Canine Influenza Virus Is Mildly Restricted by Canine Tetherin Protein

viruses

Article Canine Inﬂuenza Virus is Mildly Restricted by Canine Tetherin Protein

Yun Zheng 1,2,3,†, Xiangqi Hao 1,2,3,†, Qingxu Zheng 1,2,3, Xi Lin 1,2,3, Xin Zhang 1,2,3, Weijie Zeng 1,2,3, Shiyue Ding 1, Pei Zhou 1,2,3,* and Shoujun Li 1,2,3,* 1 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; [email protected] (Y.Z.); [email protected] (X.H.); [email protected] (Q.Z.); [email protected] (X.L.); [email protected] (X.Z.); [email protected] (W.Z.); [email protected] (S.D.) 2 Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, Guangzhou 510642, China 3 Guangdong Provincial Pet Engineering Technology Research Center, Guangzhou 510642, China * Correspondence: [email protected] (P.Z.); [email protected] (S.L.) † These authors contributed equally to this work.

Received: 10 July 2018; Accepted: 10 October 2018; Published: 16 October 2018

Abstract: Tetherin (BST2/CD317/HM1.24) has emerged as a key host-cell ·defence molecule that acts by inhibiting the release and spread of diverse enveloped virions from infected cells. We analysed the biological features of canine tetherin and found it to be an unstable hydrophilic type I transmembrane protein with one transmembrane domain, no signal peptide, and multiple glycosylation and phosphorylation sites. Furthermore, the tissue expression profile of canine tetherin revealed that it was particularly abundant in immune organs. The canine tetherin gene contains an interferon response element sequence that can be regulated and expressed by canine IFN-α. A CCK-8 assay showed that canine tetherin was effective in helping mitigate cellular damage caused by canine influenza virus (CIV) infection. Additionally, we found that the overexpression of canine tetherin inhibited replication of the CIV and that interference with the canine tetherin gene enhanced CIV replication in cells. The impact of canine tetherin on CIV replication was mild. However, these results elucidate the role of the innate immune factor, canine tetherin, during CIV infection for the first time.

Keywords: canine tetherin; bioinformatics; canine inﬂuenza virus H3N2; canine inﬂuenza virus H5N1; innate immunity

1. Introduction In recent years, researchers have gradually discovered that humans and other mammals have various specific antiviral factors. In January 2008, the interferon-induced membrane protein tetherin (also known as CD317 or bone marrow stromal cell antigen 2 (BST2)) was identified by Neil. This protein can restrict the release of human immunodeficiency virus type 1 (HIV-1) particles from infected cells and can be counteracted by the HIV-1 protein Vpu [1]. Since then, many researchers have conducted additional studies of tetherin and have found that it exerts broad antiviral effects by targeting virion components of various enveloped viruses. Tetherin has been shown to restrict the extracellular release of retrovirus [2], filovirus (Ebola and Marburg viruses) [3,4], arenavirus (Lassa virus) [5], paramyxovirus (Nipah and Hendra viruses) [6], gamma-herpesvirus (Kaposi’s sarcoma-associated herpesvirus, KSHV) [7,8], rhabdovirus (vesicular stomatitis virus, VSV) [9], hepatitis C virus (HCV) [10], and Dengue virus [11]. Canine influenza virus (CIV) belongs to the influenza A virus (IAV) family, which is composed of enveloped viruses. CIV H3N2 circulates and causes epidemics primarily in Asian countries including

Viruses 2018, 10, 565; doi:10.3390/v10100565 www.mdpi.com/journal/viruses Viruses 2018, 10, 565 2 of 18

Korea and China [12–14]. However, in April 2015, an outbreak of CIV H3N2 occurred in Chicago and rapidly spread to several states in the United States [15]. Songserm first isolated the H5N1 virus from a dog and speculated that the virus may have been transmitted from infected ducks to dogs [16]. The highly pathogenic avian influenza (HPAI) H5N1 virus first appeared in southern China in 1996 [17]. This virus is a highly contagious and deadly pathogen that occurs mainly in birds but can cross phylogenetic barriers and infect different animal hosts [18–20]. Chen reported that dogs are highly susceptible to H5N1 and may serve as intermediate hosts before transfer to humans [21]. Some studies have shown that the release of influenza viruses from the cytomembranes of infected cells [22] occurs at the site of the antiviral activity of tetherin and that this activity restricts virus production. When this line of research was initially carried out, some researchers found that tetherin had little or no ability to inhibit influenza virus infection [23–25], although obvious inhibitory effects of tetherin were observed when virus-like particles (VLPs) were released [24]. In contrast, subsequent analyses demonstrated appreciable inhibition of the influenza virus release by tetherin [26–29] but the tetherin sensitivity of the influenza virus is believed to be strain-specific [29]. The current research on tetherin-resistant influenza viruses focuses on human tetherin and the H1N1 subtype but no reports on canine tetherin are available in this context. As IAVs differ considerably among different virus subtypes, extensive research is still needed to establish whether tetherin inhibits CIV replication. In our study, for the first time, we have analysed the bioinformatic characteristics of canine tetherin, including the properties of the sequence, secondary structure, transmembrane domains, and 3D simulation structure, using online bioanalytical software such as ProtParam, ProtScale, SOPMA, TMHMM, TargetP, SignalP, Motif Scan, InterProScan, and I-TASSER. In addition, we have shown that the release of both CIV H3N2 and CIV H5N1 from cells was inhibited by canine tetherin although tetherin’s ability to limit CIV replication is mild.

2. Materials and Methods

2.1. Cells and Viruses Human embryonic kidney cells (HEK 293T) and Madin–Darby canine kidney (MDCK) cells were obtained from the Shanghai Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences, China. Cells were propagated in a growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island-New York, NY, USA) supplemented with 10% foetal bovine serum (FBS; Biological Industries, Kibbutz Beit-Haemek, Israel) and 1% Glutamax (Gibco, Grand Island-New York, NY, ◦ USA) at 37 C and 5% CO2 until they reached ~80% confluence. The CIVs A/canine/Guangdong/02/2013 (H3N2) and A/canine/Guangdong/01/2013 (H5N1) were isolated in 2013 from dogs with severe respiratory symptoms in Guangdong, China. Virus stocks were propagated in the allantoic cavities of nine-day-old specific-pathogen-free (SPF) embryonated hens’ eggs (Merial, Beijing, China) at 37 ◦C for 48 h. Allantoic fluid containing viruses was harvested, aliquoted, and frozen at −80 ◦C until it was used in experiments. The plaque-forming units (PFU) of CIV H3N2 and CIV H5N1 were determined by plaque assays in MDCK cells. Virus titres in the supernatant were measured by the 50% egg infective dose (EID50). The Reed-Muench method was used to calculate the results. All experiments with live viruses were performed in an enhanced animal biosafety level 3 (ABSL-3) facility at the South China Agricultural University, Guangzhou, China. The protocol number was SYXK (YUE) 2016-0136.

2.2. Identiﬁcation and Cloning of the Canine Tetherin Gene Based on the NCBI data for canine tetherin (GenBank accession no. XM_860510.5), oligonucleotide primers were synthesized to correspond to the open reading frame (ORF) start (forward primer 50-ATGGCACCTACGCTTTACCAC-30) and stop (reverse primer 50-TCAGGCCAGCAGAGCC CTAAGG-30) codons of canine tetherin. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of a healthy beagle using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and subjected to Viruses 2018, 10, 565 3 of 18 real-time reverse transcription-polymerase chain reaction (RT-PCR). The amplified fragments were cloned into the plasmid p3×FLAG-CMV-10. The recombinant eukaryotic expression plasmid for canine tetherin was named FT. Then, FT was transiently transfected into 293T cells. Forty-eight hours post-transfection, the cells were lysed by P0013 (Beyotime, Shanghai, China). The cell lysates were separated on 12% gels by SDS-polyacrylamide gel electrophoresis (PAGE). The separated proteins were then transferred onto a nitrocellulose membrane and blocked with 5% dried milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 for 2 h at room temperature. The nitrocellulose membrane was incubated overnight at 4 ◦C with the appropriate primary antibodies, which included the anti-FLAG antibody (Sigma, Saint Louis, MO, USA), to detect canine tetherin and the anti-GAPDH antibody (Sigma). The membrane was washed three times with Tris-buffered saline with Tween 20 (TBST) for 5 min each time. Then, goat anti-mouse IgG (Odyssey, Lincoln, NE, USA)) was incubated with the nitrocellulose membrane for 1 h at room temperature. The results were detected using an infrared two-colour laser imaging system (Odyssey, Lincoln, NE, USA).

2.3. Bioinformatics Analysis The tetherin sequences from different species, including the chimpanzee (NM_001190480.1), domestic cat (NM_001243085.1), domestic ferret (XM_004761031.2), domestic guinea pig (XM_003464252.4), human (NM_004335.3), horse (XM_005604172.3), house mouse (NM_198095.2), pig (NM_001161755.1), rhesus monkey (NM_001161666.1), and tiger (XM_015540423.1), were downloaded from the NCBI. Through the neighbour joining method in MEGA7, a phylogenetic tree was obtained based on genetic distances. ProtScale (https://web.expasy.org/protscale/) was used to analyse the hydrophilicity of the protein. SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl) was used to analyse the secondary protein structure. The SignalP 4.1 Server (http://www.cbs.dtu.dk/ services/SignalP/) was used to analyse the protein signal peptide sequence, and the TMHMM Server v2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and InterProScan (http://www.ebi.ac.uk/ interpro/) were used to predict the transmembrane and functional domains of the protein. Motif Scan (https://myhits.isb-sib.ch/cgi-bin/motif_scan) was used to analyse various post-translational modiﬁcation sites in the protein. GPI (glycosylphosphatidylinositol) Modiﬁcation Site Prediction (http://mendel.imp.ac.at/sat/gpi/gpi_server.html) was used to predict the anchor point of canine tetherin. I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) was used to simulate the 3D structure of the protein.

