viruses Review Ubiquitin in Influenza Virus Entry and Innate Immunity Alina Rudnicka 1 and Yohei Yamauchi 1,2,3,* 1 Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, Zurich CH-8057, Switzerland; [email protected] 2 School of Cellular and Molecular Medicine, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK 3 Structural Biology Research Center, Department of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan * Correspondence: [email protected]; Tel.: +44-117-331-2067 Academic Editors: Eric Freed and Thomas Klimkait Received: 14 September 2016; Accepted: 14 October 2016; Published: 24 October 2016 Abstract: Viruses are obligatory cellular parasites. Their mission is to enter a host cell, to transfer the viral genome, and to replicate progeny whilst diverting cellular immunity. The role of ubiquitin is to regulate fundamental cellular processes such as endocytosis, protein degradation, and immune signaling. Many viruses including influenza A virus (IAV) usurp ubiquitination and ubiquitin-like modifications to establish infection. In this focused review, we discuss how ubiquitin and unanchored ubiquitin regulate IAV host cell entry, and how histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase with ubiquitin-binding activity, mediates IAV capsid uncoating. We also discuss the roles of ubiquitin in innate immunity and its implications in the IAV life cycle. Keywords: ubiquitin; unanchored ubiquitin; HDAC6; aggresome processing; influenza virus; virus entry; virus uncoating; innate immunity; virus–host interactions 1. Ubiquitin and Ubiquitination Ubiquitin is a small, 8.5 kDa protein composed of 76 amino acids expressed in different tissues and present in different subcellular compartments. Post-translational attachment of ubiquitin to other proteins, namely ubiquitination, alters the function, location, or trafficking of the protein, or targets it for destruction by the 26S proteasome. The ability of ubiquitin to form structurally and functionally distinct polymers greatly increases the complexity of ubiquitination. Ubiquitin has a globular shape with the last four C-terminal residues (LRGG) extending from the compact structure. C-terminal glycine (G) can be covalently conjugated to proteins by isopeptide linkage to the "-amino group of lysine (K) residues or less frequently to the N-terminal α-amino group or the thiol group of cysteine residues. Ubiquitin itself contains eight amino groups to which another ubiquitin molecule can be conjugated: the "-amino groups of seven K residues (K6, 11, 27, 29, 33, 48 and 63) and the α-amino group of the N-terminal methionine residue. All of the eight ubiquitin chain types are present in the cell, among which the K48- and K63-linked chains are most abundant and the best described. K48-based linkages lead mainly to the proteasome-mediated degradation of the ubiquitinated protein, while K63-based chains control primarily protein endocytosis, trafficking, and enzyme activity [1–4]. Mechanistically, the process of protein ubiquitination involves a three-step enzymatic cascade, which starts with the ubiquitin-activating enzyme E1, followed by the ubiquitin-conjugating enzyme E2, and the ubiquitin ligase E3. Ubiquitin is activated in an ATP-dependent manner, when a high-energy thioester bond is formed between the C-terminus of ubiquitin and an internal cysteine residue of the ubiquitin-activating enzyme E1. Activated ubiquitin is then transferred onto the active site cysteine Viruses 2016, 8, 293; doi:10.3390/v8100293 www.mdpi.com/journal/viruses VirusesViruses2016 2016, 8,, 2938, 293 2 of2 16 of 15 ubiquitin‐activating enzyme E1. Activated ubiquitin is then transferred onto the active site cysteine ofof one one of of E2-conjugating E2‐conjugating enzymes. enzymes. Finally,Finally, the formation of of an an isopeptide isopeptide bond bond is iscatalyzed catalyzed by by E3 E3 ubiquitinubiquitin ligases, ligases, which which link link ubiquitin ubiquitin moietiesmoieties to target proteins proteins or or elongate elongate a apolyubiquitin polyubiquitin chain chain (Figure(Figure1)[ 1)5– [5–7].7]. Figure 1. Enzymatic pathway of protein ubiquitination. The attachment of ubiquitin (Ub) to proteins involves Figure 1. Enzymatic pathway of protein ubiquitination. The attachment of ubiquitin (Ub) to consecutiveproteins involvesaction of three consecutive classes of action enzymes: of three ubiquitin classes‐activating of enzymes: enzyme ubiquitin-activating E1, ubiquitin‐conjugating enzyme enzyme E1, E2,ubiquitin-conjugating and ubiquitin ligase E3. enzyme First, E2, the and C‐terminus ubiquitin of ligase ubiquitin E3. First, binds the to C-terminus E1 in an ATP of ubiquitin‐dependent binds manner. to UbiquitinE1 