Functional Comparison of Mx1 from Two Different ... - Journal of Virology

Functional Comparison of Mx1 from Two Different Mouse Species Reveals the Involvement of Loop L4 in the Antiviral Activity against Influenza A Viruses Judith Verhelst,a,b Jan Spitaels,a,b Cindy Nürnberger,c,d,e Dorien De Vlieger,a,b Tine Ysenbaert,a,b Peter Staeheli,c Walter Fiers,a,b Xavier Saelensa,b Medical Biotechnology Center, VIB, Ghent, Belgiuma; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgiumb; Institute of Virology, University Medical Center Freiburg, Freiburg, Germanyc; Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germanyd; Faculty of Biology, University of Freiburg, Freiburg, Germanye

ABSTRACT

The interferon-induced Mx1 gene is an important part of the mammalian defense against influenza viruses. Mus musculus Mx1 inhibits influenza A virus replication and transcription by suppressing the polymerase activity of viral ribonucleoproteins (vRNPs). Here, we compared the anti-influenza virus activity of Mx1 from Mus musculus A2G with that of its ortholog from Mus spretus. We found that the antiviral activity of M. spretus Mx1 was less potent than that of M. musculus Mx1. Comparison of the M. musculus Mx1 sequence with the M. spretus Mx1 sequence revealed 25 amino acid differences, over half of which were present in the GTPase domain and 2 of which were present in loop L4. However, the in vitro GTPase activity of Mx1 from the two mouse species was similar. Replacement of one of the residues in loop L4 in M. spretus Mx1 by the corresponding residue of A2G Mx1 increased its antiviral activity. We also show that deletion of loop L4 prevented the binding of Mx1 to influenza A virus nucleoprotein and, hence, abolished the antiviral activity of mouse Mx1. These results indicate that loop L4 of mouse Mx1 is a determinant of antiviral activity. Our findings suggest that Mx proteins from different mammals use a common mechanism to inhibit influenza A viruses. IMPORTANCE

Mx proteins are evolutionarily conserved in vertebrates and inhibit a wide range of viruses. Still, the exact details of their antiviral mechanisms remain largely unknown. Functional comparison of the Mx genes from two species that diverged relatively recently in evolution can provide novel insights into these mechanisms. We show that both Mus musculus A2G Mx1 and Mus spretus Mx1 target the influenza virus nucleoprotein. We also found that loop L4 in mouse Mx1 is crucial for its antiviral activity, as was recently reported for primate MxA. This indicates that human and mouse Mx proteins, which have diverged by 75 million years of evolution, recognize and inhibit influenza A viruses by a common mechanism.

T

he Mx proteins are interferon (IFN)-induced GTPases that inhibit a wide range of viruses, including Orthomyxoviridae, Bunyaviridae, and Rhabdoviridae (reviewed in references 1 and 2). The gene encoding mouse Mx1, the founder member of this family of antiviral proteins, was discovered almost 30 years ago on the basis of the resistance of the A2G mouse strain to influenza A virus infection (3, 4). This resistance is inherited as a dominant autosomal trait and depends on a single gene (Mx1) located on chromosome 16 (5). However, most inbred laboratory mouse strains contain a three-exon deletion or a nonsense mutation in the Mx1 locus and are susceptible to influenza viruses (6). In contrast, Mx1⫹ and Mx1⫺ alleles can be found at similar frequencies in wild mice. This suggests that there is a selective advantage of heterozygosity at the Mx1 locus, as one would expect that the Mx1⫹ allele would otherwise be fixed in wild mouse strains (7). The mouse Mx locus contains Mx1 and Mx2. Remarkably, Mx2 is also nonfunctional in laboratory mouse strains but functional in wild mouse strains (8, 9). It is unclear why laboratory mouse strains lack functional Mx genes. One possibility is a founder effect, as most laboratory strains are derived from a small number of mice. Other possibilities are the absence of positive selection for a functional Mx locus or a selective advantage for an Mx⫺ locus in laboratory mice (6, 7). Mx1 expression is induced by type I and type III interferons

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and can protect mice against influenza A virus infection (10–13). However, Mx1 can protect cells against influenza A virus infection in the absence of interferons (14, 15). The molecular details of the anti-influenza virus mechanism of mouse Mx1 are only partially resolved. There is strong evidence that Mx1 inhibits the activity of the viral polymerase, which is present in viral ribonucleoproteins (vRNPs) (16–18). These vRNPs are the minimal units required for viral transcription and replication. They contain the viral RNA (vRNA) genome complexed with multiple nucleoprotein (NP) molecules and one RNA-dependent RNA polymerase complex containing polymerase basic protein 1 (PB1), PB2, and polymerase acid protein (PA) (19). We recently showed that Mx1 interacts