2.4. G418 Screening for Cell Lines with Stable Expression The plasmid FT was transiently transfected into MDCK cells using LipofectamineTM 3000 transfection reagent (Thermo Fisher Scientific, Carlsbad, CA, USA), according to the manufacturer’s instructions. The empty vector p3×FLAG-CMV-10 (F) was transfected into another set of MDCK cells to generate a control cell line. First, we determined the optimal concentrations of G418 to screen cells. In brief, the control cell suspension was diluted to 1000 cells/mL and 100 µL of the cell suspension was added to each well of a 24-well plate. Complete culture medium, containing G418, was pre-added to each well. The concentration of G418 was diluted to 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, and 1100 µg/mL in separate wells. At 10–14 days after transfection, the lowest concentration at which all cells died was defined as the baseline concentration. The optimal concentration was one level above the baseline concentration. Then, we began to screen cells. The culture medium was changed every 3–5 days according to the colour of the culture medium and the degree of cell growth. When a large number of cells die, the G418 concentration can be reduced by half to maintain the screening pressure. After 14 days of screening, resistant clones were observed. After gradual expansion, a single clonal cell line was prepared into a cell suspension; the cells were counted, and the suspension was diluted to 1 cell/10 µL of medium. Fresh medium (150 µL/well) was added to the 96-well plate and then the cell suspension was added at a volume of 10 µL/well. After the single clone was gradually expanded further, it was transferred to a 48-well plate for proliferation. The normal expression of the Viruses 2018, 10, 565 4 of 18 target protein was detected through an indirect immunofluorescence assay (IFA), and the mRNA of canine tetherin was measured by real-time fluorescence quantitative PCR (RTFQ-PCR).

2.5. CCK-8 Assay Cells were seeded in a 96-well plate at a density of 104–105 cells/well in 100 µL of culture medium for ◦ testing. The cells were cultured in a CO2 incubator at 37 C for 24 h. For CIV infection, the culture medium was removed and the cells were gently washed with pre-warmed phosphate-buffered saline (PBS) and then infected with CIV H3N2 at a multiplicity of infection (MOI) of 0.1 and with CIV H5N1 at an MOI of 0.01. After 1 h of incubation at 37 ◦C, the medium containing the viruses was removed, the cells were gently washed with pre-warmed PBS, and fresh infection medium was added. Both blank (no cells) and negative (uninfected cells) groups were established. At 6, 12, 18, 24, 30, 36, and 48 h after infection, 10 µL of CCK-8 solution was added to each well of the plate, which was then incubated for 2 h in an incubator. Importantly, before the plate was read, it was agitated gently on an orbital shaker for 1 min to ensure a homogeneous distribution of colour. The absorbance was measured at 450 nm using a microplate reader.

2.6. Tetherin Interference Experiment For downregulation of canine tetherin mRNAs, small interfering RNAs (siRNAs) with a custom-designed target sequence or a scrambled sequence were purchased from RiboBio Biotechnology. The MDCK cells were seeded in 12-well plates at a density of 1.1 × 105 cells/well. After 24 h, the cells were transfected with 50 nM siRNA specific for canine tetherin or with nonsense scrambled siRNA as a control using Lipofectamine® 3000 according to the manufacturer’s protocol. At 24, 36, and 48 h after transfection, the effect of siRNA interference was examined by RTFQ-PCR. For this purpose, the canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was quantified in parallel. The programme used on the Roche 480 LightCycler included an initial denaturation step at 95 ◦C for 15 min and a melting step after amplification (65 ◦C to 97 ◦C; temperature transition rate, 0.11 ◦C/s). Tetherin cDNA was amplified using 500 nM sense primer and 500 nM antisense primer. PCR amplification and analysis were performed at 94 ◦C for 20 s and 60 ◦C for 34 s for 40 cycles with a temperature transition rate of 4.4 ◦C/s. The samples were measured in triplicate. The relative expression levels of tetherin mRNA were calculated by the 2−∆∆CT method.

2.7. Indirect Immunofluorescence and Laser Scanning Confocal Microscopy Localization experiments were performed using IFAs and laser scanning confocal microscopy. Briefly, MDCK cells grown on glass coverslips were transiently transfected with the appropriate tetherin expression constructs by employing Lipofectamine® 3000. At 24 h after transfection, the cells were infected with CIV H3N2 at an MOI of 0.1, as described previously. At 48 h after infection, the cells were fixed with 4% paraformaldehyde-PBS. The cells were incubated with an anti-hemagglutinin (HA) antibody (Sigma, Saint Louis, MO, USA) to detect the HA protein and with anti-FLAG antibody (Sigma, Saint Louis, MO, USA) to detect canine tetherin and then they were stained using secondary antibodies conjugated with fluorescein isothiocyanate (FITC) and cyanine-3 (Cy3). After mounting on slides using antifade 40,6-diamidino-2-phenylindole (DAPI) mounting solution (Sigma), the cells were visualized with a Leica DM-IRE2 confocal microscope.

3. Results

3.1. Bioinformatic Analysis of Canine Tetherin The amplified canine tetherin gene (see Figure1A) was completely consistent with the NCBI reference sequence (XM_860510.5). EditSeq software analysis showed that the 567 nucleotides of the canine tetherin coding sequence (CDS) encode 188 amino acids (aa) whose total molecular weight is 20,626.84 daltons. MegAlign software analysis (see Figure1B) showed that canine tetherin had 42, 42, 60.4, 54, 30.5, 41.9, 37.3, 42.6, 42.2, and 58.3% amino acid sequence identity with the human, Viruses 2018, 10, 565 5 of 18 chimpanzee, domestic cat, domestic ferret, domestic guinea pig, horse, house mouse, pig, rhesus monkey, and tiger homologues, respectively. Phylogenetic trees (see Figure1C) showed that the canine tetherinViruses gene 2018 was, 10, x closely FOR PEER related REVIEW to the cat and tiger genes but was far from those of5 humans of 18 and other species. To further analyse the characteristics of the canine tetherin gene sequence, we used MegAlign60.4, software 54, 30.5, to41.9, identify 37.3, 42.6, key 42.2, amino and acids 58.3% by amino comparing acid sequence them withidentity the with human the sequence.human, The chimpanzee, domestic cat, domestic ferret, domestic guinea pig, horse, house mouse, pig, rhesus results (see Figure1D) showed that canine tetherin has three conservative cysteine residues, two monkey, and tiger homologues, respectively. Phylogenetic trees (see Figure 1C) showed that the glycosylationcanine tetherin sites, and gene a was GPI closely anchor related point, to the which cat and is consistent tiger genes but with was the far human from those sequence. of humans These are the keyand positions other species. required To further for tetherin analyse the to characterist maintainics its of specific the canine topological tetherin gene structure sequence, andwe used to exert its antiviralMegAlign function software and undergo to identify post-translational key amino acids by modification comparing them [30]. with To further the human analyse sequence. the constitutiveThe characteristicsresults (see of canineFigure 1D) tetherin showed protein, that canine we conducted tetherin has a three secondary conservative structure cysteine prediction. residues, two The results showedglycosylation that the secondary sites, and a structureGPI anchor ofpoint, canine which tetherin is consistent contains with the 52.13% humanα sequence.-helices, These 6.91% areβ -turns, the key positions required for tetherin to maintain its specific topological structure and to exert its 17.55% extended strands, and 23.4% random coils. More α-helices exist in the secondary structure antiviral function and undergo post-translational modification [30]. To further analyse the of humanconstitutive tetherin, characteristics which includes of canine 57.22% tetherinα-helices, protein, we 6.11% conducted beta-turns, a second 18.33%ary structure extended prediction. strands, and 18.33%The random results coils.showed The that transmembranethe secondary structure and of functional canine tetherin domains contains of 52.13% tetherin α-helices, were predicted6.91% by TMHMMβ-turns, and 17.55% InterProScan. extended Thestrands, results and 23.4% (see Figure random2B) coils. show More that α-helices in human exist in tetherin, the secondary amino acids (aa) 1–21structure constitute of human the cytoplasmic tetherin, which domain, includes aa 57.22% 22–44 α-helices, constitute 6.11% the beta-turns, transmembrane 18.33% extended region, and aa strands, and 18.33% random coils. The transmembrane and functional domains of tetherin were 45–180 constitute the noncytoplasmic domain, including a coil consisting of aa 99–147. This protein predicted by TMHMM and InterProScan. The results (see Figure 2B) show that in human tetherin, is a typeamino II transmembrane acids (aa) 1–21 constitute protein. the However, cytoplasmi caninec domain, tetherin aa 22–44 has constitute an opposite the transmembrane membrane topology comparedregion, to humanand aa 45–180 tetherin. constitute In canine the noncytoplasmic tetherin, aa domain, 1–31 constitute including a the coil noncytoplasmicconsisting of aa 99–147. domain, aa 32–53 constituteThis protein the is a transmembranetype II transmembrane region, protein. and However, aa 54–188 canine constitute tetherin has the an cytoplasmic opposite membrane domain (see Figure2topologyA). Canine compared tetherin to human is a type tetherin. I transmembrane In canine tetherin, protein. aa 1–31 Due constitute to the differencethe noncytoplasmic in membrane topologydomain, between aa 32–53 canine constitute and human the transmembrane tetherin, the simulatedregion, and 3Daa 54–188 structure constitute of tetherin the cytoplasmic was analysed by domain (see Figure 2A). Canine tetherin is a type I transmembrane protein. Due to the difference in I-TASSER. In this study, the most reliable model was selected as the simulated 3D structure of tetherin membrane topology between canine and human tetherin, the simulated 3D structure of tetherin was protein.analysed The spatial by I-TASSER. structures In ofthis canine study, tetherinthe most (seereliable Figure model2C) was and selected human as tetherin the simulated (see Figure3D 2D) were foundstructure to be of similar;tetherin protein. both had The multiple spatial structures ligand binding of canine sites tetherin able (see to bindFigure ions 2C) orand nucleic human acids or becometetherin phosphorylated (see Figure 2D) to exertwere found important to be similar; biological both functions.had multiple ligand binding sites able to bind ions or nucleic acids or become phosphorylated to exert important biological functions.

