Mx GTPases: dynamin-like antiviral machines of innate immunity

Review

Mx GTPases: dynamin-like antiviral § machines of innate immunity Otto Haller, Peter Staeheli, Martin Schwemmle, and Georg Kochs Institute of Virology, University Medical Center Freiburg, Freiburg, Germany

The Mx dynamin-like GTPases are key antiviral effector proteins of the type I and type III interferon (IFN) systems. They inhibit several different viruses by blocking early steps of the viral replication cycle. We focus on new structural and functional insights and discuss recent data revealing that human MxA (MX1) provides a safeguard against introduction of avian influenza A viruses (FLUAV) into the human population. The related human MxB (MX2) serves as restriction factor for HIV-1 and other primate lentiviruses. Importance of the antiviral Mx system Mouse Mx1 (for myxovirus resistance protein 1) was identified and molecularly cloned some 30 years ago as the first Mx protein family member (see Glossary) [1,2]. The starting point was a chance observation made in 1961 by Jean Lindenmann who, together with Alick Isaacs, had only recently discovered IFN [3]. He found that mice of a particular inbred strain (A2G) survived an otherwise lethal dose of FLUAV [4]. The circumstances of the Mx discovery were described by Lindenmann in a most enjoyable personal recollection [5]. The resistance phenotype is controlled by a single gene on chromosome 16 which is functional in wild mice [6] but crippled in most mouse laboratory strains by large deletions or nonsense mutations [7]. It is now clear that Mx1 is the main IFN-induced intracellular restriction factor against influenza and influenza-like viruses in mice, and that Mx homologs in other species seem to serve similar functions. Their expression is strictly controlled by virus-induced type I (a/b) and type III (l) IFNs ([8–10] for recent reviews). In recent comprehensive screens the contribution of individual IFN-stimulated gene products to the restriction of FLUAV were studied in detail: the IFN-induced transmembrane (IFITM) proteins and MX1 were the most potent antiviral effectors [11]. IFITM3 was shown to have a beneficial effect on the course of influenza virus infection in humans [12]. The antiviral power of the Mx system is best illustrated in Mx1-congenic or Mx1-transgenic mice [1,13– 15]. When standard BALB/c mice or congenic BALB/c mice carrying functional Mx1 alleles are infected with highly §

This review is dedicated to Jean Lindenmann on the occasion of his 90th birthday. Corresponding authors: Haller, O. ([email protected]); Staeheli, P. ([email protected]). Keywords: innate immunity; interferon; dynamin-like GTPase; primate evolution; influenza; HIV-1. 0966-842X/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.12.003

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pathogenic FLUAV, dramatically different outcomes are noted (Figure 1): a much higher virus dose is required to induce lethal disease in Mx1-bearing mice [16]. A new transgenic mouse line carrying the entire human MxA (MX1) locus is similarly protected from lethal FLUAV infection [17]. This is the best evidence so far that human MxA also acts as a key influenza virus restriction factor in humans. Recently, the paramount role of Mx1 in limiting FLUAV replication and disease progression was nicely verified in the collaborative cross mouse model [18] which accurately reflects the breadth of host responses seen in outbred populations, such as humans. In this review we focus on mammalian Mx proteins and discuss recent advances in understanding their biochemical, structural, and antiviral properties. Glossary BSE: bundle signaling element in Mx proteins and dynamin. It consists of a bundle of a-helices and signals conformational changes induced by GTP hydrolysis in the G domain to the rigid stalk. It is also involved in intermolecular contacts and oligomerization. Bunyaviruses: a family of zoonotic viruses with a three-segmented, negativesense, single-stranded RNA genome that includes members causing severe human disease such as Rift Valley Fever virus (RVFV) or La Crosse virus (LACV). Congenic: a congenic mouse strain is produced by introducing a desired allele (e.g., a resistance allele) from a donor mouse into the genetic background of a specific inbred strain by repeated backcrossing. Inbred congenic strains are identical at all gene loci except for the transferred locus (e.g., the resistance allele) and a linked chromosome segment. Fitness: replicative capacity of a virus variant relative to a reference virus. G domain: domain in GTPases that binds and hydrolyses GTP. Interferon (IFN): type I IFNs (13 IFN-a subtypes, a single IFN-b, and a few minor subtypes in humans) bind to a receptor expressed on most somatic cells. Type III IFNs (four human IFN-l subtypes) bind to a distinct receptor selectively expressed on epithelial cells (e.g., in the respiratory tract). Both types of IFN are induced by virus infection. Mx (MX): short for myxovirus (from Greek myxa, ‘slime’, ‘mucus’). Old nomenclature for influenza and other respiratory viruses that was used before differentiation in ortho- and paramyxoviruses was introduced. Ortholog: related gene in another species that evolved from a common ancestor gene. Orthomyxovirus: influenza and influenza-like viruses, such as Thogoto virus (THOV) or infectious salmon anemia virus (ISAV), that have a negative-strand RNA genome consisting of six to eight segments. Paramyxovirus: parainfluenzavirus, measles virus, and others having a nonsegmented negative-strand RNA genome. Paralog: related gene that originated by duplication within a genome. Primary transcription: is performed by the genome-associated RNA polymerase of negative-strand RNA viruses which becomes catalytically active in infected cells and synthesizes the first viral mRNAs. Replication: amplification of the viral genome by the viral polymerase. The term is also often used for the full virus growth cycle including mRNA transcription and genome amplification. Secondary transcription: mRNA synthesis by the RNA polymerase of negativestrand RNA viruses that occurs after translation of primary viral transcripts and a first round of viral genome replication. Stalk: extended bundle of tightly packed a-helices that mediate oligomerization in Mx GTPases and dynamin.