Received 10 July 2015 Accepted 10 August 2015 Accepted manuscript posted online 19 August 2015 Citation Verhelst J, Spitaels J, Nürnberger C, De Vlieger D, Ysenbaert T, Staeheli P, Fiers W, Saelens X. 2015. 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 89:10879 –10890. doi:10.1128/JVI.01744-15. Editor: T. S. Dermody Address correspondence to Xavier Saelens, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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with two components of these vRNPs, i.e., PB2 and NP, and that the interaction between these two viral proteins is strongly reduced in the presence of Mx1 (18). Prevention or disruption of the PB2-NP interaction could explain how Mx1 inhibits viral polymerase activity. The importance of the Mx1-NP interaction is in line with the observation that the sensitivity of different influenza virus strains to inhibition by Mx1 is determined by the origin of their NP protein, with viruses carrying avian influenza virus-derived NP typically being more sensitive to human MxA and mouse Mx1 (14, 18, 20). Mouse Mx1 belongs to the family of large GTPases which also includes dynamins (21, 22). These proteins contain three domains, a GTPase domain, a bundle-signaling element (BSE), and a stalk domain, all of which have specific functions in antiviral activity. The GTPase domain is the most conserved part of Mx proteins, as GTPase activity is usually required for antiviral activity (1, 18, 23). The stalk is important for oligomerization, which is mediated by three interfaces and one loop region (loop L4). These interfaces mediate the formation of a crisscross interaction pattern, which ultimately results in ring formation. In these Mx rings, the stalk domains point inwards and the GTPase domains are located at the periphery. An appealing but as yet unproven model is that the viral targets, e.g., the vRNPs, could occupy the inside of the ring and interact with loop L4 at the tip of the stalk domains of multiple Mx molecules. The BSE that separates the GTPase domain from the stalk is believed to be crucial for transmitting conformational changes caused by GTPase activity from the GTPase domain to the stalk (24, 25). In this model, Mx proteins cooperatively inhibit or disturb the function of their viral targets. The Mx1 region(s) that is important for interaction with influenza virus PB2 and NP is unknown. Recent studies on human MxA showed that loop L4, at the end of the stalk, is important for recognition of the vRNPs of Thogoto virus, a mouse orthomyxovirus. Specific residues in this loop are also important for MxA’s antiviral activity against influenza and La Crosse viruses. Replacement of this loop in mouse Mx1 with the human MxA counterpart enables mouse Mx1 to recognize and inhibit Thogoto virus vRNPs, suggesting that this loop is an independent entity that can recognize its viral target (26, 27). However, it has not been formally demonstrated that loop L4 in mouse Mx1 is important for recognition of influenza virus RNPs or any other viral target. In this work, we studied the antiviral activity of the Mx1 protein from Mus spretus, which diverged from M. musculus about 1.5 million years ago (28). We reasoned that the evolutionary divergence of these two mouse species might be reflected in differences in the antiviral activity of their Mx1 proteins due to exposure to different pathogens. Based on a minireplicon assay and infection with different influenza A viruses, we found that the antiviral activity of M. spretus Mx1 (SMx1) is weaker than that of M. musculus A2G Mx1 (A2G Mx1). By exchanging regions of these related Mx1 proteins, we identified one residue in loop L4 that significantly contributes to the weaker antiviral activity of SMx1. Furthermore, we revealed that loop L4 is essential for the antiviral activity of mouse Mx1. MATERIALS AND METHODS Cells. HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.4 mM Na-pyruvate, nonessential amino acids, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. MDCK cells were maintained in

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DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, nonessential amino acids, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Mouse embryonic fibroblasts (MEFs) were derived at 13.5 days postcoitum from embryos from congenic BL6.spretus-Mx1 mice and were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.4 mM Na-pyruvate, nonessential amino acids, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 ␮M ␤-mercaptoethanol. The ethical committee of Ghent University approved the use of these laboratory mice for the isolation of MEFs. Viruses. Mouse-adapted A/Puerto Rico/8/34 (maPR8; H1N1) (29) and mouse-adapted A/Swine/Ontario/42729A/01 (maSwO; H3N3) (30) were grown on MDCK cells and isolated from the culture supernatant by centrifugation at 25,000 ⫻ g for 16 h at 4°C. Virions were resuspended in phosphate-buffered saline containing 20% glycerol, aliquoted, and stored at ⫺80°C until use. Isolation of SMx1 cDNA. MEFs were prepared from congenic BL6.spretus-Mx1 mouse embryos, and the cells were treated with 400 U/ml mouse beta interferon (IFN-␤; catalog no. I-9032; Sigma) for 16 h to induce Mx1 expression. The total RNA fraction was purified with an RNeasy kit (catalog no. 74104; Qiagen) and cDNA was generated with a first-strand reverse transcriptase kit (catalog no. 04379012-001; Roche) according to the manufacturers’ instructions. SMx1 cDNA was isolated with Mx1-specific primers (forward primer, CCGCTCGAGACGATGGA TTCTGTGAATAATCT; reverse primer, ACGACGCGTAGCCTGGTTA ATCGGAGAATT). The sequences in bold correspond to the beginning and the end of the predicted Mx1 cDNA sequence in the forward and reverse primers, respectively. Plasmids. The mammalian expression plasmids pCAXL-PB1, -PB2, -PB2V5, -PA, and -NP and the pHW-NSLuc reporter plasmid have been described previously (18). pHW-NSLuc contains the firefly luciferase cDNA in a negative-sense orientation between the noncoding regions of the nonstructural (NS) protein segment (3= 23 nucleotides [nt] and 5= 26 nt) of PHW198-NS (31). The pRL-CMV plasmid (catalog no. E2261; Promega), containing a Renilla luciferase gene under the control of a cytomegalovirus (CMV) promoter, was used to normalize for transfection efficiency. The mammalian expression plasmids pCAXL-Mx1 and pCAXL-Mx1T69A, expressing mouse Mx1 cDNA from the Mus musculus A2G strain, have been described previously (18). The SMx1 cDNA was cloned in pCAXL between the XhoI and MluI restriction sites, and the SMx1T69A mutant was generated in the same way as the A2G Mx1T69A mutant (18). A plasmid containing the previously published SMx1 cDNA was a kind gift from Claude Libert (32). The region coding for this SMx1 cDNA (here referred to as SMx1 variant 1 [SMx1 v1]) was cloned into pCAXL by conventional PCR using the XhoI and MluI restriction sites. A2G Mx1dL4 contains a deletion of amino acids 499 to 538 of loop L4 and was generated by fusion PCR and cloned into pCAXL between the XhoI and MluI restriction sites. The Mx1 GTPase hybrids were generated synthetically by Gen9 (Cambridge, MA, USA) and transferred from pUC19 into pCAXL. For the production of recombinant Mx1 proteins, the cDNA of Mx1T69A, SMx1, SMx1T69A, and the Mx1 GTPase hybrids, excluding the start codon, was isolated by PCR and cloned in pQE9 between the BamHI and SalI restriction sites, placing the Mx1 cDNA in frame and downstream of the His6 tag in the pQE9 vector. The pQE9-Mx1 plasmid was described before (23). The pHW192-PB1, pHW191-PB2, pHW193PA, and pHW195-NP plasmids have been described previously (31). The pHWS-maSwO-PB1, -maSwO-PB2, -maSwO-PA, and -maSwO-NP plasmids contain the respective viral genome segments, which were isolated from mouse-adapted A/Swine/Ontario/42729A/01 virus (30). In brief, viral RNA was isolated with a NucleoSpin RNA virus kit (catalog no. 740956; Macherey-Nagel) and cDNA was produced with a first-strand reverse transcriptase kit (catalog no. 04379012-001; Roche) according to the manufacturers’ instructions. The respective genome segments were amplified by PCR as described previously and cloned into pHWSccdB (33).

