key: cord-0813689-1m0uis3d
authors: Li, Ning; Zhang, Yanxu; Wu, Shuangxiu; Xu, Ruodan; Li, Zhiqing; Zhu, Jindong; Wang, Hongliang; Li, Xiao; Tian, Mingyao; Lu, Huijun; Jin, Ningyi; Jiang, Chengyu
title: Tauroursodeoxycholic acid (TUDCA) inhibits influenza A viral infection by disrupting viral proton channel M2
date: 2019-02-15
journal: Sci Bull (Beijing)
DOI: 10.1016/j.scib.2018.08.013
sha: 2a8a83200b66ae807ce70d4e86ae189d4f3dbcce
doc_id: 813689
cord_uid: 1m0uis3d

Influenza is a persistent threat to human health and there is a continuing requirement for updating anti-influenza strategies. Initiated by observations of different endoplasmic reticulum (ER) responses of host to seasonal H1N1 and highly pathogenic avian influenza (HPAI) A H5N1 infections, we identified an alternative antiviral role of tauroursodeoxycholic acid (TUDCA), a clinically available ER stress inhibitor, both in vitro and in vivo. Rather than modulating ER stress in host cells, TUDCA abolished the proton conductivity of viral M2 by disrupting its oligomeric states, which induces inefficient viral infection. We also showed that M2 penetrated cells, whose intracellular uptake depended on its proton channel activity, an effect observed in both TUDCA and M2 inhibitor amantadine. The identification and application of TUDCA as an inhibitor of M2 proton channel will expand our understanding of IAV biology and complement current anti-IAV arsenals.

Influenza is a persistent threat to human health and there is a continuing requirement for updating antiinfluenza strategies. Initiated by observations of different endoplasmic reticulum (ER) responses of host to seasonal H1N1 and highly pathogenic avian influenza (HPAI) A H5N1 infections, we identified an alternative antiviral role of tauroursodeoxycholic acid (TUDCA), a clinically available ER stress inhibitor, both in vitro and in vivo. Rather than modulating ER stress in host cells, TUDCA abolished the proton conductivity of viral M2 by disrupting its oligomeric states, which induces inefficient viral infection. We also showed that M2 penetrated cells, whose intracellular uptake depended on its proton channel activity, an effect observed in both TUDCA and M2 inhibitor amantadine. The identification and application of TUDCA as an inhibitor of M2 proton channel will expand our understanding of IAV biology and complement current anti-IAV arsenals.

Ó 2018 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved.

As major human pathogens, influenza A viruses (IAV) continuously induce annual outbreaks and irregular epidemics [1] . The highly pathogenic avian influenza (HPAI) A H5N1 is one plausible candidate that can cause deadly flu outbreak [2] . Though the basis for the incremental virulence of H5N1 is largely unclear, clinical data suggest high viral load and the resulting cytokine storm are primary instigating factors [3] . Therefore, identifying therapies that target viral elements involved in different steps of IAV life cycle remains a public health priority [3] .

Life cycle of enveloped IAV initiates from receptor-mediated endocytosis, inhibition of which presents an ideal approach to restrict IAV replication and spread [4] . Dynamic entry of IAV starts from binding of haemagglutinin (HA) with sialic acid [5] , followed by two pH-controlled events. The optimal acid milieu in late endosomes triggers conformational alterations of HA, which fuses IAV with host endosomal membrane. Prior to fusion, acidic activation of the M2 ion channel occurs in early endosomes, which facilitates IAV uncoating [6] . Thus, inhibiting HA or M2 constitutes the primary mechanisms to suppress IAV entry. Though M2 inhibitors, amantadine and rimantadine, can block IAV entry, the emergence of adamantane-resistant M2 and associated adverse effects temper their clinical application [7] .