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A/BM/1/18 (H1N1) 100 80

Survival (%)

60

Mx1+/+, 10 LD50 Mx1+/+, 100 LD50 Mx1–/–, 10 LD50 Mx1–/–, 100 LD50

40 20 0 0

2

4

6

8 10 12 Days p.i.

14

16

18

A/Vietnam/1203/04 (H5N1) 100 Mx1+/+, 10 LD50

Survival (%)

80

Mx1–/–, 10 LD50 60 40 20 0 0

2

4

6

8 10 12 Days p.i.

14

16

of an ancestral Mx1-like gene, whereas the ancestral Mx2 gene was lost in the rodent lineage. Thus, the present-day mouse Mx2 gene is not an ortholog of human MxB but a paralog of rodent Mx1. Moreover, like Mx1, the Mx2 gene is defective in classical inbred mouse strains [20]. Both Mx genes, however, are intact in wild mice and in some laboratory strains derived from them [6,21–23], indicating that inbred mouse strains share parts of chromosome 16 with an Mx1/2-defective founder mouse. Such a genetic bottleneck is not too surprising because most laboratory strains are derived from only a few progenitors [24]. In contrast to mammals, birds have single Mx genes and fish have multiple (up to seven) Mx paralogs that evolved by gene amplification [10]. Innate defense genes undergo dramatic expansions and gene losses driven by an ever-changing pathogen environment. It will be of interest to unravel the evolutionary pressure that shaped the Mx antiviral repertoire. Regulation of Mx gene expression Mammalian Mx genes have a complex organization with many introns and an IFN-regulated promoter region [25– 27]. Mx gene expression is induced by type I (a/b) and III (l) IFNs, but not by other cytokines. In contrast to most other IFN-stimulated genes, Mx genes are not expressed constitutively and are not induced directly by viruses, but depend on IFN signaling [28,29], making Mx genes excellent markers for IFN action [30,31]. Why is expression of Mx genes so tightly controlled? MxA has been linked to increased sensitivity of cells to apoptotic stimuli [32,33]. Tight control may be a safeguard against unfavorable effects that are only tolerated in the emergency of viral infection.

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Figure 1. Mouse Mx1 facilitates survival after infection with highly pathogenic influenza A virus (FLUAV). Conventional BALB/c (Mx1 / ) mice (open symbols) and congenic BALB.A2G-Mx1 (Mx1+/+) mice homozygous for the wild type Mx1 allele (black symbols) were infected intranasally with the indicated doses of the human pandemic 1918 strain A/BM/1/18 (H1N1) or the isolate of a human lethal infection with avian A/Vietnam/1203/04 (H5N1). Survival of the animals was monitored for 18 days post-infection (p.i.); 10 LD50 and 100 LD50 indicate 10-fold and 100-fold lethal dose 50, as determined in Mx1-negative BALB/c mice. Data from [16].