Figure 1. Bioinformatic analysis of the canine tetherin gene sequence. (A) Ampliﬁcation of the Figure 1. Bioinformatic analysis of the canine tetherin gene sequence. (A) Amplification of the canine canine tetherin gene by RT-PCR. M: DL2000 DNA Marker; (B) comparison of amino acid homology; tetherin gene by RT-PCR. M: DL2000 DNA Marker; (B) comparison of amino acid homology; (C) (C) phylogenetic tree analysis of the tetherin gene; (D) comparison of the sequences of canine tetherin phylogenetic tree analysis of the tetherin gene; (D) comparison of the sequences of canine tetherin and human tetherin. Red represents amino acid homology. Blue represents amino acid inconsistency. Green triangles represent cysteine residues. Yellow triangles represent glycosylation sites. The red box represents the GPI anchor point. Viruses 2018, 10, x FOR PEER REVIEW 6 of 18

and human tetherin. Red represents amino acid homology. Blue represents amino acid inconsistency. Green triangles represent cysteine residues. Yellow triangles represent glycosylation sites. The red Viruses 2018 10 box represents, , 565 the GPI anchor point. 6 of 18

Figure 2. Prediction of the transmembrane structures and 3D structures of human tetherin and canine Figure 2. Prediction of the transmembrane structures and 3D structures of human tetherin and canine tetherin. (A) Transmembrane structure of canine tetherin; (B) transmembrane structure of human tetherin. (A) Transmembrane structure of canine tetherin; (B) transmembrane structure of human tetherin; (C) simulated 3D structure of canine tetherin; (D) simulated 3D structure of human tetherin. tetherin; (C) simulated 3D structure of canine tetherin; (D) simulated 3D structure of human tetherin. I-TASSER uses the SPICKER programme to analyse all the protein structures in the PDB database and I-TASSER uses the SPICKER programme to analyse all the protein structures in the PDB database and report the five most likely structural cluster models. The feasibility of each model is quantified by the report the five most likely structural cluster models. The feasibility of each model is quantified by the c-score (−5, 2) A higher c-score for a model corresponds to a greater feasibility of the model. Our study c-score (−5, 2) A higher c-score for a model corresponds to a greater feasibility of the model. Our study selected the most reliable model to simulate the 3D structures of canine tetherin and human tetherin. selected the most reliable model to simulate the 3D structures of canine tetherin and human tetherin. 3.2. Analysis of Canine Tetherin Expression Profiles in Tissues 3.2. Analysis of Canine Tetherin Expression Profiles in Tissues To understand the distribution of tetherin in the tissues and organs of dogs, we collected samples fromTo several understand sites (including the distribution the heart, of tetherin liver, spleen, in the tissues lung, kidney,and organs brain, of do stomach,gs, we collected trachea, samples skeletal muscle,from several thymus, sites small (includi intestine,ng the largeheart, intestine, liver, spleen, and lymph lung, kidney nodes),from brain, three stomach, healthy trachea, beagles. skeletal Gene expressionmuscle, thymus, in the small heart wasintestine, selected large as intestine, a reference an tod calculatelymph nodes) fold changes.from three The healthy relative beagles. quantitative Gene resultsexpression were in calculatedthe heart was using selected the 2− as∆∆ aC referenceT method. to Thecalculate results fold (see changes. Figure 3The) show relative that quantitative the canine tetherinresults were gene wascalculated expressed using in allthe of 2 the−ΔΔC collectedT method. tissues The results and organs (see butFigure at different 3) show levels. that Thethe canine tetherin expressiongene was expressed level was in lowest all of inthe the collected brain (an tissues average and fold organs change but ofat di 0.32fferent compared levels. to The the canine heart) buttetherin it was expression especially level high inwas organs lowest of in the the immune brain (an system, average including fold change the spleen of 0.32 (average compared fold change to the 2.98),heart) thymusbut it was (average especially fold high change in organs 6.21), and of the lymph immune nodes system, (average including fold change the spleen 8.63). (average These results fold suggestchange 2.98), that caninethymus tetherin (average may fold play change an important 6.21), and rolelymph in thenodes immune (average response. fold change Low 8.63). expression These levelsresults were suggest found that in thecanine heart, tetherin kidneys, may stomach, play skeletalan important muscles, role and in large the intestine.immune Canineresponse. tetherin Low was expressed to some extent in the lung (average fold change 3.06) and trachea (average fold change 0.89), where the CIV usually infects and replicates. Viruses 2018, 10, x FOR PEER REVIEW 7 of 18 Viruses 2018, 10, x FOR PEER REVIEW 7 of 18 expressionexpression levels levels were were found found in in the the heart, heart, kidneys, kidneys, stomach, stomach, skeletal skeletal muscles, muscles, and and large large intestine. intestine. Canine tetherin was expressed to some extent in the lung (average fold change 3.06) and trachea VirusesCanine2018 ,tetherin10, 565 was expressed to some extent in the lung (average fold change 3.06) and trachea7 of 18 (average(average fold fold change change 0.89), 0.89), where where the the CIV CIV usually usually infects infects and and replicates. replicates.

Figure 3. The distribution of the canine tetherin gene in various tissues. Samples were analysed in FigureFigure 3. 3.The The distribution distribution of of the the canine canine tetherin tetherin gene gene in in various various tissues. tissues. Samples Samples were were analysed analysed in in triplicate,triplicate, andand GAPDH GAPDH (Glyceraldehyde-3-phosphate (Glyceraldehyde-3-phosphate dehydrogenase dehydrogenase gene) was gene) used was as an used endogenous as an triplicate, and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase−∆∆CT gene) was used as an control for normalization. Fold changes were calculated using the 2 method.−ΔΔC TheT error bars endogenous control for normalization. Fold changes were calculated using the 2 −ΔΔ Cmethod.T The representendogenous the standard control deviation.for normalization. Fold changes were calculated using the 2 method. The errorerror bars bars represent represent the the standard standard deviation. deviation. 3.3. Canine Tetherin Protein Expression and Cellular Localization 3.3.3.3. Canine Canine Tetherin Tetherin Protein Protein Expression Expression and and Cellular Cellular Localization Localization The recombination eukaryotic expression plasmid FT and the empty vector F were transiently transfectedTheThe recombination recombination into MDCK cellseukaryotic eukaryotic to determine expression expression the expressionplasmi plasmid dFT FT and and and subcellular the the empty empty localizationvector vector F Fwere were of transiently the transiently fusion proteintransfectedtransfected by IFA into into and MDCK MDCK western cells cells blot. to to determine Thedetermine IFA results the the expr expr (seeessionession Figure and and4A) subcellular subcellular show that localization greenlocalization ﬂuorescence of of the the fusion wasfusion observedproteinprotein by by after IFA IFA incubationand and western western with blot. blot. the The The FITC-labelled IFA IFA results results (s secondary(seeee Figure Figure 4A) antibodies. 4A) show show that that The green green western fluorescence fluorescence blot results was was (seeobservedobserved Figure after4 B)after show incubation incubation that the with with canine the the tetherinFITC-labelled FITC-labelled signal secondary was secondary heterogeneous antibodies. antibodies. due The toThe awestern complexwestern blot glycosylationblot results results (see (see patternFigureFigure 4B) that 4B) show causedshow that that tetherin the the canine canine to migrate tetherin tetherin on signal SDS-polyacrylamide signal was was heterogeneous heterogeneous gels asdue due three to to a bands acomplex complex between glycosylation glycosylation 15 kDa andpatternpattern 35 kDa. that that Thesecaused caused results tetherin tetherin are to similarto migrate migrate to on observations on SDS-po SDS-polyacrylamidelyacrylamide of human tetherin,gels gels as as three whosethree bands bands major between between signals 15 range15 kDa kDa and 35 kDa. These results are similar to observations of human tetherin, whose major signals range fromand 28 35 to kDa. 40 kDa These [26 ].results are similar to observations of human tetherin, whose major signals range fromfrom 28 28 to to 40 40 kDa kDa [26]. [26].

Figure 4. IFA (Immunoﬂuorescence assay) and western blot detection of recombinant canine tetherin proteinFigureFigure 4. expression. 4.IFA IFA (Immunofluorescence (Immunofluorescence (A) IFA results in assay) MDCK assay) and cells;and western western (B) Western blot blot detection blotdetection analysis of of recombinant recombinant of canine tetherin canine canine tetherin protein. tetherin M:proteinprotein protein expression. expression. standard (A marker;()A IFA) IFA results results FT: expression in in MDCK MDCK cells; product cells; (B ()B Western) of Western p3×FLAG-CMV10-Tetherin; blot blot analysis analysis of of canine canine tetherin tetherin F: expression protein. protein. productM:M: protein protein of the standard standard empty vector.marker; marker; FT: FT: expression expression prod productuct of of p3×FLAG-CMV10-Tetherin; p3×FLAG-CMV10-Tetherin; F: F:expression expression productproduct of of the the empty empty vector. vector. To examine the subcellular localization of canine tetherin relative to sites of CIV budding, we probedToTo surfaceexamine examine tetherin the the subcellular subcellular and HA localizationlocalization localization in of CIVof canine canine H3N2-infected tetherin tetherin relative relative MDCK to to cellssites sites usingof of CIV CIV laser budding, budding, scanning we we confocalprobedprobed surface microscopysurface tetherin tetherin (see and and Figure HA HA localization5 ).localization Anti-FLAG-tetherin in in CIV CIV H3N2-infected H3N2-infected strongly labelledMDCK MDCK cells the cells surfacesusing using laser laser of infectedscanning scanning MDCKconfocalconfocal cells. microscopy microscopy Individual (see (see cells Figure Figure showed 5). 5). Anti-FLAG-tetherin variable Anti-FLAG-tetherin tetherin levels strongly strongly after infection labelled labelled (seethe the Figuresurfaces surfaces5C). of of Infectedinfected infected cellsMDCKMDCK were cells. cells. labelled Individual Individual with an cells anti-HAcells showed showed antibody variable variable (see tetherin Figuretetherin5 levelsB). levels High-magniﬁcation after after infection infection (see (see images Figure Figure of 5C). MDCK 5C). Infected Infected cells showcellscells were that were thelabelled labelled distributions with with an an ofanti-HA anti-HA surface antibody tetherin antibody and(see (see viralFigure Figure HA 5B). overlapped5B). High-magnification High-magnification substantially images images (see Figure of of MDCK MDCK5D). cellscells show show that that the the distributions distributions of of surface surface tetherin tetherin and and viral viral HA HA overlapped overlapped substantially substantially (see (see Figure Figure 5D).5D).

