Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Insight/Outlook Functional Diversity of Mx : Variations on a Theme of Host Resistance to Infection

Seung-Hwan Lee1 and Silvia M. Vidal1,2 1 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada

In vertebrates, host defense against patho- spread (Arnheiter et al. 1996). The presence of proteins inhibit principally orthomyxovi- gens is mediated by two general systems: in- a natural resistance to influenza was in- ruses; in contrast, the cytoplasmic isoforms nate and acquired immunity. Innate immu- triguing because mice are not natural hosts inhibit RNA viruses of the most diverse fami- nity constitutes the first line of defense, pro- for orthomyxoviruses. Soon it became clear lies, whereas some isoforms lack detectable viding a rapid response by the expression of that Mx1 was the first member of a small gene antiviral function (Table 1). germ-line encoded proteins that preexist or family present in all vertebrate species from What is the structural basis of Mx func- are induced within hours of infection. Adap- fish to men, and that the spectrum of antivi- tion? Mx proteins belong to a superfamily of tive immunity is a slower, yet highly specific ral activity was much larger than initially ap- proteins, the large GTPases, which includes response mediated by B and T lymphocytes preciated. However, the exact mechanism of the dynamins, the products encoded by the that confers effective and long-lasting protec- action of Mx proteins is still a matter of de- Drosophila shibire, the yeast vacuolar sorting tion against infection. Adaptive immunity is bate, and it is not clear whether the antiviral Vps1p, and the GTP-binding protein based on the generation of a large repertoire activity of Mx is a luxurious accident of some from Arabidopsis thaliana (van der Bliek of -recognition receptors by somatic undefined cellular function or has evolved to 1999). Members of this family are present in a gene rearrangement. Diversity has been con- inhibit in each species a set of species-specific variety of cell locations where they perform a sidered the hallmark of adaptive immunity. pathogens. range of functions including endocytosis, in- In the last few years, however, evidence has In this issue, Watanabe’s group describes tracellular vesicle transport, and mitochon- accumulated supporting the importance of a series of Mx alleles in chicken breeds some dria distribution. These proteins present diversity (probably as a response to selective of which encode proteins with antiviral ac- modular organization characterized by at pressures) even within innate immunity tivity against influenza and vesicular stoma- least three distinct functional domains with (Hoffmann et al. 1999). Moreover, differences titis virus (VSV) (Ko et al. 2002). These find- varied degrees of sequence conservation un- in innate immune mechanisms have been ings are interesting from several perspectives. derlying their ability to self-assemble into shown to be critical in host susceptibility to First, they close the chain of observations ini- higher order structures that resemble rings infection (Cooke and Hill 2001), making this tiated with the discovery of the innate resis- and helical stacks of rings, as in dynamin and an area of intense research. tance in A2G mice to a mouse-adapted influ- Mx proteins (van der Bliek 1999). In particu- The -induced Mx1 protein is enza virus by demonstrating, as has been lar, Mx proteins present a highly conserved one of the best studied determinants of in- speculated to exist, the presence of an active N-terminal GTPase domain of ∼300 amino ac- nate immunity to viral infection. In 1962, Mx protein in a species that functions as a ids, a “middle” domain of ∼150 amino acids, Lindenmann showed that the inbred mouse reservoir for the virus. Second, they highlight and a GTPase effector domain (GED) of ∼100 strain A2G is resistant to doses of mouse- the versatility of the antiviral function of the amino acids including two leucine zippers adapted influenza virus that are lethal to Mx protein that is associated with a single that have the capacity to form amphipathic other inbred strains (Lindenmann 1962). Ser631Asn substitution present in about 50% ␣-helices (Fig. 1A). Studies with human MxA This was a particularly interesting observa- of the breeds studied. Third, these findings showed that the GED is able to specifically tion because innate resistance in A2G mice also trigger important questions: Is there a contact the middle domain, and that this in- was dependent on a single dominant locus, physiological function for Mx proteins? Is teraction is critical to constitute a functional named Mx1, that was expressed in a variety of there a common theme in the mechanism of GTPase domain, as well as for oligomeriza- cell types ranging from macrophages to he- action of Mx against viruses? What are the tion (Schumacher and Staeheli 1998; Di patocytes and was exquisitely specific for or- mechanisms that fashioned the Mx antiviral Paolo et al. 1999). Despite its functional and thomyxoviruses. Subsequent studies showed activity? structural role that might be expected to re- that the specific resistance of Mx1+ murine Most species have one to three Mx pro- strict the amount of sequence variation cells to influenza viruses is attributable to the tein isoforms with different antiviral activi- among the different Mx proteins, the car- IFN-induced protein Mx1, and that after virus ties and intracellular localization. The proto- boxy-terminal region shows 22% sequence infection, the Mx1 protein is rapidly ex- type protein, mouse Mx1, as well as other ro- identity as opposed to 38% identity in the pressed in the nuclei of cells in the area where dent isoforms including rat Mx1, accumulate remaining sequence (Fig. 1B). This would in- virus replication occurs, thus blocking viral in the nucleus. In contrast, other isoforms as dicate that these sequence variations underlie well as Mx proteins of humans and most functional differences between the different other species are localized in the cytoplasm. Mx isoforms as well as with other members of 2Corresponding author. Expression of Mx proteins is stimulated by the dynamin family. Indeed, divergent se- E-MAIL [email protected]; FAX (613) 562- IFN␣/␤ or by viral infection irrespective of quences in the carboxy-terminus on other 5452. Article and publication are at http://www.genome. their activity. In fact, Mx proteins present a members of the dynamin family are thought org/cgi/doi/10.1101/gr.20102. wide range of antiviral activity: nuclear Mx1 to control specific localization and functional