Mx family members in vertebrates Mx proteins are highly conserved in vertebrates and can be subdivided into five subgroups according to their sequence similarities (Figure 2). Most mammals have two Mx genes that arose from an ancient duplication event, leading to a human MX1-like and a human MX2-like lineage. In humans, MX1 (usually known as MxA, the designation used for convenience throughout this review) and MX2 (usually known as MxB) are encoded by closely linked genes on the long arm of chromosome 21 (map position 21q22.3) [19] that is syntenic with the distal part of mouse chromosome 16. The human MxA protein is more closely related to bovine, porcine, canine, feline, and bat (myotis, pteropus) Mx1 than to the human MxB protein that clusters with Mx2 proteins of these species. Interestingly, the two genes in the rodent subgroup arose by gene duplication

Mx structure at atomic resolution Sequence alignments showed that Mx GTPases have an Nterminal GTPase (G) domain, a middle domain (MD), and a C-terminal GTPase effector domain (GED) in common with dynamin, but they lack a pleckstrin homology (PH) domain and a proline-rich domain [8,34–36]. The crystal structure of nucleotide-free human MxA revealed a three-domain architecture that is characteristic of dynamin-like GTPases. It does not strictly coincide with the linear domains deduced from the primary sequence [35] (Figure 3A,B). The globular G domain is composed of a central core consisting of six bsheets surrounded by a-helices. It is connected to an elongated stalk that consists of a four-helix bundle comprising the MD and GED. The connection between the G domain and the stalk consists of a bundle signaling element (BSE). It is formed by three a-helices derived from the flanking regions of the G domain and the very C-terminal part of the molecule which folds back to the N-terminal G domain, as in dynamin [37] (Figure 3B). The BSE is thought to transmit a signal from the GED to the G domain, and probably transfers structural changes induced by GTP binding and hydrolysis to the stalk, as recently shown for MxA [38]. Two hinge-like regions provide the structural flexibility required for a molecular machine. The rigid stalk is the central assembly hub that mediates formation of dimers in a crisscross fashion via a central interface (Figure 3C). Dimers further oligomerize into extended multimers via additional 155

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Halibut Mx

Fish Mx

Rainbow trout Mx1 Salmon Mx1 Zebrafish Mx

Turkey Mx Duck Mx

Avian Mx

Chicken Mx

Agm Mx2

Myos Mx2 Pteropus Mx2 Canine Mx2 Porcine Mx2

Human MX2-like

Human MxB (MX2)

Bovine Mx2 Coon rat Mx1 Mouse Mx1 Rat Mx1 Coon rat Mx2 Mouse Mx2

Myos Mx1 Pteropus Mx1 Agm Mx1

Human MX1-like

Rat Mx2

Human MxA (MX1) Woolly monkey Mx1 Bovine Mx1 Porcine Mx1 Canine Mx1 Feline Mx1 0.06 TRENDS in Microbiology

Figure 2. Phylogenetic tree of vertebrate Mx proteins. The Mx proteins are grouped into five subgroups according to their similarities. Sequences were obtained from GenBank and aligned with Geneious software (version Pro 6.1.6) using the Neighbor-Joining algorithm and a Jukes–Cantor distance model. Scale bar represents genetic distance (amino acid substitutions per site). The following sequences were used for the alignment: African green monkey (agm) Mx1 [44], agm Mx2 (KJ650325), bovine Mx1 (P79135), bovine Mx2 (AF355147), canine Mx1 (AAF44684), canine Mx2 (AF239824), chicken Mx (Q90597), cotton rat Mx1 (DQ218274), cotton rat Mx2 (DQ218273), duck Mx (P33238), equine Mx1 (Q28379), equine Mx2 (XP_001491517), feline Mx1 (XM_006935878), halibut Mx (AAF66055), human MxA (A33481), human MxB (M30818), mouse Mx1 (NP_034976), mouse Mx2 (NP_038634), Myotis davidii Mx1 (XM_006754325), Myotis Mx2 (XM_006754324), porcine Mx1 (P27594), porcine Mx2 (AB258432), Pteropus alecto Mx1 (XM_006916729), Pteropus Mx2 (XM_006916730), rainbow trout Mx1 (AAA87839), rat Mx1 (NP_775119), rat Mx2 (NP_599177), salmon Mx (NP_001117165), turkey Mx (EF575607, partial sequence), woolly monkey Mx1 (JX297236), and zebrafish Mx (AF533769).