Viruses 2018, 10, 565 8 of 18 Viruses 2018, 10, x FOR PEER REVIEW 8 of 18

Figure 5. Co-localization of HA and canine tetherin in MDCK cells. (A) DAPI (40,6-Diamidino Figure 5. Co-localization of HA and canine tetherin in MDCK cells. (A) DAPI (4′,6-Diamidino-2- -2-phenylindole) -positive cells; (B) CIV H3N2 HA protein is marked by FITC; (C) Canine tetherin is phenylindole) -positive cells; (B) CIV H3N2 HA protein is marked by FITC; (C) Canine tetherin is marked by Cy3; (D) A through C merged; (E) Canine tetherin is marked by Cy3 in the uninfected marked by Cy3; (D) A through C merged; (E) Canine tetherin is marked by Cy3 in the uninfected control cells; (F) HA protein is marked by FITC in the infected cells without canine tetherin. control cells; (F) HA protein is marked by FITC in the infected cells without canine tetherin. 3.4. The Expression of Canine Tetherin Is Inducible by IFN-α and CIV Infection 3.4. The Expression of Canine Tetherin Is Inducible by IFN-α and CIV Infection Interferons (IFNs) are very important components of innate immunity. When a host is infected withInterferons a virus, cells (IFNs) detect are the very presence important of pathogen-associated components of innate molecular immunity. patterns When (PAMPs) a host is throughinfected patternwith a virus, recognition cells detect receptors the (PRRs).presence The of activatedpathogen-associated receptors then molecular recruit adaptor patterns proteins (PAMPs) and through trigger apattern series ofrecognition signalling receptors cascade reactions, (PRRs). The ultimately activate activatingd receptors the expressionthen recruit of adaptor IFNs. The proteins binding and of IFNstrigger to cella series surface of signalling receptors elicits cascade signals reactions, that induce ultimately the expression activating of the IFN-stimulated expression of genes IFNs. (ISGs). The Tetherinbinding isof aIFNs recently to cell described surface ISGreceptors with antiviral elicits signals activity that [31 induce]. To study the expression this mechanism, of IFN-stimulated we analysed thegenes nucleotide (ISGs). Tetherin sequences is of a therecently promoter described region ofISG canine with tetherinantiviral and activity the 50 -untranslated[31]. To study region this (5mechanism,0UTR) obtained we analysed from the the 50 -RACEnucleotide analysis sequences (see Figureof the 6promoterA). The sequenceregion of ofcanine the human tetherin tetherin and the promoter5′-untranslated region region (GenBank (5′UTR) accession obtained no. from KP316196.1) the 5′-RACE contains analysis several (see potential Figure 6A). transcription The sequence factor of bindingthe human sites tetherin that may promoter bind to region nuclear (GenBank factor of activatedaccession T-cellsno. KP316196.1) (NF-AT) or cont to proteinsains several of the potential signal transducertranscription and factor activator binding of transcriptionsites that may (STAT) bind to family. nuclear The factor sequence of activated also contains T-cells two(NF-AT) interferon or to responseproteins of elements, the signal namely, transducer interferon and activator regulatory of factortranscription (IRF) and (STAT) interferon-stimulated family. The sequence response also elementcontains(ISRE). two interferon We speculated response that elements, canine tetherin namely, protein interferon expression regulatory was regulatedfactor (IRF) by and IFN interferon- induction. stimulated response element (ISRE). We speculated that canine tetherin protein expression was regulated by IFN induction.

Viruses 2018, 10, 565 9 of 18 Viruses 2018, 10, x FOR PEER REVIEW 9 of 18

FigureFigure 6. The 6. expressionThe expression of canineof canine tetherin tetherin is is inducible inducible by by IFN- IFN-αα.(. (A) Nucleotide Nucleotide sequence sequence alignment alignment of 0 0 the 5 offlanking the 5′ flanking regions regions of canine of canine tetherin tetherin and human and human tetherin. tetherin. The The sequence sequence from from 5 RACE5′ RACE of of the the canine tetherincanine gene tetherin and the gene putative and the promoter putative region promoter were region compared were compared with those with of the those human of the tetherin human gene. Putativetetherin regulatory gene. Putative elements regulatory conserved elements across conserved different across species different are shadedspecies are in red.shaded The in translationalred. The start codonstranslational are shadedstart codons in purple; are shaded (B) induction in purple; of (B tetherin) induction expression of tetherin by expression IFN-α at different by IFN-α dilution at ratios;different (C) the dilution level of canineratios; ( tetherinC) the level expression of canine in tetherin infected expr MDCKession cells; in infected (D) the MDCK level ofcells; canine (D) the tetherin expressionlevel of in canine infected tetherin canine expression lungs. Nine in infected uninfected canine beagles lungs. Nine were uninfected randomly beagles divided were into randomly the following divided into the following groups: H3N2, H5N1 and control (mock-infected dogs). In the H3N2 and groups: H3N2, H5N1 and control (mock-infected dogs). In the H3N2 and H5N1 groups, dogs were 6.0 H5N1 groups, dogs were inoculated6.0 intranasally with 10 -times the 50% tissue culture infective dose inoculated intranasally with 10 -times the 50% tissue culture infective dose (TCID50) of the virus. (TCID50) of the virus. Control dogs received 1 mL of phosphate-buffered saline (PBS). The dogs were Controleuthanized dogs received at 3 days 1 mLpost-infection of phosphate-buffered (dpi) in each group saline based (PBS). on The previous dogswere studies. euthanized Samples were at 3 days post-infectionanalysed in (dpi) triplicate in each and group GAPDH based was on used previous as an endogenous studies. Samples control werefor normalization. analysed in triplicateFold and GAPDHchanges were was usedcalculated as an using endogenous the 2−ΔΔC controlT method. for Each normalization. experiment was Fold conducted changes werethree calculatedtimes − usingindependently the 2 ∆∆CT method. and the data Each analysis experiment was conducted was conducted in SPSS (Statistical three times Program independently for Social Sciences) and the data analysiswith was independent conducted t-tests; in SPSS a p value (Statistical less than Program 0.05 was for considered Social Sciences) statistically with significant independent (** p < t0.01).-tests; a p valueThe less error than bars 0.05 represent was considered the standard statistically deviation. significant (** p < 0.01). The error bars represent the standard deviation. To confirm that the expression of canine tetherin is induced by type I IFNs, we treated MDCK Tocells confirm with different that dilutions the expression of canine of IFN- canineα (Kingfisher tetherin Biotech, is induced Saint Paul, by MN, type USA) I IFNs, for 24 we h. The treated MDCKexpression cells with level different of canine dilutions tetherin was of caninemeasured IFN- by αRTFQ-PCR.(Kingfisher IFN- Biotech,α was found Saint to Paul, significantly MN, USA) increase canine tetherin expression in MDCK cells (see Figure 6B). However, an obvious dose- for 24 h. The expression level of canine tetherin was measured by RTFQ-PCR. IFN-α was found to dependent relationship was observed between IFN-α and canine tetherin expression. The average significantly increase canine tetherin expression in MDCK cells (see Figure6B). However, an obvious fold changes were 56.5, 26.8, 6.2, 3.4, 2.4, and 1.7 at canine IFN-α dilutions of 10,000, 5000, 2500, 1000, dose-dependent100, and 10 units/mL, relationship respectively. was observed Altogether, between thes IFN-e resultsα and confirmed canine tetherin that the expression.expression of The canine average fold changestetherin is were inducible 56.5, by 26.8, IFN- 6.2,α. 3.4, 2.4, and 1.7 at canine IFN-α dilutions of 10,000, 5000, 2500, 1000, 100, and 10In units/mL,addition, CIV respectively. infection results Altogether, in secretion these of type results I IFNs confirmed [32]. Tetherin that is the a stimulus-response expression of canine tetheringene is of inducible IFNs. Therefore, by IFN- αwe. verified the changes in canine tetherin expression in MDCK cells in Inresponse addition, to CIV CIV infection. infection We results found in(see secretion Figure 6C) of th typeat the I IFNsexpression [32]. levels Tetherin of canine is a stimulus-response tetherin were genesignificantly of IFNs. Therefore, elevated in we both verified CIV theH3N2-infec changested in and canine CIV tetherin H5N1-infected expression cells inand MDCK that the cells in responseexpression to CIV of infection. canine tetherin We found increa (seesed with Figure the6 durationC) that theof infection. expression In addition, levels of a statistical canine tetherin analysis were showed that the ability of CIV H5N1 to induce canine tetherin expression was significantly stronger significantly elevated in both CIV H3N2-infected and CIV H5N1-infected cells and that the expression than that of CIV H3N2 at 36 h and 48 h (p < 0.01). of canine tetherin increased with the duration of infection. In addition, a statistical analysis showed that the ability of CIV H5N1 to induce canine tetherin expression was significantly stronger than that of CIV H3N2 at 36 h and 48 h (p < 0.01). Viruses 2018, 10, 565 10 of 18

