An evolutionarily conserved N-terminal leucine is essential for MX1 GTPase antiviral activity against different families of RNA viruses
2022; Elsevier BV; Volume: 299; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2022.102747
ISSN1083-351X
AutoresJoe McKellar, Mary Arnaud‐Arnould, Laurent Chaloin, Marine Tauziet, Charlotte Arpin-André, Oriane Pourcelot, Mickaël Blaise, Olivier Moncorgé, Caroline Goujon,
Tópico(s)RNA and protein synthesis mechanisms
ResumoMyxovirus resistance protein 1 (MX1) and MX2 are homologous, dynamin-like large GTPases, induced upon interferon exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus, by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with a GTPase domain and a carboxy-terminal stalk domain, connected by a bundle signaling element. These three elements are known to be essential for antiviral activity. HsMX1 has two unstructured regions, the L4 loop, also essential for antiviral activity, and a short amino (N)-terminal region, which greatly varies between MX1 proteins of different species. The role of this N-terminal domain in antiviral activity is not known. Herein, using mutagenesis, imaging, and biochemical approaches, we demonstrate that the N-terminal domain of HsMX1 is essential for antiviral activity against influenza A virus, Vesicular Stomatitis Virus, and La Crosse virus. Furthermore, we pinpoint a highly conserved leucine within this region, which is absolutely crucial for human, mouse, and bat MX1 protein antiviral activity. Importantly, mutation of this leucine does not compromise GTPase activity or oligomerization capabilities but does modify MX1 protein subcellular localization. The discovery of this essential and highly conserved residue defines this region as key for antiviral activity and may reveal insights as to the mechanism(s) of action of MX1 proteins. Myxovirus resistance protein 1 (MX1) and MX2 are homologous, dynamin-like large GTPases, induced upon interferon exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus, by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with a GTPase domain and a carboxy-terminal stalk domain, connected by a bundle signaling element. These three elements are known to be essential for antiviral activity. HsMX1 has two unstructured regions, the L4 loop, also essential for antiviral activity, and a short amino (N)-terminal region, which greatly varies between MX1 proteins of different species. The role of this N-terminal domain in antiviral activity is not known. Herein, using mutagenesis, imaging, and biochemical approaches, we demonstrate that the N-terminal domain of HsMX1 is essential for antiviral activity against influenza A virus, Vesicular Stomatitis Virus, and La Crosse virus. Furthermore, we pinpoint a highly conserved leucine within this region, which is absolutely crucial for human, mouse, and bat MX1 protein antiviral activity. Importantly, mutation of this leucine does not compromise GTPase activity or oligomerization capabilities but does modify MX1 protein subcellular localization. The discovery of this essential and highly conserved residue defines this region as key for antiviral activity and may reveal insights as to the mechanism(s) of action of MX1 proteins. Influenza A virus (IAV) is a member of the Orthomyxoviridae family and the causative agent of the disease commonly known as the flu. Upon infection of target epithelial cells within the respiratory tract, IAV is sensed by pattern recognition receptors, including retinoic acid-inducible gene I, which induce a signaling cascade leading to the production and secretion of type 1 and type 3 interferons (IFNs). The IFNs act in a paracrine and autocrine manner and, through binding to their cognate receptors and activation of the Janus Kinase/Signal transducer and activator of transcription pathway, leads to the regulation of hundreds of IFN-stimulated genes. Among these IFN-stimulated genes, many antiviral restriction factors have been described (1Schoggins J.W. Interferon-stimulated genes: what do they all do?.Annu. Rev. Virol. 