12:527–530 ©2002 by Cold Spring Harbor Laboratory Press ISSN 1088-9051/01 $5.00; www.genome.org Genome Research 527 www.genome.org Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Lee and Vidal

Table 1. Intracellular Localization and Antiviral Spectrum for Selected Mx Proteins

Antiviral specificity Accession Size Intracellular Gene no. (aa) localization Virus Family

MxA P20591 661 Cytoplasm Influenza virus, Thogoto virus Orthomyxoviridae1 Vesicular stomatitis virus Rhabdoviridae2 Measles virus Paramyxoviridae3 Hantaan virus Bunyaviridae4 Coxsackievirus B4 Picornaviridae5 Semliki Forest virus Togaviridae6 MxB P20592 715 Nucleus and cytoplasm Inactive Mx1 P09922 631 Nucleus Influenza virus, Thogoto virus, Dhori virus Orthomyxoviridae1 Mx2 NP_38634 655 Cytoplasm Vesicular stomatitis virus Rhabdoviridae2 Hantann virus Bunyaviridae4 Mx1 P18588 652 Nucleus Influenza virus, Thogoto virus Orthomyxoviridae1 Mx2 P18589 659 Cytoplasm Vesicular stomatitis virus Rhabdoviridae2 LaCrosse virus, Rift Valley fever virus Bunyaviridae4 Mx3 P18590 659 Cytoplasm Inactive Mx Q92597 705 Cytoplasm Influenza virus Orthomyxoviridae1 Vesicular stomatitis virus Rhabdoviridae2 Mx P33238 721 Nucleus and cytoplasm Inactive