We also verified that canine tetherin expression changed in canine lungs infected with CIV H3N2 and CIVViruses H5N1. 2018, 10 We, x FOR found PEER REVIEW that CIV significantly increased canine tetherin expression in all10 of infected 18 lungs (p < 0.01) compared with the lungs of the control group. Moreover, the increase in the canine tetherin expressionWe also verified level inthat lungs canine infected tetherin with expression CIV H5N1 changed was in greatercanine lungs than infected that in lungswith CIV infected H3N2 with CIV H3N2.and CIV This H5N1. difference We found may that be CIV related significantly to the differentincreased virulencecanine tetherin and pathogenicityexpression in all of infected CIV H5N1 and H3N2.lungs (p Therefore, < 0.01) compared CIV infection with thecan lungs trigger of the tetherincontrol group. expression Moreover, in susceptible the increase cells in the containing canine a functionaltetherin IFN expression system. level in lungs infected with CIV H5N1 was greater than that in lungs infected with CIV H3N2. This difference may be related to the different virulence and pathogenicity of CIV 3.5. CCK-8H5N1 Assayand H3N2. Therefore, CIV infection can trigger tetherin expression in susceptible cells containing a functional IFN system. CCK-8 provides a tool for studying the induction and inhibition of cell proliferation in any in vitro model.3.5. In CCK-8 this study, Assay we used CCK-8 to determine whether MDCK cells that expressed tetherin had an increasedCCK-8 cell viability provides and a increasedtool for studying resistance the toinduction the CIV. and We inhibition acquired of cell cell lines proliferation with stable in expressionany in throughvitro G418 model. selection In this study, in advance we used (see CCK-8 Figure to de7A–C).termine After whether the MDCK virus infectedcells that theexpressed MDCK tetherin cells that stablyhad expressed an increased tetherin cell andviability the controland increased MDCK resistance cells, cell to viabilitythe CIV. wasWe acquired evaluated cell at lines 6, 12, with 18, stable 24, 30, 36, and 48expression h. In the through H3N2 group G418 selection (see Figure in advance7D), the (see results Figure showed 7A–C). that After the the cell virus viability infected increased the MDCK from 0 to 12cells h and that then stably gradually expressed decreased. tetherin and In the control control cells, MDCK the cells, maximum cell viability average was valueevaluated of cell at 6, viability 12, was 1.3318, 24, at 30, 12 36, h and and the48 h. minimum In the H3N2 average group (see value Figure of cell 7D), viability the results was showed 0.46 at that 48 the h. Incell the viability cells that increased from 0 to 12 h and then gradually decreased. In control cells, the maximum average value stably expressed tetherin, however, the maximum average value of cell viability was 1.35 at 12 h and of cell viability was 1.33 at 12 h and the minimum average value of cell viability was 0.46 at 48 h. In the minimumthe cells that average stably valueexpressed of cell tetherin, viability however, was 0.7 the at maximum 48 h. In theaverage H5N1 valu groupe of cell (see viability Figure 7wasE), the viability1.35 of at cells12 h withand the stable minimum tetherin average expression value of was cellhigher viability than was that 0.7 at of 48 control h. In the cells H5N1 at 12, group 18, and(see 24 h (p < 0.05).Figure After 7E), 30the h,viability no difference of cells with was observed;stable tether likelyin expression due tothe was rapid higher replication than that of of control H5N1. cells Overall, both CIVat 12, H3N2 18, and and 24 h CIV (p < H5N1 0.05). After successfully 30 h, no difference infected cells. was ob Theserved; cells likely with stabledue to tetherinthe rapid expressionreplication had a greaterof H5N1. viability Overall, and CIVboth resistanceCIV H3N2 thanand CIV the cellsH5N1 transfected successfully with infected an empty cells. The vector, cells suggestingwith stable that caninetetherin tetherin expression can help had cells a greater effectively viability resist and damageCIV resistance caused than by the viral cells invasion. transfected Uninfected with an empty control cells (vector,±tetherin) suggesting were usedthat tocanine confirm tetherin that can the differenceshelp cells effectively in cell viability resist damage were indeed caused dependent by viral on the virusinvasion. (see FigureUninfected S1). control cells (±tetherin) were used to confirm that the differences in cell viability were indeed dependent on the virus (see Figure S1).

Figure 7. MDCK cells stably expressing tetherin protein were generated and the levels of tetherin Figure 7. MDCK cells stably expressing tetherin protein were generated and the levels of tetherin expression and cell viability after CIV infection were assessed at different time points. (A) The expression and cell viability after CIV infection were assessed at different time points. (A) The expressionexpression product product of canineof canine tetherin tetherin was was detected detected through IFA IFA in in cells cells stably stably expressing expressing tetherin; tetherin; (B) little(B) little or no or no expression expression product product was was found found in cells in cells transfected transfected with an with empty an vector; empty (C) vector; the mRNA (C) the mRNAlevel level of ofcanine canine tetherin tetherin expression expression in instable-expression stable-expression cell celllines lines was wasmeasured measured by real-time by real-time ﬂuorescencefluorescence quantitative quantitative PCR PCR (RTFQ-PCR). (RTFQ-PCR). ComparedCompared to to the the control control cells, cells, the the tetherin tetherin gene gene was was signiﬁcantlysignificantly increased increased in the in stable-expression the stable-expression cells (averagecells (average fold changefold change 1500). 1500). Samples Samples were were analysed in triplicateanalysed and in GAPDHtriplicate wasand usedGAPDH as an was endogenous used as an control endogenous for normalization. control for normalization. Fold changes Fold were

Viruses 2018, 10, 565 11 of 18

Viruses 2018, 10, x FOR PEER REVIEW 11 of 18 calculated using the 2−∆∆CT method; (D) the control MDCK cells and the stable-expression MDCK cellschanges were were infected calculated with CIV using H3N2; the (E )2 the−ΔΔCT control method; MDCK (D) cellsthe andcontrol the stable-expressionMDCK cells and MDCK the stable- cells wereexpression infected MDCK with CIVcells H5N1.were infected The data with analysis CIV wasH3N2; conducted (E) the control in SPSS MDCK with an cells independent and the stable-t-test, andexpression a p value MDCK less than cells 0.05 were was infected considered with statisticallyCIV H5N1. significantThe data analysis (* p < 0.05). was Theconducted error bars in SPSS represent with thean independent standard deviation. t-test, and a p value less than 0.05 was considered statistically significant (* p < 0.05). The error bars represent the standard deviation. 3.6. Tetherin-Abundant MDCK Cells Exhibit Decreased CIV Production 3.6. Tetherin-Abundant MDCK Cells Exhibit Decreased CIV Production To investigate the effect of canine tetherin on CIV production, MDCK cells that stably expressed To investigate the effect of canine tetherin on CIV production, MDCK cells that stably expressed tetherin and control MDCK cells were infected with CIV H3N2 at an MOI of 0.1 and with CIV H5N1 tetherin and control MDCK cells were infected with CIV H3N2 at an MOI of 0.1 and with CIV H5N1 at an MOI of 0.01. The virus titre of the cellular supernatant was measured by the EID50 method at at an MOI of 0.01. The virus titre of the cellular supernatant was measured by the EID50 method at selected time points. The results (see Figure8A) showed that canine tetherin was not restricted to CIV selected time points. The results (see Figure 8A) showed that canine tetherin was not restricted to H3N2 before 36 h but a difference in the virus titre was observed at 48 h. In the stable-expression cells, CIV H3N2 before 36 h but a difference in the virus titre was observed at 48 h. In the stable-expression the average virus titre of CIV H3N2 was 4.6 logEID50/mL at 48 h. In comparison, the average virus cells, the average virus titre of CIV H3N2 was 4.6 logEID50/mL at 48 h. In comparison, the average titre in the control cells (5.4 logEID50/mL) was slightly higher than that in the stable-expression cells virus titre in the control cells (5.4 logEID50/mL) was slightly higher than that in the stable-expression at 48 h (p < 0.05). However, canine tetherin had a limited effect on CIV H5N1 at 12 h (see Figure8B); cells at 48 h (p < 0.05). However, canine tetherin had a limited effect on CIV H5N1 at 12 h (see Figure the average virus titre was 3.4 logEID50/mL in control cells and 2.8 logEID50/mL in stable-expression 8B); the average virus titre was 3.4 logEID50/mL in control cells and 2.8 logEID50/mL in stable- cells (p < 0.05). In addition, the virus titre of CIV H5N1 was different between the stable-expression expression cells (p < 0.05). In addition, the virus titre of CIV H5N1 was different between the stable- cells (6.2 logEID50/mL) and the control cells (7.3 logEID50/mL) at 24 h (p < 0.05). No difference was expression cells (6.2 logEID50/mL) and the control cells (7.3 logEID50/mL) at 24 h (p < 0.05). No observed in subsequent virus titres. We speculated that the ability of CIV H5N1 to replicate in the later difference was observed in subsequent virus titres. We speculated that the ability of CIV H5N1 to stages of infection was greater than the ability of the host innate immune factor tetherin to restrict replicate in the later stages of infection was greater than the ability of the host innate immune factor viral replication. RTFQ-PCR was used to measure intracellular virus levels. The results (see Figure8C) tetherin to restrict viral replication. RTFQ-PCR was used to measure intracellular virus levels. The showed that the mRNA expression level of H3N2 HA was significantly higher in the stable-expression results (see Figure 8C) showed that the mRNA expression level of H3N2 HA was significantly higher cells than that in the control cells at 36 and 48 h (p < 0.05). The results in the H5N1 group were the in the stable-expression cells than that in the control cells at 36 and 48 h (p < 0.05). The results in the same (see Figure8D). These results may have been caused by canine tetherin trapping a large number H5N1 group were the same (see Figure 8D). These results may have been caused by canine tetherin of influenza virus particles on the cell surface. trapping a large number of influenza virus particles on the cell surface.

Figure 8. The effect of canine tetherin on CIV production in MDCK cells overexpressing tetherin. (FigureA) Replication 8. The effect and growthof canine curve tetherin of CIV on H3N2; CIV produc (B) replicationtion in MDCK and growth cells over curveexpressing of CIV H5N1; tetherin. (C) the(A) mRNAReplication expression and growth level of curve CIV H3N2of CIV HA H3N2; in cells; (B) replication (D) the mRNA and expressiongrowth curve level of of CIV CIV H5N1; H5N1 ( HAC) the in cells.mRNA Each expression experiment level was of conductedCIV H3N2 threeHA in times cells; independently (D) the mRNA and expression the data analysislevel of wasCIV conductedH5N1 HA inin SPSScells. using Each anexperiment independent wast-test. conducted A p value three less times than 0.05independently was considered and statisticallythe data analysis signiﬁcant was (*conductedp < 0.05). in The SPSS error using bars an represent independent the standard t-test. A deviation. p value less than 0.05 was considered statistically significant (* p < 0.05). The error bars represent the standard deviation. 3.7. Interference with Tetherin Expression is Associated with Increased CIV Production 3.7. InterferenceTo further analysewith Tetherin the effects Expression of endogenous is Associated canine with tetherinIncreased on CIV CIV Production production, we used siRNA to reduceTo further canine analyse tetherin the levels effects in of MDCK endogenous cells. First, canine we tetherin used RTFQ-PCR on CIV production, to verify thewe interferenceused siRNA to reduce canine tetherin levels in MDCK cells. First, we used RTFQ-PCR to verify the interference efficiency of the siRNA. The results (see Figure 9A) showed that transfection of siRNA significantly inhibited the mRNA level of canine tetherin at 24, 36, and 48 h, with the strongest interference effect