2019; 6: 567-584Crossref PubMed Scopus (372) Google Scholar). These factors establish a so-called antiviral state, powerfully limiting IAV replication (2McKellar J. Rebendenne A. Wencker M. Moncorgé O. Goujon C. Mammalian and avian host cell influenza A restriction factors.Viruses. 2021; 13: 522Crossref PubMed Scopus (7) Google Scholar). The study of the IFN response against IAV in mice led to the discovery of the Myxovirus resistance (MX) proteins, which were later identified in humans (3Lindenmann J. Resistance of mice to mouse-adapted influenza A virus.Virology. 1962; 16: 203-204Crossref PubMed Scopus (142) Google Scholar, 4Horisberger M.A. Staeheli P. Haller O. Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus.Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1910-1914Crossref PubMed Scopus (182) Google Scholar, 5Staeheli P. Haller O. Boll W. Lindenmann J. Weissmann C. Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus.Cell. 1986; 44: 147-158Abstract Full Text PDF PubMed Scopus (359) Google Scholar, 6Haller O. Arnheiter H. Pavlovic J. Staeheli P. The discovery of the antiviral resistance gene Mx: a story of great ideas, great failures, and some success.Annu. Rev. Virol. 2018; 5: 33-51Crossref PubMed Scopus (22) Google Scholar). There are two homologous MX genes in humans, MX1 and MX2. Human MX1, also called MxA (and hereafter referred to as HsMX1), inhibits a wide range of RNA and DNA viruses, replicating either in the cytoplasm, such as bunyavirus La Crosse Virus (LACV) and rhabdovirus Vesicular Stomatitis Virus (VSV), or in the nucleus, such as IAV (7Haller O. Staeheli P. Schwemmle M. Kochs G. Mx GTPases: dynamin-like antiviral machines of innate immunity.Trends Microbiol. 2015; 23: 154-163Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Human MX2 (HsMX2), or MxB, has notably been shown to potently inhibit HIV-1 and Herpes viruses (8Goujon C. Moncorgé O. Bauby H. Doyle T. Ward C.C. Schaller T. et al.Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection.Nature. 2013; 502: 559-562Crossref PubMed Scopus (427) Google Scholar, 9Kane M. Yadav S.S. Bitzegeio J. Kutluay S.B. Zang T. Wilson S.J. et al.MX2 is an interferon-induced inhibitor of HIV-1 infection.Nature. 2013; 502: 563-566Crossref PubMed Scopus (372) Google Scholar, 10Liu Z. Pan Q. Ding S. Qian J. Xu F. Zhou J. et al.The interferon-inducible MxB protein inhibits HIV-1 infection.Cell Host Microbe. 2013; 14: 398-410Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 11Schilling M. Bulli L. Weigang S. Graf L. Naumann S. Patzina C. et al.Human MxB protein is a Pan-herpesvirus restriction factor.J. Virol. 2018; https://doi.org/10.1128/JVI.01056-18Crossref PubMed Scopus (48) Google Scholar, 12Crameri M. Bauer M. Caduff N. Walker R. Steiner F. Franzoso F.D. et al.MxB is an interferon-induced restriction factor of human herpesviruses.Nat. Commun. 2018; 9: 1980Crossref PubMed Scopus (74) Google Scholar, 13Serrero M.C. Girault V. Weigang S. Greco T.M. Ramos Nascimento A. Anderson F. et al.The interferon-inducible GTPase MxB promotes capsid disassembly and genome release of herpesviruses.Elife. 2022; 11e76804Crossref PubMed Scopus (4) Google Scholar). Interestingly, mice also possess two Mx proteins: MmMx1 and MmMx2. The latter is more closely related to HsMX1 than the former, but interestingly, MmMx1 is a more potent inhibitor of IAV than HsMX1 (14Zimmermann P. Mänz B. Haller O. Schwemmle M. Kochs G. The viral nucleoprotein determines Mx sensitivity of influenza A viruses.J. Virol. 2011; 85: 8133-8140Crossref PubMed Scopus (137) Google Scholar). However, to our knowledge, MmMx1 only restricts orthomyxoviruses, whereas HsMX1 is broadly antiviral and MmMx2 inhibits VSV and Hantaan River Virus (HTNV) (7Haller O. Staeheli P. Schwemmle M. Kochs G. Mx GTPases: dynamin-like antiviral machines of innate immunity.Trends Microbiol. 2015; 23: 154-163Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). This difference could be due partly to the fact that MmMx1 is mainly localized inside the nucleus (15Engelhardt O.G. Ullrich E. Kochs G. Haller O. Interferon-induced antiviral Mx1 GTPase is associated with components of the SUMO-1 system and promyelocytic leukemia protein nuclear bodies.Exp. Cell Res. 2001; 271: 286-295Crossref PubMed Scopus (59) Google Scholar, 16Engelhardt O.G. Sirma H. Pandolfi P.