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(A)

L4

MxA

B

G domain

B

Stalk

B

G domain

B

Stalk

B

NLS

L4

MxB (B)

B

G domain

MxA Stalk N

BSE

C

L4 N

MxB

C L4 (C) 1

3

4

5 G domains 2

BSE

BSE

Stalk filament

Stalk filament Loop L4

6

L4

2

1

L4

4

3

L4

6

5

L4

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Figure 3. Structure of human Mx proteins. (A) Domain structures of human MxA and MxB. The N-terminal extension of MxB contains a putative nuclear localization sequence (NLS, light green). B indicates the bundle-signaling element, BSE (red). The G (GTPase) domain is colored in orange, the middle domain (MD) in green and the GTPase effector domain (GED) in blue. (B) Crystal structure of human MxA and structural model of human MxB. MxA data [35] are from protein database 3SZR, with modeled N terminus and loop L4 shown in grey. The structure of MxB was predicted by I-TASSER (http://zhanglab.ccmb.med.umich.edu/) and PyMOL (http://www. pymol.org) software. The color code is the same as in panel A. The N-terminal helix in MxB (light green) contains the putative NLS. (C) View of a linear oligomer in which six monomers are connected by stalk–stalk and BSE–stalk interactions as seen in MxA crystals. The color code of monomer 1 corresponds to that of panel B. Monomers 1 and 2, 3 and 4, 5 and 6 dimerize via a central stalk interface indicated by the broken lines. These dimers further oligomerize into tetramers and an extended multimer via additional interfaces (adapted from [35]). The predicted positions of the unstructured loop L4 are indicated by broken ellipses.

interfaces formed between the stalks and BSEs of neighboring molecules, as predicted by previous biochemical studies [39,40]. Purified Mx oligomers are detectable by electron microscopy and appear to form ring-like structures [41,42]. Cryo-transmission electron microscopy revealed that the G domains are directed to the outer side of the multimeric rings, whereas the stalks are directed to the opposite inner side [43]. A 40 amino acid loop (residues 533– 572), termed L4, protrudes from the tip of the stalk (Figure 3B,C) at the same sequence position as the PH domain of dynamin, and mediates membrane interaction and viral target recognition [43–45]. Human MxB shares 63% amino acid sequence identity with MxA. The 3D structure is almost superimposable with the MxA structure [38], revealing identical folds and architecture of both GTPases (Figure 3A,B). The greatest

differences between the two proteins are in the relatively unstructured N-terminal regions that extend from the BSE and in the unstructured L4 loop of the stalk. MxB exists in two isoforms, a long 78 kDa and a short 76 kDa molecule [46]. The long 78 kDa form has a nuclear localization sequence (NLS)-like stretch of basic amino acids that localizes the protein preferentially to the nuclear pores [47]. Because translation of the short 76 kDa MxB isoform starts from an alternative methionine codon at position 26, it is lacking the NLS and is cytoplasmic [46]. Subcellular localization of Mx GTPases and antiviral profiles The antiviral profile of Mx proteins is influenced to some extent by their subcellular localization (Table 1). Different Mx proteins associate with distinct intracellular compartments in the cytoplasm and nucleus. The significance of these associations for antiviral activity is not always obvious. Nuclear rodent Mx1 proteins inhibit viruses that have a nuclear replication phase (influenza and influenza-like viruses) [48,49], but not viruses that replicate exclusively in the cytoplasm, whereas cytoplasmic rodent Mx2 proteins, that exhibit about 70% amino acid sequence identity to human MxA, inhibit viruses that replicate in the cytoplasm, such as vesicular stomatitis virus (VSV) and bunyaviruses [22,50]. The cytoplasmic human MxA protein is able to inhibit a broad set of viruses, irrespective of their replication site (Table 1). Based on the high sequence identity and overlapping antiviral specificity, we speculate that the antiviral mechanism of rodent Mx2 is similar to that of MxA. The long form of human MxB localizes to the cytoplasmic face of nuclear pores via its amino terminus and inhibits the import of the HIV-1 pre-integration complex into the nucleus (see below). Rodent Mx1 proteins are translocated into the nucleus owing to a C-terminal NLS that is located in the third helix of the BSE [35]. Nuclear localization of mouse Mx1 is required for antiviral activity [51]. It accumulates in distinct nuclear dots which represent specialized Mx nuclear domains often associated with, but functionally independent of, promyelocytic leukemia nuclear bodies (PML NBs) [52,53]. Interestingly, mouse Mx1 interacts with almost all components of the SUMO-1 protein modification system in addition to PML NB components [54]. It is conceivable that SUMOylation determines intracellular localization and function of Mx proteins, but this remains to be demonstrated. Cytoplasmic Mx proteins are known to associate with intracellular membranes [41]. A substantial fraction of human MxA localizes to coat protein I (COPI)-positive membranes of the smooth endoplasmic reticulum/Golgiintermediate compartment [55]. Purified MxA protein oligomerizes in ring-like structures around negatively charged liposomes and transforms them into tubes, whereby a lysine-rich stretch in loop L4 serves as the lipidbinding moiety [43]. At present, the role of membrane binding for Mx action is still poorly understood. It may be essential for inhibiting viruses that rely on membranes for replication, such as bunyaviruses, positive-strand RNA viruses, poxviruses, and African swine fever virus (ASFV) [56–58]. It also may play a role in protecting Mx from 157

