as reconstructing various stages of assembly and budding by electron tomography. The hypothesis is that elevated acetylated tubulin in HDAC6-inhibited cells enhances influenza trafficking towards the plasma membrane where assembly and budding occur. Cells that are high-pressure frozen preserves ultrastructures in a vireous-like state, which is superior to chemical fixation (Murata, and Wolf, 2018). This enables viewing of host-virus interactions in a near-native state and therefore elucidating the role of HDAC6 during infection.
MDCK (Madin-Darby canine kidney) and A549 (adenocarcinomic alveolar basal epithelial) cells were grown in complete minimal medium (MEM) supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin, and L-glutamine (Invitrogen) at 37°C under 5% CO2 atmosphere. The cells were subcultured every two to three days in T75 flasks using trypsin (0.25%) – EDTA (1mM) (Life Technologies). The influenza virus Puerto Rico/8/1934 H1N1 (PR8) strain was propagated in 10-day old embryonated chicken eggs and titrated on MDCK cells via plaque assay.
MDCK and A549 cells were infected with influenza virus PR8 strain at a multiplicity of infection (MOI) as indicated. A virus stock of 4×108 PFU/ml was diluted in serum-free MEM and 1µg/mL trypsin, which were used to inoculate cell monolayers previously washed twice with serum-MEM. After 1 hour incubation at 35°C, 5% CO2 the inoculum was removed and cells were washed twice with serum-free MEM. Fresh serum-free MEM was added and cells were incubated at 35°C, 5% CO2 for 24 hours.
MDCK cells (5x 104) or A549 cells (1×105) were seeded on 13 mm coverslips and incubated at 37°C, 5% CO2 for 24 hours in 10% FBS MEM to achieve a monolayer. The cells were fixed in 4% formaldehyde (Sigma-Aldrich) and permeabilized with 0.5% Triton X-100. Cells were simultaneously incubated with primary goat anti-HA polyclonal antibody (1:100) and mouse anti-acetylated tubulin monoclonal antibody (1:100; Sigma) for 1 hour at room temperature. This was subsequently incubated in secondary Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (1:300; Invitrogen) and Alexa Fluor 594-conjugated donkey anti-mouse IgG antibody (1:300; Invitrogen). Additionally, cells were stained with Hoechst DNA-binding dye (1:1000; Invitrogen). Coverslips were mounted onto SlowFade gold antifade reagent (Thermofisher), then allowed to dry in absence of light and sealed. Fluorescent images were acquired by sequential scanning of entire cells on confocal laser scanning microscope.
MDCK cells (5×104) were seeded in 6 well transwells containing 0.4µm polyester pores and incubated at 37°C, 5% CO2 for 24 hours in 10% FBS MEM to achieve a monolayer. Low melting 2% agarose in serum-free MEM, 15% BSA, and one drop of toluidin blue were added to wells, creating a solid platform before punching out sections with 1mm biopsy punch. Cells were loaded on specimen carriers then high-pressure frozen in liquid nitrogen (approximately 2000 bar) at specified time points of infection (Leica EMPACT 2 High-Pressure Freezer). Samples underwent freeze-substitution in solution (2% osmium tetroixide, 1% anhydrous glutaradehyde, 10% H2O in acetone) in a Leica EM AFS2 (automatic freeze-substitution device). Samples were washed in acetone then incrementally embedded in epoxy resin embed 812 mixture (Embed 812, dodecenyl succinic anhydride, nadic methyl anhydride,N-Benzyldimethylamine,). This was achieved by firstly adding 3 parts acetone: 1part resin, 2 parts acetone: 1 part resin, 1 part acetone: 1 part resin, 1 part acetone, 2 parts resin, 1 part acetone: 3 parts resin, then finally full resin infiltration and polymerized at 60°C for 48 hours to harden. Excess resin was trimmed, and carriers removed to reveal embedded sample in resin. Samples of 90nm and 250nm sections were sliced and mounted on copper mesh grids. Slices were then stained in uranyl acetate and lead citrate (Leica EM stain). Images of samples were taken on Philips CM100 transmission electron microscope (TEM) and JEM-2200FS cryo-TEM.
Culture medium from infected cells were harvested and mixed with 0.3% bovine serum albumin (BSA) and titrated on confluent MDCK cells for microplaque assay. MDCK cells were infected with 10-fold serial dilutions of the culture medium, inoculum removed, and overlaid with equal volumes of serum-free MEM and 1.6% Avicel containing 1µg/mL trypsin. After 24 hour incubation, overlay was removed and cells were fixed in 4% formaldehyde and subsequently permeabilized with 0.5% Triton X-100. Cells were stained with mouse anti-NP antibody (1:1000) followed by HRP-conjugated anti-mouse antibody (1:1000). Plaques were developed using Trueblue peroxidase substrate (KPL). Labelled plaques were counted manually and plaque-forming units per mL (PFU/mL) was calculated.