ped, negative-sense segmented RNA virus ped, negative-sense non-segmented RNA virus positive-sense non-segmented RNA virus ped, positive-sense, non-segmented RNA virus characteristics of individual proteins, includ- into the cytoplasm, indicating that the sites molecular weight MxA oligomers represent a ing interactions with cellular proteins (Floyd for VSV or influenza recognition are distinct. storage form, whereas GTP-bound MxA and De Camilli 1998). It is conceivable that a The cytoplasmic protein human MxA has a monomers are the active form of MxA (Jan- specialized function of Mx proteins in viral broader spectrum of antiviral activity. As zen et al. 2000). In contrast, a number of defense has evolved as a result of a direct in- Mx1, MxA inhibits orthomyxoviruses replica- single point mutations, summarized in Figure teraction between the carboxy-terminus of tion. However, in this case the block is at the 1B, result in loss of antiviral activity despite individual Mx proteins and species-specific posttranscriptional level through an interac- maintaining adequate subcellular localiza- viral pathogens. Such evolutionary mecha- tion with the nucleocapsid NP protein of tion and the predicted overall helical struc- nism for host-resistance has been Thogoto virus that prevents the infecting par- ture of the carboxy domain. A noteworthy shown to prevail in plants, in which resis- ticles from entering the cell nucleus (Kochs MxA mutant is the human E645R protein tance to bacterial and viral pathogens is me- and Haller 1999a,b). MxA also inhibits the that has lost activity against VSV while re- diated by gene-for-gene interactions between multiplication of measles virus; however, in taining activity against influenza and corresponding genes for resistance and aviru- this case the protective effect of MxA is cell- Thogoto virus, further supporting the notion lence in host and pathogen, respectively type specific blocking either viral RNA syn- of a direct interaction of the protein with dif- (Erickson et al. 1999; Bergelson et al. 2001). thesis or synthesis of viral glycoproteins, de- ferent viruses via the carboxy-terminus. Also The antiviral properties of Mx proteins pending on the cell line used (Schneider- remarkable is the fact that the active (Mx2) differ and are influenced by the intracellular Schaulies et al. 1994). Remarkably, the and inactive (Mx3) isoforms of the murine localization of the individual proteins as well antiviral specificity of MxA is extended to Mx protein differ by eight amino acids, how- as by the pathogen, underscoring their func- positive strand RNA viruses, including Sem- ever, antiviral activity is only abrogated by tional diversity (Table 1). In addition, several liki Forest virus (Togaviridae). These patho- changes occurring in the GED do- reports support a direct interaction of Mx gens are inhibited early in their replicative main. Moreover, Ko et al. (2002) report that proteins and various viral targets. Mx1 pro- cycle by a block that targets viral components among a number of naturally occurring mu- teins accumulate in the nucleus, which is the other than structural proteins (Landis et al. tations in chicken Mx only the N631S muta- site of orthomyxovirus transcription and rep- 1998). tion in the GED domain resulted in loss of lication. Here they block primary transcrip- What are the determinants of antiviral antiviral function providing further evidence tion of influenza virus and Thogoto virus, activity? GTP-binding and proper subcellular of the key role of the GED domain in antiviral probably via an interaction of mouse Mx1 localization are two requisites, but they are activity. These findings, together with the ob- and the PB2 subunit of the influenza poly- not sufficient. Extensive mutation analysis servation that GTP-bound MxA cosediments merase (Stranden et al. 1993). Rodent Mx2 and, now functional analysis of Mx variants with viral nucleocapsids, are consistent with proteins are cytoplasmic. They inhibit vesicu- emphasize the critical role of the GED do- a model in which the antiviral activity of Mx lar stomatitis virus (VSV, Rhabdoviridae) and main in GTPase activity, polymer formation, proteins depends on specific interactions be- several members of the Bunyaviridae family and antiviral activity (Fig. 1B). Interestingly, tween the GED and specific viral structures that typically replicate in the cytoplasm, at introduction of a L612K substitution in hu- (Janzen et al. 2000; Kochs et al. 2002). These an early stage of the viral cycle. However, man MxA abolishes both GTPase activity and varied interactions do not preclude, however, when Mx2 proteins are translocated into the the ability to form polymers while retaining a common antiviral mechanism of action. nucleus, they acquire activity against influ- its antiviral activity against Thogoto virus Some exciting insights into a possible mecha- enza whereas Mx1 becomes inactive if moved and VSV. This would indicate that high- nism of action have been provided from the