-P. Haller O. Mx1 GTPase accumulates in distinct nuclear domains and inhibits influenza A virus in cells that lack promyelocytic leukaemia protein nuclear bodies.J. Gen. Virol. 2004; 85: 2315-2326Crossref PubMed Scopus (43) Google Scholar), contrary to HsMX1 which is cytosolic. Dynamin-like GTPases all share a very similar general organization and members of this family of proteins can present almost superimposable 3D crystal structures (17Cao Y.-L. Meng S. Chen Y. Feng J.-X. Gu D.-D. Yu B. et al.MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion.Nature. 2017; 542: 372-376Crossref PubMed Scopus (180) Google Scholar, 18Ramachandran R. Schmid S.L. The dynamin superfamily.Curr. Biol. 2018; 28: R411-R416Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The 3D structure of HsMX1 has been partially solved (19Gao S. von der Malsburg A. Paeschke S. Behlke J. Haller O. Kochs G. et al.Structural basis of oligomerization in the stalk region of dynamin-like MxA.Nature. 2010; 465: 502-506Crossref PubMed Scopus (194) Google Scholar, 20Gao S. von der Malsburg A. Dick A. Faelber K. Schröder G.F. Haller O. et al.Structure of myxovirus resistance protein a reveals intra- and intermolecular domain interactions required for the antiviral function.Immunity. 2011; 35: 514-525Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) (Fig. 1A). HsMX1 sports a globular head, which contains the GTPase module, and possesses a stalk domain, attached to the head by a tripartite bundle signaling element (BSE), composed of three separate alpha-helices (α-helices) that allow the fold-back of the stalk towards the GTPase domain, which is akin to dynamin (21Chen Y. Zhang L. Graf L. Yu B. Liu Y. Kochs G. et al.Conformational dynamics of dynamin-like MxA revealed by single-molecule FRET.Nat. Commun. 2017; 815744Google Scholar) (Fig. 1A). The major difference between HsMX1 and dynamin is the lack in the former of the Pleckstrin Homology domain, essential for the PI(4,5)P2 binding capacity of dynamin (22Zheng J. Cahill S.M. Lemmon M.A. Fushman D. Schlessinger J. Cowburn D. Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity.J. Mol. Biol. 1996; 255: 14-21Crossref PubMed Scopus (219) Google Scholar). In place of the Pleckstrin Homology domain, HsMX1 has a flexible loop, termed loop L4, of which the structure is unresolved (Fig. 1A). Another unstructured loop, the L2 loop, is also found at the extremity of the stalk and in the vicinity of the L4 loop (Fig. 1A). In addition to these domains, HsMX1 and all MX proteins possess an amino-terminal (N-terminal) extension of unknown structure, which highly varies in length and sequence (Figs. 1, A, B and S1). HsMX1 has been shown to homodimerize through the stalk domain and further oligomerizes through numerous other interfaces on the stalk and the GTPase domains (19Gao S. von der Malsburg A. Paeschke S. Behlke J. Haller O. Kochs G. et al.Structural basis of oligomerization in the stalk region of dynamin-like MxA.Nature. 2010; 465: 502-506Crossref PubMed Scopus (194) Google Scholar, 20Gao S. von der Malsburg A. Dick A. Faelber K. Schröder G.F. Haller O. et al.Structure of myxovirus resistance protein a reveals intra- and intermolecular domain interactions required for the antiviral function.Immunity. 2011; 35: 514-525Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 23Kochs G. Haener M. Aebi U. Haller O. Self-assembly of human MxA GTPase into highly ordered dynamin-like oligomers.J. Biol. Chem. 2002; 277: 14172-14176Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 24Dick A. Graf L. Olal D. von der Malsburg A. Gao S. Kochs G. et al.Role of nucleotide binding and GTPase domain dimerization in dynamin-like myxovirus resistance protein A for GTPase activation and antiviral activity.J. Biol. Chem. 2015; 290: 12779-12792Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This may suggest the possible cohabitation of different types of HsMX1 oligomers within the cell. Of note, MmMx1 possesses a nuclear localization signal localized in the third α-helix of the BSE (25Zürcher T. Pavlovic J. Staeheli P. Nuclear localization of mouse Mx1 protein is necessary for inhibition of influenza virus.J. Virol. 