Journal of Virology


Anti-Inﬂuenza Virus Activity of Mus spretus Mx1

Chemical reagents and antibodies. N-Ethylmaleimide was obtained from Sigma (catalog no. E-3876) and dissolved in ethanol at a concentration of 2 M. Protease inhibitor cocktail tablets were from Roche (catalog no. 11 873 580 001). A polyclonal antiserum raised against the C-terminal part of mouse Mx1 has been described previously (18). The monoclonal anti-V5-horseradish peroxidase (HRP) antibody used for Western blotting was from Invitrogen (catalog no. R96125). The monoclonal anti-V5 antibody used for coimmunoprecipitation was from Pierce (catalog no. MA5-15253). The following reagents were obtained from the NIH Biodefense and Emerging Infections Research Resources Repository (NIAID, NIH): monoclonal anti-influenza A virus NP clones A1 and A3 (ascites blend, mouse; catalog no. NR-4282) and polyclonal anti-influenza virus RNP from A/Scotland/840/74 (H3N2) (antiserum, goat; catalog no. NR3133). The fluorescently labeled antibodies Alexa Fluor 488 donkey antirabbit IgG (catalog no. A21206) and Alexa Fluor 633 donkey anti-goat IgG (catalog no. A21082) were from Life Technologies Europe B.V. Mx1 protein purification. SMx1, SMx1T69A, A2G Mx1, A2G Mx1T69A, and hybrid Mx1 proteins were expressed as N-terminal His6 fusion constructs from a pQE9 plasmid in Escherichia coli strain M15(pREP4) in LB medium containing 20 ␮g/ml kanamycin and 100 ␮g/ml ampicillin. Protein expression was induced by addition of 300 ␮M isopropyl-␤-D-1-thiogalactopyranoside (catalog no. I6758-1G; Sigma) at an A600 of 0.3 and carried out overnight at 18°C. After centrifugation, bacterial pellets were suspended in ice-cold lysis buffer (50 mM Tris HCl [pH 8.0], 500 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 2 mM imidazole, 7 mM ␤-mercaptoethanol, cOmplete proteinase inhibitor [Roche]) and lysed by 7 cycles of sonication (Branson sonifier 250) for 30 s with breaks of 30 s on ice in between. Following centrifugation at 4°C to remove insoluble cell debris, the bacterial supernatant was incubated with Ni-nitrilotriacetic acid (NTA) agarose beads (catalog no. 1018244; Qiagen) that had been equilibrated with lysis buffer on a rotating wheel for 1 h at 4°C. After retention in a column, the beads were washed extensively with lysis buffer containing 30 mM imidazole, following an equilibration step with elution buffer (20 mM Tris HCl [pH 8.0], 100 mM NaCl, 5 mM MgCl2, 20% glycerol, 0.1% NP-40, 7 mM ␤-mercaptoethanol). The Mx1 proteins were eluted from the Ni-NTA agarose beads with elution buffer containing 250 mM imidazole. Protein-containing fractions were pooled, dialyzed against elution buffer overnight at 4°C, and frozen in aliquots at ⫺80°C. The purity and relative amounts of Mx1 were visualized on a Coomassie brilliant blue-stained SDS-polyacrylamide gel. ImageJ software (version 1.47g) was used to determine the relative amount of the recombinant Mx1 proteins from the stained SDS-polyacrylamide gel. GTPase activity assay. The GTPase activity of the recombinant Mx1 proteins was measured using a Transcreener GDP FI assay kit (BellBrook Labs). Briefly, proteins were diluted to the same Mx1 concentrations with elution buffer, and each reaction mixture was supplemented with a GTP master mix with a final reaction volume (25 ␮l) containing 4 ␮M GTP and 40 nM adenosine 5=-(␤,␥-imido)triphosphate lithium salt hydrate (AMP PNP). After 1 h of incubation at 37°C, the GTPase reaction was stopped by adding 25 ␮l stop and detect buffer (20 mM HEPES, pH 7.5, 40 mM EDTA, 0.02% Brij 35, 8 nM GDP-Alexa Fluor 594 tracer, 4.56 ␮g/ml GDP-IRDye QC-1 antibody) and incubated for another hour at room temperature in the dark before detection of the fluorescent signal with a Tecan Infinite M200 plate reader (excitation wavelength, 580 nm; emission wavelength, 620 nm). Influenza A virus PR8 minireplicon system based on pCAXL vectors. HEK293T cells (seeded at 5 ⫻ 104 cells per well in 24-well plates) were transfected in triplicate with the expression plasmids pCAXL-PB1, -PB2, -PA, and -NP (25 ng each), luciferase reporter pHW-NSLuc (100 ng), and pRL-CMV (25 ng) using the calcium phosphate precipitation method. To assess the effects of the different Mx1 variants, the respective Mx1 expression plasmids were cotransfected in the amounts mentioned in the figure legends. Cells were lysed 48 h later with luciferase lysis buffer (25 mM Tris phosphate, 2 mM dithiothreitol, 2 mM CDTA [trans-1,2cyclohexanediaminetetraacetic acid], 10% glycerol, 1% Triton X-100).