528 Genome Research www.genome.org Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Insight/Outlook

Figure 1 A: Domain structure and function of Mx proteins. Red stripes indicate the tripartite GTP-binding consensus elements, whereas the pink square indicates the ‘dynamin family signature’ consensus sequence that is present in all members of the dynamin family. GED stands for GTPase effector domain. B: Sequence alignment of the GED domain in selected Mx proteins. Red dots indicate conserved leucine residues participating in leucine-zipper domains. Sequences were aligned using the Clustal V program and the presence of seven of nine identical residues was highlighted using the Boxshade program. Essential residues for Mx activity identified in the GED domain are numbered 1 to 7. recent discovery that Mx1 protein is associ- of Mx1 or MxA proteins were shown to con- antiviral activities. Again, in plant systems, ated with components of the SUMO-1 system fer resistance to viral infections (Kolb et al. allelic variability has been proposed to under- of promyelocytic leukemia protein nuclear 1992; Pavlovic et al. 1995). It was clear, how- lie the adaptive acquisition of novel resis- bodies (PML NBs) (Engelhardt et al. 2001). It ever, that expression of the proteins was not tance alleles, and correlations between the is conceivable that subsequent to binding, tolerated in some cell populations including geographical distribution of resistance allele Mx proteins bring viral targets to PML NBs hepatocytes, probably due to inadequate ex- with specific pathogens have been shown known to play an important role in protea- pression of the transgene in these cells or em- (Bergelson et al. 2001). Other models ac- some-mediated degradation of ubiquitinated bryonic lethality. More recently, it was re- counting for the maintenance of susceptibil- proteins and to be involved in the cell de- ported that MxA is overexpressed in lympho- ity alleles in a population are described as the fense against varied viral infections (Everett blastoid cell lines from patients with Fanconi “hitch-hiking effect”, in which the presence et al. 1997; Anton et al. 1999). anemia (FA) (Li and Youssoufian 1997). FA is of a favorable gene conferring a heterozygous The presence of inactive variants of host- a group of genetic disorders characterized by advantage maintains tightly linked suscepti- resistance genes is always a matter of debate. congenital defects, bone marrow failure, can- bility genes in the population (Smith and In wild mice that possess two Mx genes, the cer susceptibility, and abnormal genomic in- Haigh 1974; Malo et al. 1994). However, the frequency of an inactive Mx1 allele is 50% stability in cells. All FA subtypes seem to con- possibility that Mx proteins fulfill a cellular (Haller et al. 1987), as it is for chicken Mx in verge to a common pathway characterized by function involved in cellular trafficking and/ the 18 breeds investigated by Ko et al. (2002). overexpression of MxA that is sufficient to or in stress responses (Horisberger 1992) apart In terms of evolution, it has been proposed induce apoptosis, indicating that loose con- from their documented antiviral effects re- that increased resistance to infection would trol of Mx is deleterious for mains valid. be costly in terms of other traits otherwise, the organism. Alternative possibilities for the Currently, major efforts are underway to the frequency of susceptibility alleles might presence of Mx inactive variants would be the identify and catalog single-nucleotide poly- be expected to decrease and resistance would acquisition of advantages against different morphism in humans and economically im- be monomorphic in the presence of disease pathogens not yet characterized. In that re- portant species. Mining for variations in Mx (Boots and Bowers 1999). Possible detrimen- spect, it would be interesting to retrieve the genes together with systematic screening of tal effects of Mx gene expression in specific geographical location of the susceptible lines, their antiviral properties in in vitro systems cell populations was suspected in the produc- as well as the prevalence of pathogens in the seems a promising avenue to further our un- tion of transgenic mice in which high levels respective areas, to test the hypothesis of new derstanding of the functional diversity found