1992; 66: 5059-5066Crossref PubMed Google Scholar) allowing transport into the nucleus as opposed to HsMX1 and MmMx2, which are uniquely cytoplasmic. The detailed antiviral mechanism(s) of action of MX1 proteins remain largely misunderstood, although certain intrinsic antiviral determinants have been well characterized. Four essential determinants have been identified to this day, the first being the binding/hydrolysis of GTP by the GTPase domain (26Pitossi F. Blank A. Schröder A. Schwarz A. Hüssi P. Schwemmle M. et al.A functional GTP-binding motif is necessary for antiviral activity of Mx proteins.J. Virol. 1993; 67: 6726-6732Crossref PubMed Google Scholar). Interestingly, HsMX2 does not require a functional GTPase domain for HIV-1 inhibition (8Goujon C. Moncorgé O. Bauby H. Doyle T. Ward C.C. Schaller T. et al.Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection.Nature. 2013; 502: 559-562Crossref PubMed Scopus (427) Google Scholar, 9Kane M. Yadav S.S. Bitzegeio J. Kutluay S.B. Zang T. Wilson S.J. et al.MX2 is an interferon-induced inhibitor of HIV-1 infection.Nature. 2013; 502: 563-566Crossref PubMed Scopus (372) Google Scholar, 27Goujon C. Moncorgé O. Bauby H. Doyle T. Barclay W.S. Malim M.H. Transfer of the amino-terminal nuclear envelope targeting domain of human MX2 converts MX1 into an HIV-1 resistance factor.J. Virol. 2014; 88: 9017-9026Crossref PubMed Scopus (66) Google Scholar) and in the case of HsMX1, this might also be true for Hepatitis B Virus inhibition (28Yu Z. Wang Z. Chen J. Li H. Lin Z. Zhang F. et al.GTPase activity is not essential for the interferon-inducible MxA protein to inhibit the replication of hepatitis B virus.Arch. Virol. 2008; 153: 1677-1684Crossref PubMed Scopus (17) Google Scholar). The second determinant is the presence of an intact BSE (29Ponten A. Sick C. Weeber M. Haller O. Kochs G. Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity.J. Virol. 1997; 71: 2591-2599Crossref PubMed Google Scholar), the third being the possibility to oligomerize via the stalk domain (19Gao S. von der Malsburg A. Paeschke S. Behlke J. Haller O. Kochs G. et al.Structural basis of oligomerization in the stalk region of dynamin-like MxA.Nature. 2010; 465: 502-506Crossref PubMed Scopus (194) Google Scholar), and the fourth requirement involves the extremity of the stalk, loops L2 (30Arnheiter H. Haller O. Antiviral state against influenza virus neutralized by microinjection of antibodies to interferon-induced Mx proteins.EMBO J. 1988; 7: 1315-1320Crossref PubMed Scopus (64) Google Scholar, 31Flohr F. Schneider-Schaulies S. Haller O. Kochs G. The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures.FEBS Lett. 1999; 463: 24-28Crossref PubMed Scopus (118) Google Scholar, 32Kochs G. Haller O. Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2082-2086Crossref PubMed Scopus (171) Google Scholar) and L4 (33Garber E.A. Hreniuk D.L. Scheidel L.M. van der Ploeg L.H. Mutations in murine Mx1: effects on localization and antiviral activity.Virology. 1993; 194: 715-723Crossref PubMed Scopus (20) Google Scholar, 34Patzina C. Haller O. Kochs G. Structural requirements for the antiviral activity of the human MxA protein against Thogoto and influenza A virus.J. Biol. Chem. 2014; 289: 6020-6027Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 35Verhelst J. Spitaels J. Nürnberger C. De Vlieger D. Ysenbaert T. Staeheli P. et al.Functional comparison of Mx1 from two different mouse species reveals the involvement of loop L4 in the antiviral activity against influenza A viruses.J. Virol. 2015; 89: 10879-10890Crossref PubMed Scopus (22) Google Scholar). Indeed, deletions or point mutations of the L4 loop of HsMX1 and MmMx1 abrogated antiviral activity against IAV and Thogoto virus (33Garber E.A. Hreniuk D.L. Scheidel L.M. van der Ploeg L.H. Mutations in murine Mx1: effects on localization and antiviral activity.Virology. 