Luciferase activity was measured with a dual-luciferase reporter assay system (catalog no. E-1960; Promega) and a GloMax 96 microplate luminometer (Promega). The relative luciferase activity was calculated as the ratio between the activities of firefly and Renilla luciferases [(firefly luciferase activity/Renilla luciferase activity) ⫻ 1,000]. Influenza A virus minireplicon systems based on pHW vectors. HEK293T cells (seeded at 5 ⫻ 104 cells per well in 24-well plates) were transfected in triplicate by the calcium phosphate transfection method with 80 ng of pHW192-PB1, pHW191-PB2, pHW193-PA, and pHW195-NP (from PR8) (31) or 80 ng of pHWS-maSwO-PB1, -maSwOPB2, -maSwO-PA, and -maSwO-NP (from maSwO). The luciferase reporter pHW-NSLuc (80 ng) and pRL-CMV (25 ng) were cotransfected. In addition, increasing concentrations (5 ng, 10 ng, 20 ng, or 40 ng) of pCAXL-SMx1, -SMx1E540G, -Mx1, -Mx1G540E, or -Mx1dL4 were cotransfected. Cells were lysed for 24 h after transfection with luciferase lysis buffer, and luciferase activity was measured as described above. Coimmunoprecipitation analysis of the interaction between Mx1 and NP. HEK293T cells (seeded at 1.2 ⫻ 106 cells per 9-cm dish) were transfected using calcium phosphate with pCAXL-PB1, -PB2, -PA, and -NP and pHW-NSLuc (0.5 ␮g each) and 1 ␮g of pCAXL-SMx1, -Mx1, or -Mx1dL4. Total lysates were prepared 24 h later in low-salt lysis buffer (pH 7.2) containing N-ethylmaleimide buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 25 mM N-ethylmaleimide, protease inhibitor cocktail). The cells were lysed for 20 min on ice and centrifuged for 3 min at 16,000 ⫻ g to remove insoluble proteins. NP was immunoprecipitated from the cleared lysates with monoclonal anti-NP antibody for 3 h at 4°C. Immune complexes were collected by incubation for 1 h at 4°C in the presence of protein A-Sepharose beads (catalog no. 17-1279-01; GE Healthcare) followed by centrifugation. Immunoprecipitates were washed 4 times with high-salt lysis buffer, pH 7.2 (50 mM Tris-HCl, pH 7.2, 500 mM NaCl, 5 mM EDTA, 1% NP-40). Proteins were eluted from the beads by boiling for 10 min at 95°C in 2⫻ Laemmli buffer. The samples were separated by SDS-PAGE (8%), and immunoprecipitated proteins were visualized by Western blotting with antibodies directed against NP (polyclonal goat anti-RNP) or Mx1. Coimmunoprecipitation analysis of the PB2-NP interaction. HEK293T cells (seeded at 1.2 ⫻ 106 cells per 9-cm dish) were transfected using calcium phosphate with expression plasmids pCAXL-PB1, -PB2V5, -PA, and -NP and pHW-NSLuc (1 ␮g each) and increasing amounts of cotransfected pCAXL-SMx1 or pCAXL-Mx1 (0 ␮g, 0.5 ␮g, or 1 ␮g). Total lysates were prepared 24 h after transfection in low-salt lysis buffer, pH 8.0 (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, protease inhibitor cocktail). The cells were lysed for 20 min on ice and centrifuged for 3 min at 16,000 ⫻ g to remove insoluble proteins. V5tagged PB2 (PB2V5) and NP were immunoprecipitated from the cleared lysates with monoclonal anti-V5 and anti-NP antibodies, respectively, for 3 h at 4°C. Immune complexes were collected by incubation for 1 h at 4°C in the presence of protein A-Sepharose beads (catalog no. 17-1279-01; GE Healthcare) followed by centrifugation. Immunoprecipitates were washed 4 times with high-salt lysis buffer, pH 8.0 (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM EDTA, 1% NP-40). Proteins were eluted from the beads by boiling for 10 min at 95°C in 2⫻ Laemmli buffer. The samples were separated by SDS-PAGE (8%), and immunoprecipitated proteins were visualized by Western blotting with antibodies directed against the V5 tag (anti-V5-HRP), NP (polyclonal goat anti-RNP), or Mx1. Immunofluorescence microscopy. HEK293T cells (5 ⫻ 104 cells per well seeded on glass coverslips in 24-well plates) were transfected using calcium phosphate with 50 ng of pCAXL-Mx1, -SMx1, -Mx1T69A, or -SMx1T69A. After 24 h, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained for Mx1 with antiMx1 (diluted 1/1,000) and Alexa Fluor 488-labeled donkey anti-rabbit IgG (diluted 1/600). Cell nuclei were visualized with Hoechst (diluted 1/1,000; catalog no. H21492; Invitrogen). Images were recorded with a confocal microscope (Leica Sp5 AOBS confocal system) using a 63⫻ HCX PL Apo (numerical aperture, 1.4) oil immersion objective. A bandpass