MDCK cells were lysed in lysis buffer (50mM Tris-HCI, pH 7.4, 150mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100 and 1x protease inhibitor cocktail). Proteins of equal mass were resolved by 8 or 10% Tris-glycine SDS-PAGE gels. Proteins were subsequently transferred to nitrocellulose membrane. Membranes were probed with primary rabbit anti-HDAC6 (D2E5) (1:1000; Cell signalling), mouse anti-acetylated tubulin monoclonal antibody (1:1000; Sigma-Aldrich) or rabbit anti-β actin antibody (1:10000). This was followed by secondary horseradish peroxidase-conjugated (HRP) goat anti-mouse antibody (1:2000) for acetylated tubulin, HRP-conjugated donkey anti-rabbit antibody (1:2000) for HDAC6, or HRP-conjugated donkey anti-rabbit antibody (1:5000) for β actin. Blots were developed using SuperSignal West Pico PLUS Substrate kit (Thermoscientific) and then visualized by chemiluminescence. Scanning was performed on an Odyssey Fc Imagining system (Li-Cor).
Indicated concentrations of small interfering RNA (siRNA) targeted to human HDAC6 mRNA and non-targeting siRNA were introduced into A549 cells. The siRNA and 2µL Lipofectamine RNAiMAX (Invitrogen) were separately diluted in 100µL OptiMEM medium (Invitrogen?) for 5 minutes before being mixed together and incubated at room temperature for 30 minutes. A549 cells were reverse transfected with RNAiMAX-siRNA mixture by seeding together into 12 well plates and incubated at 37°C, 5% CO2 for 72 hours. Cells were then harvested for western blotting.
Viability of siRNA transfected A549 cells were determined via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Following 72 hour post siRNA transfection, media was removed and 400µL plus 100µL MTT reagent (Sigma-Aldrich) was added. The cells were then incubated at 37°C, 5% CO2 for 1 hour. After incubation, 1mL DMSO was added and cells were gently rocked in absence of light for 15 minutes at room temperature. The media was dispensed in 96 well plate and absorbance was measured at 570nm in 680 Microplate Reader (Biorad).
Statistical analysis of data from three independent experimental repeats were performed on GraphPad Prism 7.05. Unpaired student t-tests were performed and p-values of 0.05 or lower were considered significant.
To test the hypothesis that HDAC6 inhibits viral trafficking, assembly and budding, conditions for microscopy were first established. MDCK cells were uninfected or infected with PR8 strain at a multiplicity of infection (MOI) of 1 for 24 hours. Cells were then incubated in primary anti-NP (1:50) goat anti-HA (1:50) antibodies to see which was best suited for visualising infection. Cells were then visualised under fluorescent microscope. Figure 1a) Anti-HA antibody was utilized for further experiments, as this gave more distinct cell boundary staining and furthermore, HA represents viral envelope proteins that initiate virion assembly at the plasma membrane to initiate assembly and budding. Due to profound background staining, anti-HA was concentration was reduced to 1:100. Optimal concentrations of tubacin (Sigma-Aldrich) to inhibit HDAC6 deacetylase activity were then set. Tubacin was titrated onto MDCK cells at various concentrations to assess the level of inhibition on HDAC6 deacetylase activity. HDAC6 deacetylase substrate, tubulin was evaluated via western blot to illustrate this effect (Figure 1c). The concentration optimised was 20µM of tubacin indicated by enhanced acetylated tubulin and utilized for the duration of experiments involved.
Optimisation . To establish the optimal anti-HA antibody concentration tubacin concentration, total cell lysates of MDCK cells were prepared. Following tubacin treatment at various concentrations, acetylated tubulin expression was detected via western blot. Loading control used was β-actin. Data shown is from one experiment.
5.2 HDAC6 deacetylase inhibition effect on trafficking of viral proteins towards the plasma membrane
We next investigated the hypothesis that inhibition of HDAC6 deacetylase activity results in increased viral trafficking towards the plasma membrane during infection. MDCK cells were infected with influenza PR8 strain at a multiplicity of infection (MOI) of 1 and 10 for confocal and electron microscopy analysis, respectively. Following 1 hour infection at 35°C, 5% CO2, cells were subsequently treated with 20µM tubacin and incubated for a further 24 hours. Uninfected mock (DMSO only) and infected mock treated cells were used as controls. To visualise the effect of tubacin treatment on influenza trafficking towards the plasma membrane through confocal microscopy, cells were probed with antibodies to detect acetylated tubulin and viral HA. Acetylated tubulin was labelled to verify that tubacin treatment worked. Initially, 2D images were captured to analyse HA distribution (Figure 2). Upon further investigation, Z stacks depicting 3D representation of the cells revealed that HA was predominantly distributed at the apical surface relative to basolateral after 24 hour infection (Figure 2b), consistent with current literature (Influenza A: Understanding the Viral Life Cycle-review original). HA fluorescent intensity was therefore quantified and results indicate a mean 1.14 fold increase in viral HA distribution at the apical surface of the plasma membrane following tubacin treatment compared to mock treatment. This was not however statistically significant.