Genome Research 529 www.genome.org Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Lee and Vidal

within Mx proteins. This will provide a rich Di Paolo, C., Hefti, H.P., Meli, M., Landis, H., and Kochs, G., Haener, M., Aebi, U., and Haller, O. opportunity for the study of the evolution of Pavlovic, J. 1999. J. Biol. Chem. 2002. J. Biol. Chem. In press. 274: 32071–32078. Kochs, G. and Haller, O. 1999a. J. Biol. Chem. host-virus interactions and a unique chance Engelhardt, O.G., Ullrich, E., Kochs, G., and 274: 4370–4376. to study both evolutionary and genetic as- Haller, O. 2001. Exp. Cell Res. 271: 286– ———. 1999b. Proc. Natl. Acad. Sci. 96: 2082–2086. pects of the host antiviral functions. 295. Kolb, E., Laine, E., Strehler, D., and Staeheli, P. Erickson, F.L., Dinesh-Kumar, S.P., Holzberg, S., 1992. J. Virol. 66: 1709–1716. Ustach, C.V., Dutton, M., Handley, V., Corr, Landis, H., Simon-Jodicke, A., Kloti, A., Di Paolo, C., and Baker, B.J. 1999. Philos. Trans. R. Soc. C., Schnorr, J.J., Schneider-Schaulies, S., Hefti, AKNOWLEDGMENTS Lond. B. Biol. Sci. 354: 653–658. H.P., and Pavlovic, J. 1998. J. Virol. We are grateful to Dennis Bulman and Doug- Everett, R.D., Meredith, M., Orr, A., Cross, A., 72: 1516–1522. las Gray for critical reading of the manuscript Kathoria, M., and Parkinson, J. 1997. EMBO J. Li, Y. and Youssoufian, H. 1997. J. Clin. Invest. 16: 1519–1530. 100: 2873–2880. and Danielle Malo for stimulating discussions Floyd, S. and De Camilli, P. 1998. Trends Cell Biol. Lindenmann J. 1962 Virology 16: 203–204. throughout the preparation of this work. 8: 299–301. Malo D, Vogan K, Vidal S, Hu J, Cellier M, Schurr Haller, O., Acklin, M., and Staeheli, P. 1987. J. E, Fuks A, Bumstead N, Morgan K, and Gros P. Interferon Res. 7: 647–656. 1994. Genomics 23: 51–61. REFERENCES Hoffmann, J.A., Kafatos, F.C., Janeway, C.A., and Pavlovic, J., Arzet, H.A., Hefti, H.P., Frese, M., Anton, L.C., Schubert, U., Bacik, I., Princiotta, Ezekowitz, R.A. 1999. Phylogenetic Rost, D., Ernst, B., Kolb, E., Staeheli, P., and M.F., Wearsch, P.A., Gibbs, J., Day, P.M., perspectives in innate immunity. Science Haller, O. 1995. J. Virol. 69: 4506–4510. Realini, C., Rechsteiner, M.C., Bennink, J.R., et 284: 1313–1318. Schneider-Schaulies, S., Schneider-Schaulies, J., al. 1999.. J. Cell Biol. 146: 113–124. Horisberger, M.A. 1992. J. Virol. 66: 4705–4709. Schuster, A., Bayer, M., Pavlovic, J., and Arnheiter, H., Frese, M., Kambadur, R., Meier, E., Janzen, C., Kochs, G., and Haller, O. 2000.. J. Meulen, V. 1994. J. Virol. 68: 6910–6917. and Haller, O. 1996. Curr. Top. Microbiol. Virol. 74: 8202–8206. Schumacher, B. and Staeheli, P. 1998. J. Biol. Immunol. 206: 119–147. Johannes, L., Kambadur, R., Lee-Hellmich, H., Chem. 273: 28365–28370. Bergelson, J., Dwyer, G., and Emerson, J.J. 2001. Hodgkinson, C.A., Arnheiter, H., and Meier, E. Smith, J.M. and Haigh, J. 1974. Genet. Res. Annu. Rev. Genet. 35: 469–499. 1997. J. Virol. 71: 9792–9795. 23: 23–35. Boots, M. and Bowers, R.G. 1999. J. Theor. Biol. Ko, J.H., Jin, H.K., Asano, A., Takada, A., Stranden, A.M., Staeheli, P., and Pavlovic, J. 1993. 201: 13–23. Ninomiya, A., Kida, H., Hokiyama, H., Ohara, Virology 197: 642–651. Cooke, G.S. and Hill, A.V. 2001. Nat. Rev. Genet. M., Tsuzuki, M., Nishibori, M., et al. 2002 van der Bliek, A.M. 1999. Trends Cell Biol. 2: 967–977. Genome Res. 12: pp: 595–601. 9: 96–102.