1993; 194: 715-723Crossref PubMed Scopus (20) Google Scholar, 34Patzina C. Haller O. Kochs G. Structural requirements for the antiviral activity of the human MxA protein against Thogoto and influenza A virus.J. Biol. Chem. 2014; 289: 6020-6027Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 35Verhelst J. Spitaels J. Nürnberger C. De Vlieger D. Ysenbaert T. Staeheli P. et al.Functional comparison of Mx1 from two different mouse species reveals the involvement of loop L4 in the antiviral activity against influenza A viruses.J. Virol. 2015; 89: 10879-10890Crossref PubMed Scopus (22) Google Scholar) and this loop was elegantly shown to have been under positive selection during evolution (36Mitchell P.S. Patzina C. Emerman M. Haller O. Malik H.S. Kochs G. Evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor MxA.Cell Host Microbe. 2012; 12: 598-604Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Taken together, these studies showed that a number of intrinsic elements are needed for MX1 protein antiviral activity; however, to our knowledge, no study has so far addressed in depth the importance of the N-terminal domain of MX1 proteins. The N-terminal domain of HsMX2, however, which is longer than that of HsMX1 (91 amino acids compared to 43 amino acids) has been shown to be a crucial determinant for HIV-1 restriction (27Goujon C. Moncorgé O. Bauby H. Doyle T. Barclay W.S. Malim M.H. Transfer of the amino-terminal nuclear envelope targeting domain of human MX2 converts MX1 into an HIV-1 resistance factor.J. Virol. 2014; 88: 9017-9026Crossref PubMed Scopus (66) Google Scholar, 37Goujon C. Greenbury R.A. Papaioannou S. Doyle T. Malim M.H. A triple-arginine motif in the amino-terminal domain and oligomerization are required for HIV-1 inhibition by human MX2.J. Virol. 2015; 89: 4676-4680Crossref PubMed Scopus (47) Google Scholar, 38Busnadiego I. Kane M. Rihn S.J. Preugschas H.F. Hughes J. Blanco-Melo D. et al.Host and viral determinants of Mx2 antiretroviral activity.J. Virol. 2014; 88: 7738-7752Crossref PubMed Scopus (114) Google Scholar). Transferring the N-terminal domain of HsMX2 onto HsMX1 resulted in a chimeric protein able to inhibit HIV-1, without losing the anti-IAV activity (27Goujon C. Moncorgé O. Bauby H. Doyle T. Barclay W.S. Malim M.H. Transfer of the amino-terminal nuclear envelope targeting domain of human MX2 converts MX1 into an HIV-1 resistance factor.J. Virol. 2014; 88: 9017-9026Crossref PubMed Scopus (66) Google Scholar). These data prompt that the N-terminal domain of MX proteins may generally be important for antiviral activity. In this study, we examined the role of the N-terminal domain of several MX1 proteins, showing that this domain is essential for antiviral activity. Indeed, deletion of the N-terminal region abrogates antiviral activity against IAV and this effect was mapped to a single essential residue, leucine 41 (L41) in HsMX1. This residue was also essential for the inhibition of rhabdoviruses and bunyaviruses. We further show that this residue is highly conserved between MX1 proteins of different origins, and we show that the corresponding leucines in MmMx1 (leucine 7, L7) and little yellow-shouldered bat, Sturnira lilium MX1 (SlMX1) (leucine 39, L39) are also essential for anti-IAV activity. Finally, we demonstrate that mutation of this highly conserved leucine did not seem to impact lower- or higher-order oligomerization status of HsMX1 or MmMx1 in cells, the propensity to hydrolyze GTP in vitro, or the natural structure of the first BSE α-helix in silico. However, we show that this residue is essential for correct subcellular localization of MX1 proteins. This study therefore confirms the complex multimodular nature of MX1 proteins and defines their N-terminal region as another important antiviral module governed by an essential and highly conserved leucine. The loop L4 and all 43 amino acids of the N-terminal domain are absent from the published crystal structures of HsMX1 (19Gao S. von der Malsburg A. Paeschke S. Behlke J. Haller O. Kochs G. et al.Structural basis of oligomerization in the stalk region of dynamin-like MxA.Nature. 