Viruses 2018, 10, 565 12 of 18

Viruses 2018, 10, x FOR PEER REVIEW 12 of 18 efﬁciency of the siRNA. The results (see Figure9A) showed that transfection of siRNA signiﬁcantly inhibited the mRNA level of canine tetherin at 24, 36, and 48 h, with the strongest interference effect observed at 36 h. After siRNA interference, the MDCK cells infected with CIV H3N2 were at an MOI observed at 36 h. After siRNA interference, the MDCK cells infected with CIV H3N2 were at an MOI of 0.1 and the cells infected with CIV H5N1 were at an MOI of 0.01. In the CIV H3N2 group (see of 0.1 and the cells infected with CIV H5N1 were at an MOI of 0.01. In the CIV H3N2 group (see Figure 9B), no difference was observed in the replication ability of the virus between the MDCK cells Figure9B), no difference was observed in the replication ability of the virus between the MDCK cells with tetherin siRNA and the control cells before 36 h but the virus titre was different between the with tetherin siRNA and the control cells before 36 h but the virus titre was different between the tetherin siRNA cells (5.7 logEID50/mL) and the control cells (5.0 logEID50/mL) at 36 h (p < 0.05). In tetherin siRNA cells (5.7 logEID /mL) and the control cells (5.0 logEID /mL) at 36 h (p < 0.05). In comparison, the replication ability50 of CIV H5N1 (see Figure 9C) was slightly50 higher in the tetherin comparison, the replication ability of CIV H5N1 (see Figure9C) was slightly higher in the tetherin siRNA MDCK cells than that in the control cells from 12 to 24 h (p < 0.05). However, the virus titre of siRNA MDCK cells than that in the control cells from 12 to 24 h (p < 0.05). However, the virus titre of CIV H5N1 in the control cells was similar to that in the tetherin siRNA MDCK cells due to the rapid CIV H5N1 in the control cells was similar to that in the tetherin siRNA MDCK cells due to the rapid proliferation of the virus. These data demonstrate that canine tetherin can mildly limit CIV proliferation of the virus. These data demonstrate that canine tetherin can mildly limit CIV production. production.

Figure 9. Interference with tetherin expression in MDCK cells. (A) Analysis of the effect of tetherin Figure 9. Interference with tetherin expression in MDCK cells. (A) Analysis of the effect of tetherin siRNA. The samples were measured in triplicate. The relative expression levels of tetherin mRNA were siRNA. The samples were measured in triplicate. The relative expression levels of tetherin mRNA calculated according to the 2−∆∆CT method; (B) replication and growth curves of CIV H3N2 in MDCK were calculated according to the 2−ΔΔCT method; (B) replication and growth curves of CIV H3N2 in cells with silenced tetherin expression; (C) replication and growth curves of CIV H5N1 in MDCK cells MDCK cells with silenced tetherin expression; (C) replication and growth curves of CIV H5N1 in with silenced tetherin expression. Each experiment was conducted three times independently and MDCK cells with silenced tetherin expression. Each experiment was conducted three times the data analysis was conducted in SPSS with an independent t-test. A p value less than 0.05 was independently and the data analysis was conducted in SPSS with an independent t-test. A p value less considered statistically significant (* p < 0.05). The error bars represent the standard deviation. than 0.05 was considered statistically significant (* p < 0.05). The error bars represent the standard 4. Discussiondeviation. The innate immune response is the first line of defence in organisms and can respond to infection 4. Discussion within minutes to hours. Therefore, this response plays an important role in preventing virus invasionThe innate [33]. Tetherin,immune response as a host is restriction the first line factor, of defence has a uniquein organisms ability and to inhibitcan respond virus to replication infection withinand therefore minutes plays to hours. a pivotal Therefore, role in defencethis respon againstse plays viral invasionan important and infectionrole in preventing of host cells virus [34]. invasionThe unique [33]. bridge-like Tetherin, as topology a host restriction of tetherin factor, consists has a of unique several ability domains, to inhibit including virus replication an N-terminal and thereforecytoplasmic plays tail, a apivotal transmembrane role in defence domain, against an extracellular viral invasion coiled-coil and infection domain, of and host a C-terminalcells [34]. The GPI uniqueanchor. bridge-like All of these topology domains togetherof tetherin allow consists tetherin of toseveral effectively domains, tether theincluding viral membrane an N-terminal to the cytoplasmichost membrane tail, a [35 transmembrane]. Tetherin is the domain, first known an extracellular host protein coiled-coil that can domain, completely and stopa C-terminal a virus fromGPI anchor.exiting aAll cell of andthese infecting domains the together next cell. allow If a tetherin virus such to effectively as the CIV tether takes the hold, viral the membrane infected cell to willthe host membrane [35]. Tetherin is the first known host protein that can completely stop a virus from exiting a cell and infecting the next cell. If a virus such as the CIV takes hold, the infected cell will become a workshop producing new virions. The virions will then be released from the cell, infecting

Viruses 2018, 10, 565 13 of 18 become a workshop producing new virions. The virions will then be released from the cell, infecting other cells nearby and increasing the virus’s infection rate. Tetherin activation is one of the methods through which the immune system responds to viral infections. Normally, tetherin stops infected cells from releasing newly produced virions, thereby blocking the virus from infecting other cells. Previous research has found that tetherin plays a role in the immune system’s response to HIV-1 [36]. Other studies have revealed that host cells also use this defence against other types of viruses, such as Ebola [37], the Lassa virus [4], and IAV [38], which are not closely related to HIV-1. Such a broad range of viruses affected by tetherin indicates that all enveloped viruses may be targets of this antiviral system. Therefore, understanding tetherin’s mechanism of action is critical. With such information, a therapeutic approach can be designed to inhibit a virus using tetherin. Using this approach, cells’ natural defence system can slow down the replication of the virus, giving the animal or human patient time to launch more effective antiviral responses and restore health. In our study, we found that the amino acid sequence of canine tetherin is significantly different from that of human tetherin, with a similarity of only 42%. The characteristics of the canine tetherin gene sequence were further analysed, which was found to have three cysteine residues and two glycosylation sites in positions matching the human sequence, along with a GPI anchor point. Three cysteine residues are involved in the formation of disulfide bonds between tetherin molecules. Tetherin forms a dimer, which is key to its antiviral effect. Studies have reported that mutation of any of the three cysteine residues completely strips tetherin of its antiviral activity, although the residues are located in the cell membrane [30]. Modification of N-linked glycosylation sites facilitates the correct folding and transport of proteins. However, whether the glycosylation site is a crucial site for tetherin’s antiviral activity remains debatable. Some studies have found that human tetherin loses the ability to limit HIV-1 when the protein is mutated such that it cannot be modified by glycosylation. Other studies suggest that glycosylation is not the key to tetherin’s antiviral activity [39]. Therefore, the importance of glycosylation in the function of tetherin remains to be determined. The C-terminal end of canine tetherin contains a GPI anchor domain, which is a key functional area for the protein’s antiviral activity. Although the amino acid sequences of tetherin’s GPI anchor domain vary substantially across species, this domain is the core component of tetherin’s antiviral function. Tetherin’s antiviral activity is lost in the absence of GPI anchorage [40]. In addition, transmembrane domain prediction and functional domain analysis have revealed that canine tetherin and human tetherin have only one transmembrane domain each. However, the sequence before the transmembrane region of canine tetherin is the extracellular domain, and the long sequence after the transmembrane region is the cytoplasmic region, contrasting with the order of the domains in human tetherin (these results are consistent across two websites, TMHMM Server v2.0 and InterProScan). The conformation of canine tetherin is strange and rather unlikely since such a conformation would result in cytoplasmic localization of the N-linked glycosylation sites; therefore, the putative structure should be validated in future experiments. Upon comparing the simulated 3D structures of human and canine tetherin proteins, we found that the two proteins had similar spatial structures. Moreover, multiple ligand binding sites exist, which can bind with ions or nucleic acids or accept phosphate groups. Therefore, we believe that although canine tetherin has a substantially different amino acid sequence from that of human tetherin and has its extracellular and intracellular domains in the opposite order, the key to tetherin’s inhibition of virion budding is its unique and complex tertiary topological structure rather than its simple primary structure. When exploring the capacity of canine tetherin to limit CIV H3N2 and CIV H5N1 infection in MDCK cells, we found that the protein did not limit CIV H3N2 before 36 h, but some restriction of CIV H5N1 was observed at 12 h. This difference may be due to the infective dose (MOI = 0.1 for CIV H3N2 and MOI = 0.01 for CIV H5N1). The virus has acquired an antagonistic ability during its long evolution because of the selective pressure exerted by type I interferons [41]. Studies have shown that influenza viruses can antagonize and evade the effects of IFNs and replicate in host cells, mainly due to the crucial role of influenza virus nonstructural protein 1 (NS1). Through various mechanisms, NS1 Viruses 2018, 10, 565 14 of 18 can inhibit the generation and release of the mRNA encoded by the IFN gene [42]. Previously, Stephan found that NS1 could inhibit activation of the AP-1 transcription factor. Activation of AP-1 plays an important role in the immune response to viral infection since the factor can transcribe and activate genes encoding various antiviral factors [43]. Su found that CIV H3N2 can inhibit the IFN-β promoter by inhibiting activation of the NF-κB and IRF3 recognition elements, and NS1 is the key gene that inhibits IFNs [44]. Therefore, the ability of the influenza virus to induce cells to produce IFNs and inhibit their antiviral effect also plays a key role in influenza virus replication. In the early stage of infection, CIV H3N2 was at 10-times the dose of CIV H5N1; therefore, to some extent, CIV H3N2 may inhibit the response of type I IFNs. Although IFNs themselves have no antiviral effect, inhibition of IFNs delayed the normal transcription and expression of the interferon stimulant gene tetherin. Thus, the antiviral effect cannot occur rapidly. Another possibility is that CIV H5N1 induces higher mRNA expression levels of IFN-α and canine tetherin than CIV H3N2. CIV H5N1 is a highly pathogenic influenza virus, while CIV H3N2 is mild and has low pathogenicity. When invading a host, CIV H5N1 appears to be more capable of causing cytokine storms that lead to a strong immune response in the host compared to CIV H3N2. Our results also show that CIV H5N1 is more capable of inducing canine tetherin mRNA in both lung tissues and MDCK cells compared to CIV H3N2. In addition, some studies have found that tetherin reduces the number of virions released into the supernatant at the same time that it increases the number of virions in the cell, but other studies have shown that the virus particles that are anchored to the surface of the cell membrane by tetherin are transported back into the cell or internalized to lysosomes and degraded. Consequently, the number of virus particles trapped in the cell surface is reduced [45]. Our results showed that regardless of infection with either CIV H3N2 or CIV H5N1, cells with stable expression of canine tetherin had significantly higher mRNA expression levels of viral HA at 36 and 48 h than the control cells. These results suggest that many influenza virus particles may have been trapped on the cell surface by canine tetherin. Whether these virions will be degraded by lysosomes through the endocytosis pathway is unclear. Tetherin can significantly inhibit the matrix proteins of the Lassa fever virus, the Ebola virus, and the Marburg virus to form VLPs, whose target can be the matrix protein of the virus or the components of the host cell but not the glycoprotein on the surface of the virus. Therefore, tetherin may use some common mechanism to inhibit the release of multiple enveloped viruses from host cells. The virus particles are bound to the cell surface and cannot be released because tetherin can connect to the cholesterol-rich lipid rafts of the cytoplasmic membrane and physically link the serous lipid rafts and the viral envelope [46]. Therefore, the role of tetherin is obvious for many enveloped viruses such as the influenza virus, which needs to accumulate and bud in a serous area rich in lipid rafts. Hu found that the M2 protein of the influenza virus A/WSN/33 (H1N1) strain interacted with tetherin and led to downregulation of cell surface tetherin via the proteasomal pathway [38]. In our study, by using co-IP after 48 h of co-transfection, we also found that the matrix protein M1 and membrane protein M2 of CIV H5N1 can interact with canine tetherin (see Figure S2). In addition, we found that the level of M2 protein decreased significantly as the canine tetherin dose increased, while the level of M1 protein did not change significantly (see Figure S3). M1, the matrix protein expressed by the M gene of the influenza virus, is involved in the nuclear export of the viral ribonucleoprotein (vRNP) complex. M1 also plays a central role in the process of virus packaging and morphogenesis [47]. The M2 protein acts as a proton channel that promotes the dissociation of the vRNP complex and other components of the virus in addition to causing viral uncoating. The vRNP complex is released into the cytoplasm and then enters the nucleus, thus initiating transcription and replication of the viral genome [48]. These two proteins have very important roles in the replication of influenza viruses. Tetherin not only affects the production of virus particles but also reduces the infectivity of Vpu-deficient strains. Accordingly, tetherin has been speculated to interfere with the processing of the viral Gag protein, which affects the shape of the virus [48]. Therefore, we hypothesize that canine tetherin can affect the replication of influenza viruses by degrading the viral M2 protein and thus causing a deficiency of influenza viral protein. However, these findings are preliminary, and more research is needed on topics such as VLPs. Viruses 2018, 10, 565 15 of 18