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filter of 510 to 555 nm was positioned before the detector to measure the fluorescence of Alexa Fluor 488 after excitation with a 488-nm argon laser. Flow cytometric analysis of cells infected with influenza A virus. HEK293T cells (5 ⫻ 105 cells per 6-cm dish) were transfected in triplicate with 100 ng of pCAXL-SMx1, -SMx1T69A, -SMx1E540G, -Mx1, -Mx1T69A, or -Mx1dL4. The Fugene HD reagent (catalog no. E-2311; Promega) was used for transfection according to the manufacturer’s instructions. After 24 h (for maPR8) or 30 h (for maSwO), the cells were infected with maPR8 at a multiplicity of infection (MOI) of 0.5 for 16 h or with maSwO at an MOI of 1 for 8 h. Next, the cells were collected, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. The cells were stained for Mx1 (with anti-Mx1, diluted 1/400) and for influenza virus NP (with anti-RNP, diluted 1/2,000). We used Alexa Fluor 488-labeled donkey anti-rabbit IgG (diluted 1/600) and Alexa Fluor 633labeled donkey anti-goat IgG (diluted 1/1200) as secondary antibodies, respectively. The number of Mx1- and RNP-positive cells was determined on an LSR-II flow cytometer (BD, San Jose, CA) using FACSDiva (BD) and FlowJo X (TreeStar) software. Statistics. The data were analyzed with Genstat (v16) software (34). For the infection assays, the data analysis of infected cell proportions was performed by logistic regression, as implemented in Genstat (v16). Infected cell proportions were logit transformed to provide additivity and stabilize the variance. The significance of the Mx1 activity was assessed by an F test. The significance levels of pairwise comparisons between Mx1 protein mean proportions were assessed by a Fisher’s protected least significant difference test. In the GTPase experiment, the GTPase activities of the various Mx1 proteins were compared using a one-way analysis of variance (ANOVA), with the replicates of the experiment being set as blocks. Significance was assessed by an F test. For the PR8 and maSwO minireplicon assays, analysis of firefly luciferase activity was performed by a hierarchical generalized linear mixed model (HGLM) regression implemented in Genstat (v16). HGLMs provide another way of modeling nonnormal data when there are several sources of error variation. Unlike generalized linear mixed models (GLMMs), HGLMs do not constrain the additional error terms added to the linear predictor to follow a normal distribution and to have an identity link (as in the GLMMs). For example, if the basic generalized linear model is a log-linear model (with a Poisson distribution and a log link), a more appropriate assumption or the additional random terms might be a gamma distribution and a log link. A log-linear model (with a Poisson distribution and a log link) containing the Mx1 protein and concentration as fixed terms was fitted to the firefly luciferase values normalized to those for Renilla luciferase. The additional random terms experiment and replicates within an experiment were assumed to follow a gamma distribution (and a log link), and the dispersion parameter was set as free. The significances of pairwise comparisons between Mx1 protein effects at a fixed concentration were assessed by a Fisher’s protected least significant difference test. Nucleotide sequence accession number. The Mus spretus Mx1 sequence was submitted to GenBank under accession number KT591117.

RESULTS

Isolation and sequence analysis of SMx1. Congenic mice expressing M. spretus Mx1 (SMx1) are equally if not better protected against influenza A virus infection than congenic mice expressing M. musculus A2G Mx1 (A2G Mx1) (C57BL/6 background) (32). To determine if this reported stronger in vivo protection of mice carrying SMx1 can be explained by a difference in the intrinsic antiviral activities of SMx1 and A2G Mx1, we compared their antiviral activities in cell culture. First, we cloned the Mx1 cDNA from the congenic BL6.spretus-Mx1 mice by reverse transcriptionPCR using RNA isolated from MEFs that had been pretreated with 400 U/ml mouse IFN-␤ to induce Mx1 gene expression. The predicted SMx1 sequence contains 25 amino acid substitutions compared to the sequence of A2G Mx1 (Fig. 1). These differences are