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Table 1. Localization and antiviral profile of human and mouse Mx proteinsa Mx protein Human MxA

Localization Cytoplasm

Human MxB (78 kDa) Human MxB (76 kDa) Mouse Mx1

Nuclear pore Cytoplasm Nucleus

Mouse Mx2

Cytoplasm

Virus FLUAV, THOV LACV, RVFV, Hantaan virus Puumala virus, Tula virus, Dugbe virus, Crimean-Congo hemorrhagic fever virus VSV Measles virus Human parainfluenza virus Coxsackievirus B SFV Reovirus HBV ASFV Monkey pox virus HIV-1

Virus family Orthomyxoviridae Bunyaviridae

Refs [48,76–78] [83,100] [101,102] [102,103]

Rhabdoviridae Paramyxoviridae Paramyxoviridae Picornaviridae Togaviridae Reoviridae Hepadnaviridae Asfarviridae Poxviridae Retroviridae

[78,81] [90] [82] [104] [89] [105] [66,84] [57] [56] [61,70,71]

FLUAV, THOV, Dhori virus, Bakten virus

Orthomyxoviridae

VSV Hantaan virus

Rhabdoviridae Bunyaviridae

[2,49,97] [106,107] [22,50] [22]

a

For a comprehensive listing of Mx proteins from other species consult a recent review article by Verhelst and coworkers [10]. See main text and glossary for abbreviations of virus names.

degradation and stabilize the intracellular Mx pool after induction by IFN [59]. Finally, it may have some general function in intracellular protein transport and sorting. In agreement with this view, MxB was shown to be involved in regulating nucleocytoplasmic transport and cell cycle progression [47]. Canine Mx1 was recently identified as a new component of non-raft protein transport from the trans-Golgi network to the apical plasma membrane in polarized Madin–Darby canine kidney (MDCK) cells [60]. Future work will hopefully shed new light on the relationship between intracellular localization, transport function, and antiviral activity. Antiviral specificity Mx family members have distinct antiviral profiles against a diverse range of viruses, among them pathogens of great importance in human and veterinary medicine (Table 1), as summarized in a recent comprehensive review [10]. Human MxA is inhibitory to an increasing number of RNA and DNA viruses, but not against many other viruses, including HIV-1. A broad screen for IFN-induced anti-HIV-1 factors revealed that human MxB might have such properties [11]. Later work demonstrated that human MxB inhibits HIV-1 and other primate lentiviruses but none of the MxA-sensitive viruses [61,70,71]. Atlantic salmon Mx1 inhibits influenza-like viruses of fish [62] as well as an aquatic birnavirus [63], which are both threatening the aquaculture industry. The molecular mechanism behind this broad yet specific antiviral action is not fully understood, but recent work on Mx structure and evolution is providing first insights. Mutational analysis demonstrated that the antiviral activity of mouse and human Mx1/MxA GTPases depends on (i) GTP binding/hydrolysis, (ii) an intact BSE, and (iii) oligomerization via the stalk [35,40,64,65]. An exception is the inhibition of hepatitis B virus (HBV) by human MxA, which does apparently not require GTPase activity [66]. 158