2010; 465: 502-506Crossref PubMed Scopus (194) Google Scholar, 20Gao S. von der Malsburg A. Dick A. Faelber K. Schröder G.F. Haller O. et al.Structure of myxovirus resistance protein a reveals intra- and intermolecular domain interactions required for the antiviral function.Immunity. 2011; 35: 514-525Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) (Fig. 1A). While the L4 loop has been extensively studied in the past (34Patzina C. Haller O. Kochs G. Structural requirements for the antiviral activity of the human MxA protein against Thogoto and influenza A virus.J. Biol. Chem. 2014; 289: 6020-6027Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 36Mitchell P.S. Patzina C. Emerman M. Haller O. Malik H.S. Kochs G. Evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor MxA.Cell Host Microbe. 2012; 12: 598-604Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), to date, the importance of the N-terminal region for the antiviral activity of HsMX1 has not been evaluated. Therefore, to address this, we generated a series of N-terminal truncation mutants for HsMX1, with mutants missing either the first 14 (HsMX115-662) or 28 (HsMX129-662) amino acids or the entire N-terminal region (HsMX144-662, named hereafter HsMX1ΔNter) (Fig. 1B). In parallel to two negative controls (E2-Crimson fluorescent protein, termed CTRL, and HsMX1 inactive GTPase mutant, HsMX1T103A), the WT protein and mutants were ectopically expressed in Human Embryonic Kidney 293T (HEK293T) cells and an IAV minigenome infection reporter assay was performed, as reported previously (8Goujon C. Moncorgé O. Bauby H. Doyle T. Ward C.C. Schaller T. et al.Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection.Nature. 2013; 502: 559-562Crossref PubMed Scopus (427) Google Scholar). In this assay, a negative sense minigenome coding for the Firefly luciferase is recognized, replicated, and transcribed by IAV polymerase in IAV-infected cells. Thus, Firefly activity is used to monitor replication efficiency. Cells were also cotransfected with a Renilla luciferase coding plasmid for normalization. We observed that the deletion of the first 14 or 28 amino acids had no effect on HsMX1 antiviral activity (Fig. 2A top panel). In contrast, the deletion of the entire N-terminal region totally abrogated antiviral activity in a comparable manner to the control-inactive GTPase mutant HsMX1T103A (Fig. 2A top panel). This suggested the presence of essential residues located between positions 29 and 43 of the N-terminal domain of HsMX1. Mus musculus Mx1 (MmMx1) is another well-studied IAV-inhibiting MX1 protein. Alignment of MmMx1 and HsMX1 proteins revealed a conserved motif of four amino acids located between positions 29 and 43 of HsMX1: 39-NNLC-42 (or 5-NNLC-8 in the case of MmMx1) (Fig. 1B). Upon replacement in HsMX1 of these four amino acids with four alanines (HsMX1NNLC39-42A), we saw a complete loss of antiviral activity comparable to that of HsMX1ΔNter and HsMX1T103A (Fig. 2A, top panel). Alanine point mutations of these four residues (HsMX1N39A, HsMX1N40A, HsMX1L41A, and HsMX1C42A) revealed that leucine 41 (L41) was responsible for the loss-of-function phenotype observed with the MX1NNLC39-42A mutant, whereas the other three mutants completely retained their anti-IAV activity (Fig. 2A, top panel). While the HsMX129-662 and HsMX1ΔNter truncation mutants showed decreased expression levels compared to the WT protein, as assessed by immunoblotting, the loss of antiviral activity of the HsMX1NNLC39-42A and HsMX1L41A mutants could not be attributed to a decrease in expression levels (Fig. 2A, bottom panel). The observed phenotypes were then confirmed using a Nanoluciferase reporter–expressing version of A/Victoria/3/75 (IAV-NLuc) (39Doyle T. Moncorgé O. Bonaventure B. Pollpeter D. Lussignol M. Tauziet M. et al.The interferon-inducible isoform of NCOA7 inhibits endosome-mediated viral entry.Nat. Microbiol. 