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scattered over the entire protein, with 13 differences occurring in the GTPase domain, 10 occurring in the stalk domain, and 2 occurring in the BSE. Modeling of the three-dimensional structure of mouse Mx1 on the basis of the crystal structure of human MxA revealed that 22 of the 24 different residues (this excludes residue 10, as the first 10 amino acids are absent from the modeled structure) were surface exposed. Based on this distribution, we conclude that amino acid substitutions do not occur preferentially in a particular domain. A sequence of SMx1 has been published once before (32). We found that the SMx1 sequence that we determined (based on four independent cDNA clones) differs from this previously reported sequence at four positions: H10Q, G84E, T209I, and I547T. It is possible that these two SMx1 sequences represent allelic variants, both of which can suppress influenza A virus replication. Therefore, we compared the antiviral activity of the newly isolated SMx1 cDNA (SMx1) to the one described previously (here named SMx1 variant 1) (32) in a PR8 minireplicon assay. SMx1 variant 1 did not inhibit influenza virus PR8 polymerase activity, in contrast to SMx1 (Fig. 2). Therefore, SMx1 variant 1 represents a clone that most likely lacks anti-influenza virus activity. We continued the A2G-M. spretus comparison using the SMx1 cDNA described in this report. Mx1 from M. spretus inhibits PR8 less effectively than Mx1 from M. musculus A2G. To compare the antiviral activities of SMx1 and A2G Mx1, we first used the influenza virus PR8 minireplicon system. As expected, coexpressed A2G Mx1 strongly reduced the luciferase reporter activity in this assay in a dose-dependent way (Fig. 3A). The inhibition by SMx1 was also dose dependent but clearly weaker than that by A2G Mx1 (Fig. 3A). We previously showed that A2G Mx1 inhibits the interaction between NP and PB2 and that this inhibition correlates with the antiviral activity (18, 35). Therefore, we investigated the ability of both Mx1 proteins to suppress the interaction between PB2 and NP. We transfected all components of the minireplicon system in the absence or presence of SMx1 or A2G Mx1 and analyzed the PB2-NP interaction by coimmunoprecipitation. We performed pulldown of PB2V5 followed by analysis of coimmunoprecipitated NP, as well as pulldown of NP followed by analysis of coimmunoprecipitated PB2V5. We found that SMx1 had a weaker effect than A2G Mx1 on the interaction between PB2 and NP (Fig. 3B). This is in line with the lower inhibitory activity of SMx1 than A2G Mx1 in the minireplicon assay. Next, we compared the abilities of SMx1 and A2G Mx1 to inhibit influenza A virus replication. As a readout we used flow cytometry to determine the number of Mx1-positive cells that expressed viral RNP due to infection. HEK293T cells were first transfected with expression vectors for SMx1, A2G Mx1, or their GTPase-inactive T69A mutants, with the T69A mutants being inactive in the minireplicon assay (Fig. 3C). Thirty hours after transfection, the cells were infected with maPR8 at an MOI of 0.5 for 16 h. The number of infected cells in the Mx1-negative population was similar for all settings (unpublished results). Compared with cells expressing inactive T69A mutants, significantly fewer infected (RNP-positive) cells were detected among cells expressing wild-type Mx1 proteins (Fig. 3D and E). Although SMx1 substantially inhibited PR8 infection, this inhibition was significantly lower than that by A2G Mx1 (Fig. 3D and E). Taken together, these results indicate that SMx1 has a weaker antiviral activity than A2G Mx1 against PR8.

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FIG 1 Amino acid sequence alignment of A2G Mx1 and SMx1. (A) The predicted amino acid sequences of A2G Mx1 and SMx1 were aligned by use of the ClustalW program. The 25 amino acid differences are indicated in red. (B) Model of the three-dimensional structure of mouse Mx1, generated with SWISSMODEL (automated mode; generated in April 2013 on the basis of the three-dimensional structure of human MxA [PDB accession number 3SZR] and the sequence of A2G Mx1). The different functional domains are indicated: GTPase domain (blue), BSE (orange), stalk (green), and loop L4 (purple). The epitope of the anti-Mx1 polyclonal antibody used in this study is also indicated.

M. spretus Mx1 and M. musculus A2G Mx1 show similar subcellular localization. Nuclear localization of mouse Mx1 is important for its antiviral activity (36). A possible reason for the reduced antiviral activity of SMx1 could therefore be an altered subcellular localization. We compared the subcellular localization of SMx1 and A2G Mx1 and their inactive T69A mutants by confocal immunofluorescence microscopy. The T69A mutants of both SMx1 and A2G


Mx1 appeared as large aggregates both in the nucleus and in the cytoplasm (Fig. 4). In contrast, both wild-type Mx1 proteins showed punctate nuclear staining, typical for mouse Mx1 (18, 37) (Fig. 4). Therefore, it is unlikely that an altered localization accounts for the reduced anti-influenza viral activity of SMx1. Role of the GTPase domain in the reduced antiviral activity of M. spretus Mx1. To determine whether the amino acid differ-

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FIG 2 Antiviral activity of different SMx1 cDNA isolates. HEK293T cells were transfected in triplicate with PB1, PB2, PA, and NP expression plasmids (25 ng each), pHW-NSLuc (100 ng), and pRL-CMV (25 ng). An empty control or an increasing concentration of the expression vector for one of the different Mx1 proteins was cotransfected (50 ng, 100 ng, 200 ng, or 400 ng for the SMx1 variant 1; 5 ng, 10 ng, 25 ng, or 50 ng for SMx1). The relative luciferase (Rel Luc) activity in the lysates was determined 48 h after transfection. Bars represent the averages from three independent experiments, each of which was performed in triplicate, and the error bars depict the standard errors. The levels of Mx1 and NP expression were determined by Western blotting, of which a representative result is shown.