Importantly, residues in the C-terminal part of the L4 loop serve as major antiviral specificity determinants [44,45]. Sequence comparisons of MxA orthologs in simian primates demonstrated that L4 has a signature of strong positive selection, suggesting a role as antiviral interface during primate evolution [44]. A single amino acid (F561) was subject to recurrent mutations. It largely determines antiviral activity against orthomyxoviruses, demonstrating that a single position can be decisive for antiviral specificity. Likewise, only a few amino acids shape the corresponding interface on the viral target structure (see below). Experiments in which L4 was swapped between Mx proteins from different species showed that the loop acts as an autonomous antiviral module and that Mx GTPases tolerate a great deal of sequence diversity in L4 without losing activity [44,45]. Clearly, L4 is not the only domain that determines target specificity of MxA. Additional ‘hotspots’ of positive selection were identified in other parts of the molecule, particularly in the flexible N terminus [44]. They may serve as alternative interfaces against viruses that have not yet been studied. In this regard, the Mx GTPase resembles a Swiss Army knife, exposing different antiviral interfaces depending on varying conformations [67]. The N terminus, but not loop L4, is important for mediating antiviral activity of the human paralog MxB against HIV-1 [61,68–72]. Thus, only the long 78 kDa form of MxB containing the unique N-terminal NLS-like sequence was found to be antiviral. Interestingly, transfer of the N terminus of MxB onto human MxA or mouse Mx1 resulted in chimeric proteins with anti-HIV-1 activity [69], indicating that the N-terminal extension of MxB proximal to the BSE [36] (Figure 3A) represents an autonomous antiviral module in MxB comparable to loop L4 in MxA. A specific feature of full-length MxB and these chimeric proteins is their localization to the nuclear envelope [47], and this correlates with anti-HIV-1 activity [69]. An evolutionary analysis of MxB for positive selection in primates identified a few residues in the

Review distal N terminus that are critical for HIV-1 restriction [72], namely amino acids 37–44 located downstream of the NLS. The amino acid at position 37 was found to be a major specificity determinant [72] which is located proximal to the BSE according to the structural model (Figure 3B). In summary, the unique N terminus of MxB appears to display two separate functional elements, namely an NLS for proper subcellular localization and a downstream motif for specific viral targeting. This organization is reminiscent of the bipartite element recently identified in loop L4 of MxA [45]. The N-terminal domain is the most-divergent region of MxB proteins and may well have had a role in an evolutionary ‘arms race’ with evolving ancient pathogens, comparable to L4 of MxA. No antiviral or cellular activity has yet been described for the short 76 kDa MxB isoform lacking the HIV-1 targeting sequences. It can form oligomers with the long 78 kDa MxB isoform [73], suggesting that the short variant may modulate the function of the long variant. Mx GTPases inhibit different viruses at unique steps in their life cycle The main intracellular blocks imposed by nuclear mouse Mx1, cytoplasmic human MxA, and nuclear pore-associated MxB on virus replication are schematically illustrated in Figure 4. Mouse Mx1 inhibits FLUAV and presumably Thogoto virus (THOV) by specifically blocking viral mRNA synthesis that is catalyzed in the nucleus by the RNA polymerase residing in the incoming viral nucleocapsids (primary transcription) [48,74,75]. Human MxA traps THOV nucleocapsids in the cytoplasm and prevents their import into the nucleus [76]. Likewise, MxA keeps FLUAV nucleocapsids out of the nucleus with the help of unknown IFN-induced factor(s) [77]. In the absence of such factors, incoming FLUAV nucleocapsids can still start primary transcription in the nucleus but the viral genome is not replicated if MxA is present [78]. It is conceivable that MxA prevents nuclear import of newly synthesized viral nucleocapsid components, but this has to date not been demonstrated. Intriguingly, an artificial nuclear form of MxA blocks primary transcription akin to mouse Mx1, suggesting that identical mechanisms are at work [79]. Human MxA targets VSV and human parainfluenza virus nucleocapsids, and inhibits early viral mRNA synthesis in the cytoplasm [80–82]. MxA inhibits La Crosse virus (LACV) and other bunyaviruses by sequestering the newly synthesized viral N protein into perinuclear complexes [58,83]. This depletion of free N protein prevents the viral polymerase from replicating the genome. Likewise, MxA sequesters HBV capsid (core) protein into perinuclear compartments [84]. Further, MxA relocates to perinuclear, viral replication factories in ASFV-infected cells [57] (not shown in Figure 4). Finally, human MxB targets the HIV-1 capsid after cell entry. MxB does not affect reverse transcription but prevents integration of the viral DNA into the host genome by a still poorly defined mechanism that affects uncoating [68], nuclear uptake [61,70], or integration by the viral pre-integration complex (PIC) [70,71]. Thus, Mx GTPases appear to recognize specific viral components that enter the cell upon infection or are produced in infected cells shortly thereafter. Knockdown experiments showed that MxB substantially contributes to IFN-induced antiviral