In the long ﬁght between a virus and its host, the virus is unable to easily multiply in the host because the innate immune defence system of the host combats viral infection. Meanwhile, the virus uses an evolved mechanism to evade and resist the host’s innate immunity in the process of establishing infection and adapting to the host. Tetherin, an innate immune factor and potent broad-spectrum inhibitor of the release of enveloped viruses, is localized on the surface of infected cells, where it inhibits infection by retroviruses and other enveloped viruses by preventing the release of virus particles after budding from infected cells. However, several important factors remain unknown, such the common mechanism by which tetherin blocks the budding of a broad spectrum of enveloped viruses, the role of tetherin in the course of budding and virus assembly in membranes, and the transportation process of the conjugate formed by tetherin and its antagonistic protein. Numerous in vitro experiments have established that tetherin plays an important role in blocking the spread of enveloped viruses and limiting the replication of virus particles. However, the CIV was only mildly restricted by canine tetherin protein in this study and extensive research is still required to clarify the deﬁnite role of tetherin in the spread of viruses between cells. With more in-depth research, we believe that our understanding of the interaction between host and virus will be enhanced.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/10/10/565/ s1. Author Contributions: Y.Z., X.H., Q.Z., X.L., X.Z., W.Z. and S.D. conducted the experiments. Y.Z. and X.H. analysed the data and wrote the paper. P.Z. and S.L. designed the experiments and revised the paper. Funding: This project was supported in part by the National Natural Science Foundation of China (31872454, 31672563), The National Key Research and Development Program of China (2016YFD0501004), The Natural Science Foundation Guangdong province (2018B030311037, 2018A030313633), The Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases (2017B030314142). Acknowledgments: We would like to thank Wenbao Qi (College of Veterinary Medicine, South China Agricultural University) for giving some plasmids and antibodies. Conﬂicts of Interest: The authors declare no conﬂicts of interest.

Abbreviations

(EID50) 50% egg infective dose (aa) Amino acid (ABSL-3) Animal biosafety level 3 (DAPI) 40,6-Diamidino-2-phenylindole (CIV) Canine influenza virus (CCK-8) Cell Counting Kit-8 (CDS) Coding sequence (Cy3) Cyanine-3 (DMEM) Dulbecco’s modified Eagle’s medium (FBS) Foetal bovine serum (FITC) Fluorescein isothiocyanate (GAPDH) Glyceraldehyde-3-phosphate dehydrogenase gene (HCV) Hepatitis C virus (HPAI) Highly pathogenic avian influenza (HEK) Human embryonic kidney (cells) (ISGs) IFN-stimulated genes (IAV) Influenza A virus (IFN) Interferon (IRF) Interferon regulatory factor (ISRE) Interferon-stimulated response element (KSHV) Kaposi’s sarcoma-associated herpesvirus (MDCK) Madin–Darby canine kidney (cells) (NF-AT) Nuclear factor of activated T-cells (ORF) Open reading frame Viruses 2018, 10, 565 16 of 18

(PBMC) Peripheral blood mononuclear cells (PFU) Plaque-forming unit (RTFQ PCR) Real-time ﬂuorescence quantitative PCR (RT-PCR) Reverse transcription-polymerase chain reaction (STAT) Signal transducer and activator of transcription (siRNA) Small interfering RNA (SPF) Speciﬁc-pathogen-free (UTR) Untranslated region (VSV) Vesicular stomatitis virus (VLPs) Virus-like particles

References

1. Neil, S.J.; Zang, T.; Bieniasz, P.D. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 2008, 451, 425–430. [CrossRef][PubMed] 2. Jouvenet, N.; Neil, S.J.; Zhadina, M.; Zang, T.; Kratovac, Z.; Lee, Y.; McNatt, M.; Hatziioannou, T.; Bieniasz, P.D. Broad-spectrum inhibition of retroviral and filoviral particle release by tetherin. J. Virol. 2009, 83, 1837–1844. [CrossRef][PubMed] 3. Kaletsky, R.L.; Francica, J.R.; Agrawal-Gamse, C.; Bates, P. Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein. Proc. Natl. Acad. Sci. USA 2009, 106, 2886–2891. [CrossRef] [PubMed] 4. Sakuma, T.; Noda, T.; Urata, S.; Kawaoka, Y.; Yasuda, J. Inhibition of Lassa and Marburg virus production by tetherin. J. Virol. 2009, 83, 2382–2385. [CrossRef][PubMed] 5. Radoshitzky, S.R.; Dong, L.; Chi, X.; Clester, J.C.; Retterer, C.; Spurgers, K.; Kuhn, J.H.; Sandwick, S.; Ruthel, G.; Kota, K.; et al. Infectious Lassa virus, but not filoviruses, is restricted by BST-2/tetherin. J. Virol. 2010, 84, 10569–10580. [CrossRef][PubMed] 6. Kong, W.S.; Irie, T.; Yoshida, A.; Kawabata, R.; Kadoi, T.; Sakaguchi, T. Inhibition of virus-like particle release of Sendai virus and Nipah virus, but not that of mumps virus, by tetherin/CD317/BST-2. Hiroshima J. Med. Sci. 2012, 61, 59–67. [PubMed] 7. Mansouri, M.; Viswanathan, K.; Douglas, J.L.; Hines, J.; Gustin, J.; Moses, A.V.; Fruh, K. Molecular mechanism of BST2/tetherin downregulation by K5/MIR2 of Kaposi’s sarcoma-associated herpesvirus. J. Virol. 2009, 83, 9672–9681. [CrossRef][PubMed] 8. Pardieu, C.; Vigan, R.; Wilson, S.J.; Calvi, A.; Zang, T.; Bieniasz, P.; Kellam, P.; Towers, G.J.; Neil, S.J. The RING-CH ligase K5 antagonizes restriction of KSHV and HIV-1 particle release by mediating ubiquitin-dependent endosomal degradation of tetherin. PLOS Pathog. 2010, 6, e1000843. [CrossRef] [PubMed] 9. Weidner, J.M.; Jiang, D.; Pan, X.B.; Chang, J.; Block, T.M.; Guo, J.T. Interferon-induced cell membrane proteins, IFITM3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms. J. Virol. 2010, 84, 12646–12657. [CrossRef][PubMed] 10. Pan, X.B.; Qu, X.W.; Jiang, D.; Zhao, X.L.; Han, J.C.; Wei, L. BST2/Tetherin inhibits hepatitis C virus production in human hepatoma cells. Antiviral Res. 2013, 98, 54–60. [CrossRef][PubMed] 11. Pan, X.B.; Han, J.C.; Cong, X.; Wei, L. BST2/tetherin inhibits dengue virus release from human hepatoma cells. PLoS ONE 2012, 7, e51033. [CrossRef][PubMed] 12. Li, S.; Shi, Z.; Jiao, P.; Zhang, G.; Zhong, Z.; Tian, W.; Long, L.P.; Cai, Z.; Zhu, X.; Liao, M.; et al. Avian-origin H3N2 canine influenza A viruses in Southern China. Infect. Genet. Evol. 2010, 10, 1286–1288. [CrossRef] [PubMed] 13. Song, D.; Lee, C.; Kang, B.; Jung, K.; Oh, T.; Kim, H.; Park, B.; Oh, J. Experimental infection of dogs with avian-origin canine influenza A virus (H3N2). Emerg. Infect. Dis. 2009, 15, 56–58. [CrossRef][PubMed] 14. Li, G.; Wang, R.; Zhang, C.; Wang, S.; He, W.; Zhang, J.; Liu, J.; Cai, Y.; Zhou, J.; Su, S. Genetic and evolutionary analysis of emerging H3N2 canine influenza virus. Emerg. Microbes Infect. 2018.[CrossRef][PubMed] 15. New Virus Strain Causes Midwest Dog Flu Outbreak. Available online: http://news.cornell.edu/stories/ 2015/04/new-virus-strain-causes-midwest-dog-flu-outbreak (accessed on 16 August 2017). Viruses 2018, 10, 565 17 of 18