2018; 3: 1369-1376Crossref PubMed Scopus (35) Google Scholar) as a second infection readout, both in HEK293T (Fig. 2B) and in lung-derived A549 cells (Fig. 2C), which stably expressed the control and mutant proteins. Similarly to what was observed in the minigenome infection reporter assay (Fig. 2A), HsMX1ΔNter, HsMX1NNLC39-42A, and HsMX1L41A were completely inactive against IAV, comparable to the inactive HsMX1T103A mutant, whereas the other mutants retained antiviral activity (Fig. 2, B and C). Finally, to confirm that the HsMX1L41A mutant also showed a loss of antiviral activity in multiround infection experiments, we performed A/Victoria/3/75 WT virus growth curves in A549 cells stably expressing either HsMX1, the inactive HsMX1T103A mutant as a negative control, or HsMX1L41A. HsMX1 inhibited IAV replication by around two logs compared to the HsMX1T103A control at all time points (Fig. 2D). The HsMX1L41A mutant showed comparable virus yields to HsMX1T103A, confirming the inability of this mutant to inhibit IAV replication (Fig. 2D). Next, we investigated the subcellular localization of the N-terminal HsMX1 mutants using super-resolution Airyscan microscopy. As reported previously, HsMX1 showed a honeycomb-like, punctate cytoplasmic staining and HsMX1T103A presented a juxtanuclear accumulation (Fig. 2E) (24Dick A. Graf L. Olal D. von der Malsburg A. Gao S. Kochs G. et al.Role of nucleotide binding and GTPase domain dimerization in dynamin-like myxovirus resistance protein A for GTPase activation and antiviral activity.J. Biol. Chem. 2015; 290: 12779-12792Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 27Goujon C. Moncorgé O. Bauby H. Doyle T. Barclay W.S. Malim M.H. Transfer of the amino-terminal nuclear envelope targeting domain of human MX2 converts MX1 into an HIV-1 resistance factor.J. Virol. 2014; 88: 9017-9026Crossref PubMed Scopus (66) Google Scholar, 40Nigg P.E. Pavlovic J. Oligomerization and GTP-binding requirements of MxA for viral target recognition and antiviral activity against influenza A virus.J. Biol. Chem. 2015; 290: 29893-29906Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In contrast, the HsMX1ΔNter mutant formed cytoplasmic aggregate-like structures that varied in size and were dispersed throughout the cytoplasm (Fig. 2E). Unlike this mutant, HsMX1NNLC39-42A and HsMX1L41A accumulated at the perinuclear region into small spherical structures that were phenotypically different from those of HsMX1T103A or HsMX1ΔNter (Fig. 2E). This attests to a potential difference between the L41 point and N-terminal truncation mutants, which might be linked to differences in protein synthesis or stability as seen by immunoblot (Fig. 2, A–C). Taken together, these data showed that the N-terminal region, and more precisely, leucine 41, was essential for the anti-IAV activity and correct subcellular localization of HsMX1. To further understand the importance of this newly discovered essential residue for the antiviral activity of HsMX1, we tested the restriction abilities of aforementioned HsMX1 mutants against other RNA viruses known to be inhibited by HsMX1. HEK293T cells were cotransfected with either a control or HsMX1-WT or mutant expression constructs along with a Renilla luciferase–coding plasmid, and the infection levels of single-round rhabdovirus VSV firefly-expressing replicon particles (G-pseudotyped VSV∗ΔG-fLuc particles (41Berger Rentsch M. Zimmer G. A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon.PLoS One. 2011; 6e25858Crossref PubMed Scopus (149) Google Scholar)) were measured (Fig. 3A). Reminiscent of what was observed for IAV (Fig. 2A), the HsMX1ΔNter, HsMX1NNLC39-42A, and HsMX1L41A mutants totally lost their ability to restrict the VSV∗ΔG-fLuc replicon (Fig. 3A). To confirm these results using WT VSV, BHK-21 cells stably expressing HsMX1, inactive HsMX1T103A, or HsMX1L41A were infected at low multiplicity of infection (MOI, 0.01) for 24 h and viral production was measured by plaque assays on Vero cells. HsMX1 inhibited VSV replication by over one log compared to HsMX1T103A, and HsMX1L41A did not impact viral replication, reaching similar titers as in the presence of HsMX1T103A (Fig. 3B). This therefore demonstrates the essential role of L41 for the restriction of VSV by HsMX1. As HsMX1 is well known to inhibit the Orthobunyaviruses LACV and Bunyamwera Virus (BUNV) (42Kochs G. Janzen C. H
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