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defense against HIV-1 [61,70], possibly in conjunction with other IFN-induced effector molecules such as APOBEC3, TRIM5a, SAMHD1, or tetherin as discussed recently [85,86]. Interestingly, the human paralogs MxA and MxB have evolved antiviral specificities that reside in different and relatively unstructured regions of the otherwise highly structured molecules. They are mutually exclusive and target unique steps early in the life cycle of a surprisingly diverse population of viruses. Viral target structures and proposed mode of Mx action A large body of experimental work suggests that the known antiviral Mx GTPases have intrinsic activity and interact directly with crucial viral components. In appropriate in vitro test systems, purified human MxA protein inhibits transcription of VSV [80] and binds to the nucleocapsids of THOV in the presence of non-hydrolysable GTPgS [76]. A monoclonal antibody (2C12) that binds to a conserved nonlinear epitope (amino acids 432–471 in human MxA) prevents this binding and neutralizes the Mx antiviral effect in living cells [76,87]. Such findings do not formally exclude the participation of some as yet unknown cellular factors that may be expressed in a cell type-specific manner. If such factors are involved in inhibition of bunyaviruses, they must be conserved between mammalian and insect cells because human MxA inhibits LACV also in transfected mosquito cells [88]. Host factors have been implicated in the antiviral action of human MxA against measles virus, FLUAV, and Semliki Forest virus (SFV) [77,89–91]. The anti-HIV-1 activity of MxB appears to rely on host cyclophilins, cellular proteins containing a cyclophilin-like domain, and additional factors involved in nucleocytoplasmic transport [71]. Mx GTPases are expected to have multiple interaction partners required for optimal function and subcellular localization, and it will be a great challenge to characterize them all. Viral mutations gained during host adaptation that cause Mx resistance should be most helpful in revealing which of the viral components are targeted by Mx proteins. FLUAV strains differ in their MxA sensitivity. Human strains are less sensitive than avian strains, and sensitivity was found to be determined by the nucleoprotein NP [92,93]. A cluster of a few amino acid residues was identified on the surface-exposed body domain of NP and was shown to be responsible for MxA sensitivity [94]. Sequence comparisons revealed that the cluster of amino acids responsible for low MxA sensitivity is highly conserved in circulating human isolates but is virtually absent in avian viruses, suggesting that human FLUAV strains are under constant evolutionary pressure by MxA restriction. Experimental introduction of the low-sensitivity cluster into the NP of an avian FLUAV increased MxA resistance, but diminished overall replication fitness, indicating that MxA targets a crucial functional domain [94]. Multiple adaptive changes are presumably required by an avian virus to escape MxA restriction and to maintain fitness. MxA may therefore represent a considerable barrier against zoonotic introduction of avian influenza viruses into the human population. There is convincing experimental evidence that human and mouse Mx GTPases recognize the nucleocapsid protein 159

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(A)

Viral mRNA translaon Transcripon and replicaon

MxA vRNP

FLUAV and THOV

Mx1 Nucleus

(B)

Transcripon and replicaon

MxA

VSV

vRNP

Nucleus (C)

Nucleus MxA Transcripon

N

sER

MxA/N

MxA LACV vRNP Replicaon

(D)

MxB Uncoang Integraon

HIV-1

Reverse transcripon

Nuclear import

PIC

Provirus

Nucleus TRENDS in Microbiology

Figure 4. Mx-sensitive steps in the life cycles of various viruses. (A) The nucleocapsids (vRNPs, for viral ribonucleoprotein complexes) of FLUAV and THOV consist of genomic RNA segments associated with the viral nucleoprotein and RNA polymerase. Human MxA blocks nuclear translocation of incoming vRNPs and inhibits secondary transcription and replication of FLUAV genomes by interfering with synthesis and/or nuclear import of newly synthesized viral components. Mouse Mx1 acts in the nucleus (Figure legend continued on the bottom of the next page.)