in an is ATP-dependent then transferred manner. from the Ubiquitin E1 to E2. is Finally, then transferred the E3 binds from both the E1the to ubiquitin E2. Finally,‐bound the E3E2 bindsand the substrateboth the and ubiquitin-bound catalyzes formation E2 and of an the isopeptide substrate bond and catalyzes between formationthe C‐terminus of an of isopeptide ubiquitin bondand the between substrate lysinethe residue. C-terminus The oflysines ubiquitin on the and substrate the substrate‐conjugated lysine ubiquitin residue. can The be lysinesfurther onpolyubiquitinated. the substrate-conjugated A multitude of ubiquitincellular proteins can be furtherthat contain polyubiquitinated. different ubiquitin A multitude‐binding domains—namely of cellular proteins ubiquitin that contain binding different proteins (UBPs)—mediateubiquitin-binding the domains—namelycellular functions of ubiquitin ubiquitination. binding Deubiquitinases proteins (UBPs)—mediate (DUBs) that the also cellular contain functions ubiquitin‐ of ubiquitination. Deubiquitinases (DUBs) that also contain ubiquitin-binding domains can revert or binding domains can revert or modify ubiquitination. DUBs vary in the specificity towards different types of modify ubiquitination. DUBs vary in the specificity towards different types of polyubiquitin linkages, polyubiquitin linkages, the position of cleavage within polyubiquitin chains, the ability to separate single the position of cleavage within polyubiquitin chains, the ability to separate single ubiquitin moieties, ubiquitinetc. Some moieties, DUBs produceetc. Some unanchored DUBs produce polyubiquitin unanchored chains polyubiquitin that regulate chains aggresome that regulate processing aggresome and processinginnate immunity. and innate The immunity.red ubiquitin The red depicts ubiquitin ubiquitin depicts that ubiquitin is anchored, that is and anchored, the blue andubiquitin the blue depicts ubiquitin depictsunanchored unanchored ubiquitin ubiquitin with with a free a free C-terminus. C‐terminus. In complex with E2, the E3 ubiquitin ligase forms an isopeptide bond between ubiquitin moieties or Inbetween complex ubiquitin with E2, and the substrate E3 ubiquitin protein. ligase In formsmost cases an isopeptide the type of bond linkage between is determined ubiquitin moietiesby E2 orenzymes, between ubiquitinexcept for andthe linkage substrate between protein. the In amino most group cases theof the type N‐ ofterminal linkage methionine is determined residue, by E2 enzymes,determined except by the for E3 the ubiquitin linkage ligase between called the linear amino ubiquitin group chain of the assembly N-terminal complex methionine (LUBAC) residue, [8]. determinedThe E3 ubiquitin by the ligases E3 ubiquitin determine ligase the calledsubstrate linear specificity ubiquitin of ubiquitination, chain assembly and complex the diversity (LUBAC) of the [ 8]. Thecellular E3 ubiquitin functions ligases of ubiquitination determine the substrateis reflected specificity in the existence of ubiquitination, of hundreds and theof different diversity E3s of the cellularin mammals, functions compared of ubiquitination with roughly is reflected thirty in‐five the E2s existence and only of hundreds two E1s of in different humans. E3s E3 in enzymes mammals, comparedare currently with roughly classified thirty-five into three E2s and main only families two E1s inwith humans. different E3 enzymes structural are currentlyand functional classified intocharacteristics: three main families the homologous with different to E6AP structural C‐terminus and (HECT) functional domain characteristics: family of ubiquitin the homologous ligases, the to E6APcullin C-terminus‐really interesting (HECT) new domain gene family (RING) of family ubiquitin of ubiquitin ligases, the ligases, cullin-really and the interesting U‐box containing new gene ubiquitin ligases [2,3,9]. E3 ligases can be single‐ or multi‐subunit enzymes; in the second case (RING) family of ubiquitin ligases, and the U-box containing ubiquitin ligases [2,3,9]. E3 ligases can be ubiquitin‐binding and substrate binding domains reside on separate polypeptides brought together single- or multi-subunit enzymes; in the second case ubiquitin-binding and substrate binding domains by adaptor proteins. reside on separate polypeptides brought together by adaptor proteins. Ubiquitinated substrates are subsequently recognized by a large number of proteins that contain Ubiquitinated substrates are subsequently recognized by a large number of proteins that contain different ubiquitin‐binding domains; among these are DUBs, a group of about 100 enzymes in differentmammals ubiquitin-binding that hydrolyze isopeptide domains; linkages