M2 is a homo-tetramer, and each monomer consists of 97 amino acids, including an ectodomain, a transmembrane (TM) region and a cytosolic tail containing an amphipathic a-helix (AH) [8] . M2 proton conduction may involve interactions between protonated His37 and Trp41 of TM at an acidic pH, which is responsible for adamantane-based sensitivity [9] . M2 oligomerization, based on assessment of either tetramerization of M2 TM peptides or dimerization of full-length M2, was particularly necessary for its entire channel function [8] [9] [10] [11] . TM of M2 was sufficient to form tetrameric M2 [12] , which was unstable unless AH is present [9, 10, 13] . Though M2 structural conformation and function remains unclear [10, [14] [15] [16] [17] , deforming oligomeric M2 will pave the way for the development of anti-IAV drugs.

Bile acids (BA) are amphipathic molecules composed of a hydrophobic backbone and hydrophilic hydroxyl groups [18] . BA are usually conjugated with taurine to increase hydrophilicity. BAbased medications have evolved from hydrophobic chenodeoxycholic acid (CDCA) and its taurine-conjugate TCDCA to more hydrophilic ursodeoxycholic acid (UDCA) and its taurine-conjugate TUDCA. Both later strategies are current FDA-approved treatments for human liver disorders [19] . Since BA reduces intracellular protein aggregates as chaperone 4-phenylbutyrate (4-PBA) does, UDCA and TUDCA are considered as chemical chaperones [20] . Recently, TUDCA is implicated as a therapy for hepatitis B virus infection [21] . TUDCA also inhibits IAV replication [22] by suppressing the inositol-requiring enzyme 1 (IRE1) branch of the unfolded protein response (UPR) [22, 23] , a conserved molecular network orchestrating ER pathology.

In our current study, we observed distinct ER responses to seasonal H1N1 and highly pathogenic H5N1 infections in lung epithelium. Though lack of typical UPR signaling, the ER stress inhibitor TUDCA is sufficient to prevent H5N1-induced lung injuries in an UPR independent manner. Our results show that disrupting the M2 proton channel may account for TUDCA inhibition of IAV infection.

Human lung adenocarcinoma (A549) cell lines were obtained from ATCC. Madin-Darby canine kidney (MDCK) and human embryonic kidney (HEK293T) cells were purchased from Cell Resource Centre of Peking Union Medical College (Beijing, China). Details are given in Supplemental Experimental Procedures.

Avian influenza virus A/Jilin/9/2004 (H5N1) and seasonal influenza virus A/New Caledonia/20/1999 (H1N1) used in this study were obtained by inoculating stock virus into 10-day old specificpathogen-free (SPF) embryonated chicken eggs and harvested from allantoic fluid (AF) of eggs. For animal infection, SPF-housed C57BL/6J mice (4-week old, male, 16-18 g) from Vital River (Beijing, China) were used and infected. Lung injury was induced by intratracheal injection of live H5N1 IAV at a titre of 1 Â 10 6 TCID 50 -1.6 Â 10 6 TCID 50 or identical volume of AF (vehicle control). Body weight and survival rate were recorded every day after intratracheally challenged with either AF or H5N1 IAV. TUDCA at 150 mg/kg was intratracheally administrated to mice 24 and 6 h before IAV exposure or 6 and 24 h after IAV injection to determine prophylactic and therapeutic effects, respectively. Dose of TUDCA for in vivo tests was chosen from previous studies [20] . Histological assessment of mouse lung tissue and measurement of mouse lung wet/dry ratio were performed 4 days post infection. Lung injury scores were fulfilled as described [24] . Details are given in Supplemental Experimental Procedures. All applicable institutional and/or national guidelines for the care and use of animals were followed.

IVLPs were obtained by transfection of HEK293T cells with plasmids encoding IAV elements. IVLP-based M2 proton channel activity was measured by modifying previously published protocols [25] . Details are given in Supplemental Experimental Procedures.

Quantification of intracellular iVLP was determined by Accuri C6 Flow Cytometer (Becton Dickinson, BD, Franklin Lakes, NJ, USA) and analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Corresponding antibodies and details are in Supplemental Experimental Procedures.