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Review of particular Mx-sensitive viruses preferentially in the context of the assembled nucleocapsid structure [95– 97]. The FLUAV polymerase subunit PB2, which is associated with NP in the viral nucleocapsid, may serve as an additional target [97–99]. We have proposed a model in which initial binding of a few Mx dimers or tetramers leads to the recruitment of additional Mx molecules and selfassembly into higher-order oligomers. Oligomerization results in stable ring formation and activation of GTPase activity by G domain interactions between neighboring rings [35]. Subsequent conformational changes may lead to either mislocalization of targeted components into aggregates [58,83], blockage of nuclear translocation [77], or disruption of functional integrity [97]. The structure of Mx assemblies on viral components has not yet been visualized. Thus, other forms of Mx target interactions are still plausible. In compelling analogy to MxA, MxB appears to target the capsid (core) of HIV-1 [68] whereby a few amino acid positions determine sensitivity [61,68–72]. Resistance mutations emerge rapidly when HIV-1 is passaged in cells expressing high levels of MxB [71,72]. Some of these mutations also interfere with binding to cyclophilin A, specific nucleoporins (e.g., NUP153 and NUP358) and additional capsid-binding factors, such as the cleavage and polyadenylation specific factor 6 (CPSF6) [71,72]. Although it is likely that a direct interaction of MxB with the viral capsid is responsible for the inhibition (see above), alternative and more indirect mechanisms cannot be excluded at present. MxB may affect particular nuclear import pathways involved in HIV-1 infection or may impinge on cellular factors that normally contribute to capsid stability [71]. It is notable in this context that neither wild type MxB nor the chimeras consisting of the N terminus of MxB fused to MxA required binding or hydrolysis of GTP for their anti-HIV-1 activity [61,69–71]. This suggests that the effector mechanism of MxB against retroviruses is distinct from that of MxA or other dynamin-like GTPases. MxB action may be similar to the GTPase-independent activity of MxA against the reverse-transcribing DNA virus HBV. Concluding remarks Mx proteins are highly potent antiviral restriction factors of the innate immune system that control a diverse range of viruses. Despite of a highly conserved architecture this amazing molecular machine can adapt over time to new pathogens and maintain its broad antiviral specificity by only changing a few amino acids at selected sites. The principal target structures of most Mx-sensitive viruses are unknown and need to be characterized in more detail (Box 1). MxA and MxB are relevant for at least two infectious diseases that have a global impact on human populations, namely influenza and AIDS. The contribution of Mx

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Box 1. Outstanding questions  Do Mx resistance genes contribute to disease susceptibility in humans, as the mouse models suggest? Are there crucial genetic polymorphisms at the MX1 or MX2 locus on chromosome 21 in the human population that correlate with disease outcome? How important is the role of human MxA in providing a barrier against zoonotic introduction of avian influenza viruses? How potent is human MxB in mediating heightened resistance against HIV-1 infection as compared to other known restriction factors?  What are the driving forces for amplification or loss of Mx genes in different species?  What is the molecular mechanism by which Mx GTPases inhibit sensitive viruses? Why does human MxB not require GTP for its anti-HIV-1 activity? Is there a common denominator that renders some seemingly unrelated viruses sensitive to Mx?  Can Mx binding to its viral targets, for example nucleocapsids, be visualized? Can the structures of co-crystal between Mx and its viral targets be solved? Will such structural insights lead to new antiviral compounds?  How do viruses escape Mx restriction? Are there specific viral antagonists against Mx proteins?  Do cellular factors and/or post-translational modifications determine the antiviral selectivity of Mx proteins? Do cellular factors influence viral target recognition by binding to the viral target or by binding to and modulating the host GTPase? Does MxA have a physiological role in addition to its antiviral function?  What are the functions of the different MxB isoforms translated from the same mRNA?

proteins to disease susceptibility should be assessed by future genetic and epidemiological studies. Better understanding of resistance/susceptibility alleles in humans will facilitate risk assessment, ameliorate management of susceptible populations, and hopefully improve prophylaxis and treatment. Acknowledgments We thank Oliver Daumke (Max-Delbru¨ck Centrum for Molecular Medicine, Berlin, Germany), Harmit S. Malik and Patrick S. Mitchell (Fred Hutchinson Cancer Research Center, Seattle, USA), Jovan Pavlovic (University of Zu¨rich, Switzerland), and the members of the Freiburg laboratory for pertinent comments on the manuscript, Mirjam Schilling for help with graphical artwork, and all our collaborators for their contributions over many years. We are grateful for support from the German Science Foundation (KO 1579/8-2, KO 1579/9-1, SCHW 632/15-1, and STA 338/13-1), the Land Baden-Wu¨rttemberg, the Federal Ministry of Education and Research (FluResearchNet), and the European Union (FLUINNATE).

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