530 Genome Research www.genome.org Erratum

Genome Research 12: 527–530 (2002)

Functional Diversity of Mx Proteins: Variations on a Theme of Host Resistance to Infection Seung-Hwan Lee and Silvia M. Vidal

In the reproduction of Table 1. Intracellular Localization and Antiviral Spectrum for Selected MX Pro- teins, the first column (Species) was truncated. The corrected table has been reprinted in its entirety here.

Table 1. Intracellular Localization and Antiviral Spectrum for Selected Mx Proteins

Antiviral specificity Accession Size Intracellular Species Gene no. (aa) localization Virus Family

Human MxA P20591 661 Cytoplasm Influenza virus, Thogoto virus Orthomyxoviridae1 Vesicular stomatitis virus Rhabdoviridae2 Measles virus Paramyxoviridae3 Hantaan virus Bunyaviridae4 Coxsackievirus B4 Picornaviridae5 Semliki Forest virus Togaviridae6 MxB P20592 715 Nucleus and cytoplasm Inactive Mouse Mx1 P09922 631 Nucleus Influenza virus, Thogoto virus, Dhori virus Orthomyxoviridae1 Mx2 NP_38634 655 Cytoplasm Vesicular stomatitis virus Rhabdoviridae2 Hantaan virus Bunyaviridae4 Rat Mx1 P18588 652 Nucleus Influenza virus, Thogoto virus Orthomyxoviridae1 Mx2 P18589 659 Cytoplasm Vesicular stomatitis virus Rhabdoviridae2 LaCrosse virus, Rift Valley fever virus Bunyaviridae4 Mx3 P18590 659 Cytoplasm Inactive Chicken Mx Q92597 705 Cytoplasm Influenza virus Orthomyxoviridae1 Vesicular stomatitis virus Rhabdoviridae2 Duck Mx P33238 721 Nucleus and cytoplasm Inactive

1,4Enveloped, negative-sense segmented RNA virus 2,3Enveloped, negative-sense non-segmented RNA virus 5Naked, positive-sense non-segmented RNA virus 6Enveloped, positive-sense, non-segmented RNA virus

We apologize for any confusion this may have caused.

1012 Genome Research 12:1012 ©2002 by Cold Spring Harbor Laboratory Press ISSN 1088-9051/01 $5.00; www.genome.org www.genome.org Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Functional Diversity of Mx Proteins: Variations on a Theme of Host Resistance to Infection

Seung-Hwan Lee and Silvia M. Vidal

Genome Res. 2002 12: 527-530 Access the most recent version at doi:10.1101/gr.20102

Related Content Erratum for vol. 12, p. 527 Genome Res. June , 2002 12: 1012

References This article cites 28 articles, 15 of which can be accessed free at: http://genome.cshlp.org/content/12/4/527.full.html#ref-list-1

Articles cited in: http://genome.cshlp.org/content/12/4/527.full.html#related-urls

License

Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the Service top right corner of the article or click here.

To subscribe to Genome Research go to: https://genome.cshlp.org/subscriptions

Cold Spring Harbor Laboratory Press