ences in the GTPase domains of SMx1 and A2G Mx1 are involved in the reduced antiviral activity of SMx1, we generated Mx1 hybrids in which the GTPase domains (amino acids 35 to 306) were mutually exchanged. The antiviral activities of these hybrids were determined in the PR8 minireplicon system. Replacement of the GTPase domain of A2G Mx1 with that of SMx1 completely abolished the antiviral activity (Fig. 5A). In addition, grafting of the GTPase domain of A2G Mx1 on SMx1 reduced the inhibitory activity of the latter even further (Fig. 5A). To assess if a difference in GTPase activity could account for the different antiviral activities, we purified recombinant SMx1, A2G Mx1, the two GTPase domain hybrids, and the GTPase-inactive T69A mutant proteins from E. coli and determined their GTPase activities (Fig. 5B and C). In this GTPase assay, all Mx1 variants except the inactive T69A mutant proteins showed comparable GTPase activities (Fig. 5C). This implies that the mutations present in the GTPase domain do not influence GTPase activity. In addition, this indicates that the reduced antiviral activity of the Mx1 hybrids and SMx1 is not the result of a reduced GTPase activity. Loop L4 in Mx1 is important for NP binding and antiviral activity. Loop L4 in human MxA is important for viral target recognition, as was shown for Thogoto virus NP (26, 27). Therefore, we hypothesized that this loop in mouse Mx1 could also be important for recognition of influenza virus NP and, hence, for antiviral activity. In addition, the sequences of SMx1 and A2G Mx1 differ at positions 516 and 540 in this loop. It is thus possible that one or both of these residues are responsible for the lower antiviral activity of SMx1 against PR8 virus. First, we determined the importance of loop L4 for binding to the influenza virus NP protein. We generated a mouse Mx1 vari-

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ant, A2G Mx1dL4, in which amino acids 499 to 538 were deleted and assessed its binding to influenza virus NP by coimmunoprecipitation. Additionally, we validated if SMx1 was able to bind to influenza virus NP. For this, we transfected all components of the minireplicon system together with SMx1, A2G Mx1, or A2G Mx1dL4 and analyzed the NP-Mx1 interaction by coimmunoprecipitation with anti-NP. This experiment showed that both SMx1 and A2G Mx1 could interact with influenza virus NP. In contrast, in the absence of loop L4 (A2G Mx1dL4), no binding with influenza virus NP could be observed (Fig. 6A). This suggests that loop L4 in mouse Mx1 is important for viral target recognition. Next, we investigated if the differences in loop L4 between SMx1 and A2G Mx1 contributed to the lower antiviral activity of SMx1 against influenza virus PR8. We mutated either of these different positions alone or together in SMx1 to their equivalent in A2G Mx1, generating SMx1V516A, SMx1E540G, and SMx1V516AE540G. In addition, we included the A2G Mx1dL4 mutant to validate the importance of loop L4 for antiviral activity. On the basis of the results of the minireplicon assay, the removal of loop L4 proved to be detrimental to the antiviral activity of A2G Mx1 (Fig. 6B). The V516A substitution slightly reduced the antiviral activity of SMx1, whereas the E540G mutation increased the antiviral activity of SMx1 (Fig. 6B). The activity of the double mutant was slightly reduced compared to that of the single SMx1E540G mutant. To evaluate the importance of position 540 in the context of a viral infection, we compared the antiviral activities of SMx1, SMx1E540G, A2G Mx1, and A2G Mx1dL4 in cells infected with influenza virus PR8 (MOI ⫽ 0.5). The E540G mutation in SMx1 increased its antiviral activity (Fig. 6C). We conclude that position 540 in loop L4 is partially responsible for the lower antiviral activity of SMx1 against PR8. In addition, the difference in activity between SMx1E540G and A2G Mx1 indicates that another position(s) in Mx1 is also important for antiviral activity. M. spretus Mx1 inhibits human- and avian-type influenza A virus replication. Different influenza virus strains show different sensitivities to the antiviral activity of mouse Mx1 and human MxA (14, 18, 20). The human influenza virus PR8 strain is more resistant than avian strains to Mx1 activity (18). We compared the antiviral activity of SMx1 and A2G Mx1 against the more sensitive BALB/c mouse-adapted A/Swine/Ontario/42729A/01 strain (maSwO). The parental strain of maSwO is an avian H3N3 strain isolated from a pig (38). Previous experiments have shown that the NP of SwO is very sensitive to A2G Mx1 (18). First, we tested the antiviral activity of SMx1 against this avian-type influenza virus in an infection experiment. We transfected HEK293T cells with SMx1, SMx1E540G, A2G Mx1, or A2G Mx1dL4 expression plasmids. After 24 h, the cells were infected with maSwO (MOI ⫽ 1) for 8 h. We used an infection time shorter than that used for the PR8 infection experiments outlined above due to the faster infection kinetics of this virus. Flow cytometric analysis of the number of infected cells in the Mx1-positive populations showed that all Mx1 proteins, except A2G Mx1dL4, inhibited maSwO to a similar extent (Fig. 7). The maSwO virus is very sensitive to the inhibitory activity of Mx1. Therefore, it is possible that a difference between SMx1 and A2G Mx1 could be detected only at lower Mx1 protein concentrations. Therefore, we compared the activities of SMx1, SMx1E540G, A2G Mx1, and A2G Mx1G540E in a minireplicon assay derived from PR8 (Fig. 8A and B) or maSwO (Fig. 8C and