16. Songserm, T.; Amonsin, A.; Jam-on, R.; Sae-Heng, N.; Pariyothorn, N.; Payungporn, S.; Theamboonlers, A.; Chutinimitkul, S.; Thanawongnuwech, R.; Poovorawan, Y. Fatal avian influenza A H5N1 in a dog. Emerg. Infect. Dis. 2006, 12, 1744–1747. [CrossRef][PubMed] 17. Chen, H.; Deng, G.; Li, Z.; Tian, G.; Li, Y.; Jiao, P.; Zhang, L.; Liu, Z.; Webster, R.G.; Yu, K. The evolution of H5N1 influenza viruses in ducks in southern China. Proc. Natl. Acad. Sci. USA 2004, 101, 10452–10457. [CrossRef][PubMed] 18. Amonsin, A.; Payungporn, S.; Theamboonlers, A.; Thanawongnuwech, R.; Suradhat, S.; Pariyothorn, N.; Tantilertcharoen, R.; Damrongwantanapokin, S.; Buranathai, C.; Chaisingh, A.; et al. Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand. Virology 2006, 344, 480–491. [CrossRef] [PubMed] 19. Keawcharoen, J.; Oraveerakul, K.; Kuiken, T.; Fouchier, R.A.; Amonsin, A.; Payungporn, S.; Noppornpanth, S.; Wattanodorn, S.; Theambooniers, A.; Tantilertcharoen, R.; et al. Avian influenza H5N1 in tigers and leopards. Emerg. Infect. Dis. 2004, 10, 2189–2191. [CrossRef][PubMed] 20. Zhu, Q.; Yang, H.; Chen, W.; Cao, W.; Zhong, G.; Jiao, P.; Deng, G.; Yu, K.; Yang, C.; Bu, Z.; et al. A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens. J. Virol. 2008, 82, 220–228. [CrossRef][PubMed] 21. Chen, Y.; Zhong, G.; Wang, G.; Deng, G.; Li, Y.; Shi, J.; Zhang, Z.; Guan, Y.; Jiang, Y.; Bu, Z.; et al. Dogs are highly susceptible to H5N1 avian influenza virus. Virology 2010, 405, 15–19. [CrossRef][PubMed] 22. Medina, R.A.; Garcia-Sastre, A. Influenza A viruses: New research developments. Nat. Rev. Microbiol. 2011, 9, 590–603. [CrossRef][PubMed] 23. Bruce, E.A.; Abbink, T.E.; Wise, H.M.; Rollason, R.; Galao, R.P.; Banting, G.; Neil, S.J.; Digard, P. Release of filamentous and spherical influenza A virus is not restricted by tetherin. J. Gen. Virol. 2012, 93, 963–969. [CrossRef][PubMed] 24. Watanabe, R.; Leser, G.P.; Lamb, R.A. Influenza virus is not restricted by tetherin whereas influenza VLP production is restricted by tetherin. Virology 2011, 417, 50–56. [CrossRef][PubMed] 25. Winkler, M.; Bertram, S.; Gnirss, K.; Nehlmeier, I.; Gawanbacht, A.; Kirchhoff, F.; Ehrhardt, C.; Ludwig, S.; Kiene, M.; Moldenhauer, A.S.; et al. Influenza A virus does not encode a tetherin antagonist with Vpu-like activity and induces IFN-dependent tetherin expression in infected cells. PLoS ONE 2012, 7, e43337. [CrossRef][PubMed] 26. Leyva-Grado, V.H.; Hai, R.; Fernandes, F.; Belicha-Villanueva, A.; Carter, C.; Yondola, M.A. Modulation of an ectodomain motif in the influenza A virus neuraminidase alters tetherin sensitivity and results in virus attenuation in vivo. J. Mol. Biol. 2014, 426, 1308–1321. [CrossRef][PubMed] 27. Mangeat, B.; Cavagliotti, L.; Lehmann, M.; Gers-Huber, G.; Kaur, I.; Thomas, Y.; Kaiser, L.; Piguet, V. Influenza virus partially counteracts restriction imposed by tetherin/BST-2. J. Biol. Chem. 2012, 287, 22015–22029. [CrossRef][PubMed] 28. Dittmann, M.; Hoffmann, H.H.; Scull, M.A.; Gilmore, R.H.; Bell, K.L.; Ciancanelli, M.; Wilson, S.J.; Crotta, S.; Yu, Y.; Flatley, B.; et al. A serpin shapes the extracellular environment to prevent influenza A virus maturation. Cell 2015, 160, 631–643. [CrossRef][PubMed] 29. Gnirss, K.; Zmora, P.; Blazejewska, P.; Winkler, M.; Lins, A.; Nehlmeier, I.; Gartner, S.; Moldenhauer, A.S.; Hofmann-Winkler, H.; Wolff, T.; et al. Tetherin Sensitivity of Influenza A Viruses Is Strain Specific: Role of Hemagglutinin and Neuraminidase. J. Virol. 2015, 89, 9178–9188. [CrossRef][PubMed] 30. Hinz, A.; Miguet, N.; Natrajan, G.; Usami, Y.; Yamanaka, H.; Renesto, P.; Hartlieb, B.; McCarthy, A.A.; Simorre, J.P.; Gottlinger, H.; et al. Structural basis of HIV-1 tethering to membranes by the BST-2/tetherin ectodomain. Cell Host Microbe 2010, 7, 314–323. [CrossRef][PubMed] 31. Blasius, A.L.; Giurisato, E.; Cella, M.; Schreiber, R.D.; Shaw, A.S.; Colonna, M. Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation. J. Immunol. 2006, 177, 3260–3265. [CrossRef][PubMed] 32. Julkunen, I.; Sareneva, T.; Pirhonen, J.; Ronni, T.; Melen, K.; Matikainen, S. Molecular pathogenesis of influenza A virus infection and virus-induced regulation of cytokine gene expression. Cytokine Growth Factor Rev. 2001, 12, 171–180. [CrossRef] 33. Kumar, H.; Kawai, T.; Akira, S. Pathogen recognition in the innate immune response. Biochem. J. 2009, 420, 1–16. [CrossRef][PubMed] Viruses 2018, 10, 565 18 of 18

34. Hotter, D.; Sauter, D.; Kirchhoff, F. Emerging role of the host restriction factor tetherin in viral immune sensing. J. Mol. Biol. 2013, 425, 4956–4964. [CrossRef][PubMed] 35. Yi, E.; Oh, J.; Giao, N.Q.; Oh, S.; Park, S.H. Enhanced production of enveloped viruses in BST-2-deficient cell lines. Biotechnol. Bioeng. 2017, 114, 2289–2297. [CrossRef][PubMed] 36. Liberatore, R.A.; Bieniasz, P.D. Tetherin is a key effector of the antiretroviral activity of type I interferon in vitro and in vivo. Proc. Natl. Acad. Sci. USA 2011, 108, 18097–18101. [CrossRef][PubMed] 37. Brinkmann, C.; Nehlmeier, I.; Walendy-Gnirss, K.; Nehls, J.; Gonzalez, H.M.; Hoffmann, M.; Qiu, X.; Takada, A.; Schindler, M.; Pohlmann, S. The Tetherin Antagonism of the Ebola Virus Glycoprotein Requires an Intact Receptor-Binding Domain and Can Be Blocked by GP1-Specific Antibodies. J. Virol. 2016, 90, 11075–11086. [CrossRef][PubMed] 38. Hu, S.; Yin, L.; Mei, S.; Li, J.; Xu, F.; Sun, H.; Liu, X.; Cen, S.; Liang, C.; Li, A.; et al. BST-2 restricts IAV release and is countered by the viral M2 protein. Biochem. J. 2017, 474, 715–730. [CrossRef][PubMed] 39. Neil, S.J. The antiviral activities of tetherin. Curr. Top. Microbiol. Immunol. 2013, 371, 67–104. [PubMed] 40. Xia, C.; Vijayan, M.; Pritzl, C.J.; Fuchs, S.Y.; McDermott, A.B.; Hahm, B. Hemagglutinin of Influenza A Virus Antagonizes Type I Interferon (IFN) Responses by Inducing Degradation of Type I IFN Receptor 1. J. Virol. 2015, 90, 2403–2417. [CrossRef][PubMed] 41. Hale, B.G.; Randall, R.E.; Ortin, J.; Jackson, D. The multifunctional NS1 protein of influenza A viruses. J. Gen. Virol. 2008, 89, 2359–2376. [CrossRef][PubMed] 42. Klemm, C.; Boergeling, Y.; Ludwig, S.; Ehrhardt, C. Immunomodulatory Nonstructural Proteins of Influenza A Viruses. Trends Microbiol. 2018, 26, 624–636. [CrossRef][PubMed] 43. Su, S.; Huang, S.; Fu, C.; Wang, L.; Zheng, Y.; Zhou, P.; Li, S. Identification of the IFN-β response in H3N2 canine influenza virus infection. J. Gen. Virol. 2016, 97, 18–26. [CrossRef][PubMed] 44. Sato, K.; Yamamoto, S.P.; Misawa, N.; Yoshida, T.; Miyazawa, T.; Koyanagi, Y. Comparative study on the effect of human BST-2/Tetherin on HIV-1 release in cells of various species. Retrovirology 2009, 6, 53. [CrossRef] [PubMed] 45. Kupzig, S.; Korolchuk, V.; Rollason, R.; Sugden, A.; Wilde, A.; Banting, G. Bst-2/HM1.24 is a raft-associated apical membrane protein with an unusual topology. Traffic 2003, 4, 694–709. [CrossRef][PubMed] 46. Rossman, J.S.; Lamb, R.A. Influenza virus assembly and budding. Virology 2011, 411, 229–236. [CrossRef] [PubMed] 47. Manzoor, R.; Igarashi, M.; Takada, A. Influenza A Virus M2 Protein: Roles from Ingress to Egress. Int. J. Mol. Sci. 2017, 18, 2649. [CrossRef][PubMed] 48. Chu, H.; Wang, J.J.; Qi, M.; Yoon, J.J.; Chen, X.; Wen, X.; Hammonds, J.; Ding, L.; Spearman, P. Tetherin/BST-2 is essential for the formation of the intracellular virus-containing compartment in HIV-infected macrophages. Cell Host Microbe 2012, 12, 360–372. [CrossRef][PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).