Histone Deacetylase 6 Inhibits Influenza A Virus Release by Downregulating the Trafficking of Viral Components to the Plasma Membrane via Its Substrate, Acetylated Microtubules

Abstract

Mammalian cells produce many proteins, such as IFITM3, ISG15, MxA, and viperin, that inhibit influenza A virus (IAV) infection. Here, we show that a new class of host protein, histone deacetylase 6 (HDAC6), inhibits IAV infection. We found that HDAC6-overexpressing cells release about 3-fold less IAV progeny, whereas HDAC6-depleted cells release about 6-fold more IAV progeny. The deacetylase activity of HDAC6 played a role in its anti-IAV function as tubacin, a specific small-molecule inhibitor of HDAC6, increased the release of IAV progeny in a dose-dependent manner. Further, as visualized by electron microscopy, tubacin-treated cells showed an increase in IAV budding at the plasma membrane, the site of IAV assembly. Tubacin is a domain-specific inhibitor and binds to one of the two HDAC6 catalytic domains possessing tubulin deacetylase activity. This indicated the potential involvement of acetylated microtubules in the trafficking of viral components to the plasma membrane. Indeed, as quantified by flow cytometry, there was about a 2.0-to 2.5-fold increase and about a 2.0-fold decrease in the amount of viral envelope protein hemagglutinin present on the plasma membrane of tubacin-treated/HDAC6-depleted and HDAC6-overexpressing cells, respectively. In addition, the viral ribonucleoprotein complex was colocalized with acetylated microtubule filaments, and viral nucleoprotein coimmunoprecipitated with acetylated tubulin. Together, our findings indicate that HDAC6 is an anti-IAV host factor and exerts its anti-IAV function by negatively regulating the trafficking of viral components to the host cell plasma membrane via its substrate, acetylated microtubules. IMPORTANCEHost cells produce many proteins that have the natural ability to restrict influenza virus infection. Here, we discovered that another host protein, histone deacetylase 6 (HDAC6), inhibits influenza virus infection. We demonstrate that HDAC6 exerts its anti-influenza virus function by negatively regulating the trafficking of viral components to the site of influenza virus assembly via its substrate, acetylated microtubules. HDAC6 is a multisubstrate enzyme and regulates multiple cellular pathways, including the ones leading to various cancers, neurodegenerative diseases, and inflammatory disorders. Therefore, several drugs targeting HDAC6 are under clinical development for the treatment of a wide range of diseases. Influenza virus continues to be a major global public health problem due to regular emergence of drug-resistant and novel influenza virus strains in humans. As an alternative antiviral strategy, HDAC6 modulators could be employed to stimulate the anti-influenza virus potential of endogenous HDAC6 to inhibit influenza virus infection.

Influenza A Virus Dysregulates Host Histone Deacetylase 1 That Inhibits Viral Infection in Lung Epithelial Cells

“…However, viruses have evolved their own strategies to antagonize those factors (3). Recently, we discovered that the human enzyme histone deacetylase 6 (HDAC6) has an anti-IAV function (4) and that IAV downregulates HDAC6 activity and induces a caspase-mediated cleavage of HDAC6 polypeptide to antagonize its antiviral function (5,6). These findings led to the hypothesis that other human HDACs potentially have a similar role in IAV infection.…”

“…Two main pathways, one mediated by proteasome and other mediated by lysosome, govern the degradation of proteins in eukaryotic cells. To identify the pathway leading to the degradation of HDAC1 polypeptide in IAV-infected cells, PR8-infected cells were treated with proteasome inhibitor MG132 (20 M) or lysosome inhibitor NH 4 Cl (20 mM); at these concentrations, MG132 and NH 4 Cl have been shown to inhibit proteasomal and lysosomal activity, respectively (20,21). The levels of HDAC1 polypeptide were then analyzed and quantified as described above.…”

“…Lastly, class IV has only one member, HDAC11, which shares homology with both class I and class II HDACs (12). Previously, we found that HDAC6, a class II HDAC, possesses an anti-IAV property and IAV undermines HDAC6 activity and integrity to limit its antiviral function (4)(5)(6). Likewise, we demonstrate here that HDAC1, a class I HDAC, also possesses anti-IAV properties and is an integral part of host type I interferon (IFN)-mediated response against IAV.…”

Cellular defence or viral assist: the dilemma of HDAC6

“…In fact, IAV ribonucleoprotein complex is colocalized with acetylated MTs and HDAC6 exerts its antiinfluenza virus activity by negatively regulating the trafficking of viral components to the assembly site via its substrate, acetylated MTs. As a result, inhibiting the deacetylase activity of HDAC6 by tubacin increases the release of IAV progeny in a dose-dependent manner [90]. Mechanistically, a recent report has demonstrated that the caspase-3 cleavage motif DMAD-S exists at the C terminus of HDAC6 between the SE14 domain and BUZ domain, and such cleavage removes most of the BUZ domain from HDAC6 in influenza virus-infected cells.…”

Microtubule Regulation and Function during Virus Infection

“…To prevent this, Ad5-encoded E4orf3 protein inactivates core components of host double-strand break repair complexes by targeting them to centrosome-resident aggresomes in an MT-and dynein-dependent manner (104,105). Influenza virus exploits aggresomes to promote infection by mimicking misfolded proteins to recruit HDAC6 and other aggresome factors to viral fusion sites (106), although HDAC6 has also been suggested to negatively impact influenza virus release due to effects on acetylated MTs that likely mediate egress (107). ASFV replication sites form at the centrosome and resemble aggresomes, potentially using the aggresome pathway for virus assembly (108).…”