Confocal images, including those with acid bypass tests, were acquired using an Olympus FV1000 confocal microscope (FV-1000, Olympus, Tokyo, Japan). Quantification of signals was performed with Olympus Fluoview 3.0 (Olympus). Negative staining of iVLP and observation of infected A549 cells were performed with transmission electron microscope (JEM 1230, JEOL, Tokyo, Japan). Details are given in Supplemental Experimental Procedures.

All data shown are mean ± SEM, analyzed according to at least 3 independent experiments. Two-group comparison was analyzed by student's t-test, and statistical tests of multiple groups were performed by ANOVA followed by Turkey post-test with IBM SPSS 19.0 or GraphPad Prism 5.0 (San Diego, CA, USA). P < 0.05 was considered as statistically significant.

Previous studies showed that TUDCA constrained seasonal PR/8/34 H1N1 IAV-induced UPR in lung epithelium [22] . We consistently observed increased IRE-1a and Bip signals of UPR in A549 cells after infection with seasonal New Caledonia/20/1999 H1N1 IAV (Fig. S1a online) . UPR activation occurred in parallel with hyperplasia of smooth ER, which became striking 24 h post infection (Fig. 1a) . However, UPR responsive pathways were absent in HPAI A H5N1-challenged host ( Fig. S1b online) , including IRE-1a branch (Fig. S1a online) . Though lack of typical UPR signatures, a remarkable ER swelling was detectable 1 h after H5N1 infection (Fig. 1a ). This result confirmed ER involvement in IAV life cycle and further suggested different ER responses to seasonal and highly pathogenic IAV infection. In contrast to previous reports where UPR activation mediates pandemic IAV-induced acute lung injuries (ALI) [23] , the absence of UPR in highly pathogenic H5N1-infected cells in our study suggests alternative underlying mechanisms. In light of severe ER swelling induced 1 h after H5N1 infection, we explored the potentiality of clinically available ER stress inhibitors, TUDCA and 4-PBA, as anti-H5N1 strategies.

To investigate anti-H5N1 actions of ER chaperones, we first tested the effects of TUDCA in H5N1-infected cells. When added 12 h before and during infection, TUDCA restored A549 cell viability from 41.6% to 82.5% 48 h after H5N1 exposure (Fig. 1b) with a nearly undetectable viral load (Fig. 1c) . Notably, the anti-H5N1 effect of TUDCA was only achieved at relatively high doses, which were much greater than those used to relieve ER stress [22, 23] . In view of good tolerance and apparent benefits of high dose TUDCA against lethal IAV infection (Fig. S1c, d online) , we utilized 3 mmol/ L in our in vitro studies. Due to lack of UPR, TUDCA did not impact UPR markers in H5N1-exposed cells, though H1N1-infected cells could possess a tight association of TUDCA with IRE-1a (Fig. S1a , b online), as shown previously [22] . In contrast, 4-PBA failed to rescue H5N1-induced cell death (Fig. S1e, f online) , which supports an UPR-independent antiviral action of TUDCA. Moreover, protection of the host against H5N1 infection was unsuccessful when TUDCA was added 3 h post H5N1 challenge compared to prophylactic interventions (À6 and À3 h) or therapeutic treatments within 2 h after infection (Fig. 1d) . As 2-3 h is the critical time point corresponding to the end of HA fusion and the start of viral genome transportation [26] , our results suggest IAV entry as a target of TUDCA.

To examine the anti-entry role of TUDCA, we quantified internalized H5N1 IAV 1 and 3 h post infection (Fig. 2a, S2a) . In infected cells, NP positive staining was 1.5% and 16.7% of the total cell area after 1 and 3 h of H5N1 infection, respectively. TUDCA reduced about 90% NP signals at both 1 and 3 h post infection. Of note, blockage of IAV entry by TUDCA was not exclusive to H5N1, as we found a similar 90% suppression of IAV entry in cells infected with H1N1 (Figs. 2b and S2b (online)). Altogether, these results provide evidence that TUDCA inhibits IAV entry.