- Acetylated Tubulin Viral Haemagglutinin Merged
A B C
D E F
Infected-Tubacin Infected-Mock Uninfected-Mock
G H I
(to add scale bars)
Influenza traffics to the apical cell surface.(a) Confocal microscopy images illustrate uninfected mock treated (A-C), infected mock (D-F), and tubacin treated infected (G-I) cells labelled for nucleus (blue), influenza HA (green) and cellular acetylated tubulin (red) (A-K). (b) Images composed of horizontal Z stacks were captured at 75µm intervals from basolateral to apical surface.Images were captured at 60 times magnification and scale bars are 25µm (A-I).
- Viral Haemagglutinin Acetylated Tubulin
Enrichment at YZ axis
Mock Tubacin Mock Tubacin
Enrichment at XZ axis
Mock Tubacin Mock Tubacin
Mean Fluorescent Enrichment
Mock Tubacin Mock Tubacin
(c) Images were quantified in imageJ Fiji software by assessing the average mean fluorescent intensity for apical HA (L) and overall acetylated tubulin (M) between orthogonal sections, XZ and YZ . Fold enrichment values for HA and acetylated tubulin were standardised to uninfected control and analysed in GraphPad prism. (d) Scatterplots of data illustrates individual fold enrichment values ± SEM for each orthogonal section from three independent experimental repeats (n=3). Mean increase in HA distribution at the plasma membrane due to tubacin treatment compared to mock is not significant (p value>0.05). Mean increase in acetylated tubulin in tubacin treatment compared to mock is significant (**p value=0.006).
HA MEAN DIFF = 13.6%
HA ZY MEAN DIFF = 17.1%
HA XZ MEAN DIFF = 10%
Fold enrichment atub threshold of 2
HA MEAN DIFF= 30%
The potential that conditions were not optimised to detect a difference in viral release between mock and tubacin treatment via microscopy lead to performing virus release assays. Media from infected mock and infected tubacin treated conditions were collected and serial dilutions (10-2, 10-3, 10-4) were titrated onto MDCK cells. Plaque numbers were counted in order to calculate PFU/mL. Tubacin treatment resulted in a mean 2.1 fold increase of infectious particle release from cells compared to infected mock control. This indicates that inhibition of HDAC6 deacetylase activity results in increase in viral propagation and subsequent budding and release.
Viral Release in MDCK cells
Following 24 hour Infection
Fold Change in Virus Titre (PFU/mL)
Tubacin treatment leads to increase in viral release in MDCK cells. Virus titre from infected media of mock control and tubacin treatment were calculated by fold change in PFU/mL. Data shown is from three independent experiment repeats ± SEM (n=3). Increase in virus release due tubacin treatment compared to mock is significant (**p value=0.0071).
Optimal concentration of HDAC6 siRNA were first established in order to knockdown protein expression. A549 cells were incubated with various concentrations of HDAC6 siRNA and protein knockdown assessed via western blot (Figure 4a). The concentration optimized was 10nM indicated by complete protein knockdown and utilized for the duration of experiments involved. (b) An MTT assay was performed illustrating that 10nM siRNA does not compromise cell viability. Error bars represent ±SEM from one experiment. Following optimisation, we investigated whether HDAC6 knockdown produced the same phenotype as deacetylase inhibition. Cells were seeded for both confocal and western blot analysis. Following 72 hour knockdown, cells were infected with PR8 strain at a MOI of 1 at 35°C, 5% CO2 for 24 hours. Cells for confocal analysis were probed for viral HA and acetylated tubulin to visualise the effect of HDAC6 knockdown. Knockdown of HDAC6 was confirmed via western blot and acetylated tubulin levels was assessed to see whether this had the same effect as deacetylase inhibition.
- Cell Viability Assay
in A549 Cells
Relative Absorbance Values
CT 1 10 25 50
Acetylated Tubulin Viral Haemagglutinin Merged
Infected-Tubacin Infected-Mock Uninfected-Mock
e) Viral Haemagglutinin Acetylated Tubulin
Enrichment at YZ axis
Enrichment at XZ axis
Mean Fluorescent Enrichment
Mean Fluorescent Enrichment
Mock KD Mock KD
HDAC6KD-from 2 experiments
Mean HA DIFF: 9.8%
MEAN HA YZ DIFF: 7.4%
MEAN HA XZ DIFF: 12.4 %
* BUZ domain could be important not only for early stage but late stages.