Mechanisms of IAV entry reflect the interplay between IAV and host endocytic machineries. To clarify in which step TUDCA plays a role, we permuted TUDCA treatment into four tactics based on when TUDCA is administered to the host, namely either before or concomitant with IAV infection (Figs. 2c and S2c (online)). Intriguingly, even when cells were not pre-exposed, as long as TUDCA was applied during IAV infection (À/+), TUDCA inhibition of IAV entry was comparable to the condition where TUDCA was added both before and during infection (+/+). Meanwhile, presence of TUDCA during infection (À/+ and +/+) was critical, as removal of TUDCA during infection even after a 12 h pre-treatment of the host (+/À) failed to prevent viral infection, an effect similar to non-TUDCA-treated cells (À/À). This result suggests that the anti-IAV effects of TUDCA unlikely rely on priming host cells. We further verified this point by testing dynamics of host cell endocytosis using virus-free, fluorescence-labeled dextran (Fig. 2d) . Our results showed no inhibition of dextran uptake by TUDCA at both 20 and 60 min after dextran loading, indicating that TUDCA does not affect host endocytosis. In addition, TUDCA as a bile acid may sequester cholesterol from the cellular membrane, leading to attenuated viral entry [27] . Here, we found no decrease of internalized H5N1 when cells were pre-incubated with cholesterol-depleting methyl-bcyclodextrin (MbCD, Figs. 2e and S2d (online)), consistent with H1N1-infected cells [28] . Altogether, these data indicate TUDCA may target IAV instead of the host.

As HA mediates both receptor-binding and acid-controlled fusion during IAV entry, we tested the effects of TUDCA on HA using the acid bypass assay [6] . Briefly, acid bypass mimics the acidic environment of late endosomes in the extracellular compartment, which deceives the viral HA expose fusion-peptide without allocation in late endosomes. In response to acid, the preattached IAV should immediately fuse with the plasma membrane. As shown in Fig. 3a , inhibition of TUDCA on IAV internalization was completely abolished when acid bypass was applied, suggesting that TUDCA does not interfere with receptor-binding and fusion of HA.

To gain deeper insight into other individual elements of the IAV envelope, we utilized the influenza virus-like particles (iVLPs). Since M1 is essential for efficient iVLPs assembly, we created iVLPs containing HA, NA and M1 (iVLPhnm1), M1 and M2 (iVLPm1-2) or HA, NA, M1 and M2 (iVLP4, Fig. S3a , b online). We then determined entry of iVLPs in A549 cells (Figs. 3b and S3c-e (online)). Our results showed that TUDCA had minor effects on iVLPhnm1 uptake. However, TUDCA interrupted the internalization iVLPm1-2, even in the absence of fusion protein HA (Fig. 3b) .

To gain insight into the mechanism by which TUDCA suppresses iVLPm1-2 cell-penetration, we tested whether iVLPm1-2 was internalized via an endocytic pathway. By employing bafilomycin A1 and chloroquine (CQ), we observed elevated iVLPm1-2 signals in A549 cells (Fig. S3e online) . This pattern indicates a deficiency in degrading engulfed iVLPm1-2, suggesting endocytosis as a mechanism for iVLPm1-2 entry into cells.

To clarify which components of iVLPm1-2 determine its cellpenetration, we utilized mammalian-cell derived M1 or M2 protein to treat A549 cells. In cell-based experiments, we did not detect any endocytosed M1 in A549 cells; however, M2 from both H5N1 and H1N1 IAV could be clearly detected, which was attenuated by TUDCA (Figs. 3b and S3h (online)). Together with inhibition of iVLPm1-2 uptake by M2 inhibitor amantadine (Fig. 3b) , it is likely that entry of iVLPm1-2 is M2-dependent.

Moreover, co-incubating TUDCA with iVLPm1-2 revealed that M2 dimer and tetramer, especially tetramer, were lost when TUDCA was present, while HA, NA and M1 remained unaltered in all three iVLPs we created (Fig. 3c) . We verified bands of M2 oligomers by M2 specific antibody (Fig. S3i online) and confirmed disruption of M2 oligomers by TUDCA through co-incubating TUDCA with either authentic H5N1 IAV (Fig. 3d) or corresponding iVLPm1-2 from A/Jilin/9/2004 strain (Fig. S3j online) . Our results showed that TUDCA impaired M2 tetramer of both H5N1 IAV and iVLPm1-2 without an obvious impact on HA and M1 of authentic H5N1 IAV. Collectively, these data provide critical evidence that the effects of TUDCA are associated with M2.

Since the oligomeric stage of M2 may affect its channel activity [10] , we then examined the proton conductivity of M2. Challenging iVLPm1-2 (Fig. 3e ) or iVLP incorporated with Gag and M2 (iVLPgm2) [25] (Fig. S3k online) with acidic buffer resulted in membrane depolarization, an indication of M2 activation. In response to TUDCA injection, acid-stimulated proton translocation was largely abolished (Figs. 3e and S3k (online)), indicating M2 function requires oligomerization. However, amantadine or rimantadine treatment augmented tetrameric M2 (Fig. S3l online) , suggesting a dissimilar mechanism of action of TUDCA.

Furthermore, using TEM we observed that iVLPm1-2 formed a liposome-like structure, which was distinctive from M1 and M2 protein alone. Consistent with endocytosis, M2 protein and iVLPm1-2, but not M1 protein, were dramatically disrupted after TUDCA addition (Fig. 3f) , which was further confirmed by atomic forced microscopy (AFM, Fig. S3g online) .

Finally, we assessed whether TUDCA was a potential anti-IAV therapy. We tested the effect of TUDCA as both a prophylactic and a therapeutic approach. We administered prophylactic TUDCA or saline intraperitoneally in mice both 24 and 6 h before intratracheal challenge with H5N1 IAV. Similar to our in vitro data, TUDCA increased the survival rate of mice without detectable side effects (Fig. 4a) . Reduced body weight in infected mice improved in TUDCA-treated subjects (Fig. 4b) . Lung injury scores based on H&E staining (Fig. 4c) indicated a 50% reduction of lung impairment in TUDCA-treated mice (Fig. 4d) , and pulmonary edema in infected mice dramatically recovered in TUDCA-treated groups (Fig. 4e) . These results indicate that TUDCA is a highly effective prophylactic treatment against IAV-induced disease. Conversely, when TUDCA treatment began 6 and 24 h after H5N1 exposure, both survival rate and body weight in the TUDCA-treated group was indistinguishable from the non-treated group (Fig. S4a, b  online) . These in vivo observations confirm TUDCA targets internalization rather than replication of IAV.

The role of ER during IAV infection is well-documented [22, 23] , and the activation of UPR is not a unique feature of ER in IAVinfected cells [23] . Though mechanisms by which IAV induce ER stress have been attributed to accumulated IAV proteins in the ER lumen during IAV replication [22] , changes of ER morphology 1 h post H5N1 exposure are indicative of other interactions between ER and IAV. Besides assembling and folding newly synthesized proteins, ER as intracellular bank of Ca 2+ also regulates cytosolic Ca 2+ . Indeed, IAV infection induced cytosolic Ca 2+ oscillations in host cells, which was required for efficient IAV internalization [29] . Yet, details behind ER pathophysiology during IAV entry remain mostly unclear, it opens interesting questions -whether UPR activation induces detrimental or beneficial host responses following IAV infection and whether typical ER signals serve as indicators of viral virulence.