* Set threshold for which cells to use (tubacin).
* HA does not accumulate at the membrane.
* could do obtain phenotype from HDAC6 KD – had been
* regression HDAC6 inhibition does not lead to increased trajectory: inhibition does not increase kinetics but could take away the barrier it places.
* ratio of cyto to membrane
* cell vs animal model
* threshold of 2 fold enrichment acetylation = 27.5% increase HA and 0.063 p value.
A class IIb HDAC, HDAC6 is amongst a family of HDACs shown to be anti-IAV host factors. Previously our lab has shown that class I HDACs, HDAC1 and 2, are important regulators of host anti-IAV innate immune response. Knockdown of HDAC1 or HDAC2 in lung epithelial cells decreases IAV-induced expression of interferon-stimulated genes (ISGs) such as viperin, leading to a decrease in the innate immune response.
Few studies have proposed a pro-viral role of HDAC6. It is possible that the opposing views are context dependent due to the profound role of HDAC6 in the cell and potential to affect different stages of the viral cycle, but the details surround this requires further investigation.
6.1 Main findings and future directions
The aim of this project was to elucidate the effect of HDAC6 on assembly and budding of IAV at the plasma membrane. Various techniques were employed to research this effect. The findings obtained show that HDAC6 deacetylase activity on microtubules inhibits trafficking of IAV. Although not statistically significant, a mean increase in viral HA assembly at the plasma membrane in A549 and MDCK cells was observed following HDAC6 knockdown and inhibition of HDAC6 deacetylase activity via tubacin, respectively. Furthermore, plaque assay results confirmed that treatment by tubacin led to an increase in virus release, which follows assembly and budding.
6.2 IAV assembly and budding in HDAC6 inhibited cells
Initially we investigated the effect of HDAC6 deacetylase activity inhibition on assembly and budding of IAV via confocal microscopy. Confocal results indicated an increased 13.6% mean fold enrichment of viral HA at the plasma membrane in tubacin treated cells compared to mock/DMSO only, and a mean 9.8% increased fold enrichment in HDAC6 knockdown cells. Although not statistically significance, there was an observable trend where increased HA recruitment at the plasma membrane for assembly was associated with increased tubulin acetylation due to tubacin which inhibits HDAC6 deacetylase activity. In support of this, HDAC6 deacetylase inhibition led to an increase in virus release as illustrated by plaque assay results, with a significant 2.1 fold increase. Since viral assembly is temporary and particles do not accumulate at the plasma membrane it is possible that solely quantifying the level of HA distribution at the plasma membrane does not give an accurate depiction of the effect of HDAC6 inhibition on viral trafficking. Furthermore, it is probable that the number of viral particles entering each cell is not equal which could distort the cell-to-cell quantitation comparison of HA distribution at the plasma membrane. A more accurate quantification would be to analyse the ratio of cytoplasmic to plasma membrane HA distribution between mock versus tubacin treated group. In addition, it is likely that the efficiency of tubacin infiltration was not equal across all cells. This is apparent since fold enrichment of tubulin acetylation in tubacin treated cells ranged from 1.09 to 2.75 times compared to control, indicating that some cells chosen for quantification may not have efficiently been treated with tubacin. A set threshold of tubulin acetylation due to HDAC6 inhibition could be required to observe a quantifiable difference in HA distribution. This makes sense, since the level of tubulin acetylation is correlated with increased trafficking (figure in appendix), and HDAC6 deacetylase inhibition by tubacin increases tubulin acetylation (reference).
Although non-significant, HDAC6 KD had a mean increase of 9.8% mean fold enrichment of HA at the apical surface. HDAC KD however did not lead to an increase in acetylated tubulin as shown by previous research (reference). Due to the importance of HDACs in normal cell function, there is a functional redundancy. It is possible that other cytoplasmic HDACs compensated for knockdown of HDAC6 polypeptide, signalled through some feedback mechanism. In fact, HDAC1 knockdown leads to upregulation of HDAC2 in F9 embryonal carcinoma and mouse epithelial cells (MEFs), and vice versa. It would be interesting to investigate the effect of HDAC6 overexpression on HA enrichment at the plasma membrane, which is a complementary experiment to HDAC6 knockdown. In addition, our lab has previously shown that HDAC6 overexpression, which results in a decrease in tubulin acetylation, leads to a decrease in viral release in A549 cells.
Previously our lab has shown through electron microscopy
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FE of Haemagglutinin
Acetylated Tubulin FE
Pearson correlation coefficient significance: Slopes P value <0.001
Acetylation threshold of 2 fold.
P value: 0.037
29.6% mean increase – 45 data points (15 cells each)