In the current study, we generated a series of iVLPs, among which iVLPm1-2 was of unexpected. First, it forms a liposomelike structure with measurable M2 proton channel activity. Second, iVLPm1-2 can penetrate freely into cells, probably through endocytic pathways. Third, the penetrating potential of iVLPm1-2 may rely on M2, which is likely endowed with a cell-penetrating competence. From the first identification of proteins that can move across cellular membranes, a group of proteins with similar features was classified as cell-penetrating peptides (CPPs) [30] . One class of CPPs was identified comprising a high degree of amphipathicity, where AH is the most common structural motif [31] . In AH, hydrophilic and hydrophobic amino acids are usually grouped on separate faces of helix, with a highly hydrophobic patch on one face and a cationic patch on the other face. Penetration of CPPs containing AH is mainly due to the amphipathicity of AH [32] . Regarding M2, the existence of AH in C-terminus has been reported by several groups [15, 16, 33] and its putative function involves viral budding and scission [33, 34] . When expressed in host cells, AHs of M2, which are arranged parallel to membrane, partially penetrate themselves through its hydrophobic face mediating mem-brane deformation [35] . In this regard, it is plausible that AH of M2 utilizes a similar mechanism triggering IAV entry by inducing membrane curvature. However, due to being interior of IAV, it is more likely that AH of M2 reinforces the contact between the viral envelope and host membrane by a protruding viral envelope. The implication of M2 as CPP is by far not fully defined and additional work will be required to understand whether the CPP property of M2 is the nature of IAV entry. Besides cell-penetration, AH that is positively charged could be attracted by negatively charged BA. BA are facial amphiphiles, adopting an almost flat conformation with a rigid steroidal backbone in parallel with the interface, which allows contact of hydroxyl groups with the aqueous environment [36] . Typically, conjugated head groups of BAs are able to capture cationic species [37] . Our TEM and AFM data (Figs. 3f and S3f, g (online)) suggest that TUDCA may form a complex with M2, which inherited the flat conformation of BA. This complex may be composed of a hydrophobic interface of BA in parallel with a hydrophilic contact via electrostatic forces between positively charged M2 AH and negatively charged TUDCA. Thus, TUDCA reorganization of the M2 protein deforms the liposomal construct of iVLPm1-2, shown as strings of membranous debris. As binding features of BA usually appear above millimolar concentrations [37] , this may explain the high doses of TUDCA required in this study.

Taken together, we identified an alternative antiviral role of TUDCA. Rather than modulating the UPR of host cells, disrupting viral proton channel M2 may mediate the anti-IAV effect of TUDCA. Application of this class of inhibitors will unravel previously unidentified but important mechanisms to expand our understanding of IAV biology. As TUDCA is a clinically available medication for liver disorders and currently under clinical evaluation for metabolic syndromes, its antiviral potential to repurpose an existing pharmacological treatment may complement the current anti-IAV arsenal.

Ningyi Jin, academician of Chinese Academy of Engineering, is a professor at the Institute of Military Veterinary Medicine, Academy of Military Medicine, and Academy of Military Sciences. Dr. Jin's research interests include the isolation, identification, characteristics of virus and related mechanisms of infection and pathogenesis of virus; viral etiology, epidemiological law, distribution and harmfulness of viral diseases, early warning and scientific prevention and control of viral diseases; virus detection technology, early diagnosis and rapid detection of virus diseases; vaccine preparation technology platform, and genetically engineered vaccines and drugs.

Chengyu Jiang, is a professor and Executive Vice Dean of School of Basic Medicine at Peking Union Medical College and Chinese Academy of Medical Sciences. Her research is to elucidate molecular pathogenesis of Acute Respiratory Distress Syndrome induced by RNA viruses such as SARS-CoV, Avian and Swine Influenza, and Ebola. Dr. Jiang has proposed a number of repurposing drugs after elucidation of disease molecular targets.

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Ning Li is an associate Professor at China Academy of Chinese Medical Sciences. She has worked on gluco-lipo toxicity in type 2 diabetes during her M.D. and postdoctoral training in the Department of

This work was supported by the National Natural Science 

The authors declare that they have no conflict of interest.

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.scib.2018.08.013.