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FIG 3 SMx1 has a lower level of antiviral activity than A2G Mx1 against influenza virus PR8. (A) HEK293T cells were transfected in triplicate with PB1, PB2, PA, and NP expression plasmids (25 ng each), pHW-NSLuc (100 ng), and pRL-CMV (25 ng). In addition, different amounts of pCAXL-SMx1 or pCAXL-Mx1 (0 ng, 12.5 ng, 25 ng, 50 ng, or 125 ng) were cotransfected. The relative luciferase activity in the lysates was determined 48 h after transfection. Bars represent average Rel Luc activity from three transfections in one experiment, and the error bars depict the standard errors. This graph is a representative from at least two independent experiments. Mx1 and NP expression was analyzed by Western blotting. (B) HEK293T cells were transfected with expression plasmids for PB1, PB2V5, PA, and NP (1 ␮g each) and pHW-NSLuc (1 ␮g). In addition, different amounts of pCAXL-SMx1 or pCAXL-Mx1 (0 ␮g, 0.5 ␮g, or 1 ␮g) were cotransfected. Total lysates were made 24 h after transfection, and PB2V5 and NP were immunoprecipitated (IP) with anti-V5 and anti-NP antibodies, respectively. Proteins were visualized by Western blotting with antibodies recognizing the V5 tag (anti-V5-HRP), NP (anti-RNP), and Mx1. (C) HEK293T cells were transfected in triplicate with PB1, PB2, PA, and NP expression plasmids (25 ng each), pHW-NSLuc (100 ng), and pRL-CMV (25 ng). An empty control or an expression vector for one of the Mx1 mutants (125 ng of pCAXL or pCAXL-SMx1, -SMx1T69A, -Mx1, or -Mx1T69A) was cotransfected. The relative luciferase activity in the lysates was determined 48 h after transfection. Bars represent the averages from triplicate transfections, and the error bars depict the standard errors. This graph is a representative of the graphs from three independent experiments. Mx1 and NP expression was determined by Western blotting. (D and E) HEK293T cells were transfected in triplicate with 100 ng of pCAXL-SMx1, -SMx1T69A, -Mx1, or -Mx1T69A. After 30 h, the cells were infected with maPR8 at an MOI of 0.5. After 16 h, the cells were collected, fixed, permeabilized, and stained for Mx1 (anti-Mx1) and NP (anti-RNP). The number of infected (RNP-positive) cells in the Mx1-positive population was determined on an LSR-II flow cytometer, and the data were analyzed with FACSDiva and FlowJo software. Mx1 expression in lysates of transfected cells was determined by Western blotting. Infected cell proportions were analyzed by logistic regression (logit was used as the link function). Significance levels of pairwise comparisons were assessed by a Fisher’s protected least significant difference test. ***, P ⬍ 0.001. (E) For each group, one representative fluorescence-activated cell sorting plot is shown, and the percentage of infected cells in the Mx1-positive population is indicated. WT, wild type.


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FIG 4 SMx1 and A2G Mx1 have similar subcellular localization. HEK293T cells were transfected with pCAXL-SMx1, -Mx1, -SMx1T69A, or -Mx1T69A (50 ng each). After 24 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained for Mx1 with rabbit anti-Mx1 serum (diluted 1/1000) and Alexa Fluor 488-labeled donkey anti-rabbit IgG (diluted 1/600, depicted in green) (left). Cell nuclei were visualized with Hoechst (diluted 1/1,000, depicted in blue). Images were recorded with a confocal microscope (Leica Sp5 AOBS confocal system). (Right) Overlay of Mx1 (green) and nuclei (blue) staining. Bars ⫽ 10 ␮m.

D). As a negative control we included A2G Mx1dL4. Unlike the minireplicon experiments described above, these minireplicon assays were based on bidirectional pHW plasmids. Therefore, the expression of the viral polymerases and NP, which we monitored by Western blotting, is amplified by reconstituted influenza virus polymerase activity. We found that A2G Mx1 reduced polymerase activity more efficiently than SMx1 for both virus strains (Fig. 8A and C). For the maSwO virus, the difference between A2G Mx1 and SMx1 was significant only at low Mx1 concentrations (5 ng and 10 ng of transfected plasmid). For PR8, the difference was significant at all Mx1 concentrations tested. Again, mutation of position 540 in SMx1 slightly increased its antiviral activity, whereas the reciprocal mutation in A2G Mx1 decreased its antiviral activity. This suggests that position 540 in Mx1 is important for its antiviral activity against different influenza A virus strains. DISCUSSION

To identify Mx1 domains that are important for antiviral activity, we investigated the antiviral activity of the Mx1 protein of M.

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FIG 5 Role of the GTPase domain in the reduced antiviral activity of SMx1 against PR8. (A) HEK293T cells were transfected in triplicate with PB1, PB2, PA, and NP expression plasmids (25 ng each), pHW-NSLuc (100 ng), and pRL-CMV (25 ng). An empty control or an expression vector for one of the different Mx1 mutants was cotransfected (125 ng of pCAXL or pCAXLSMx1T69A, -SMx1, -SMx1-GTPaseA2G, -Mx1-GTPaseSMx1, -Mx1, or -Mx1T69A). The relative luciferase activity in the lysates was determined 48 h after transfection. Bars represent the averages from triplicate transfections, and the error bars depict the standard errors. This graph is a representative of the graphs from three independent experiments. Mx1 and NP expression was determined by Western blotting. (B) Mx1, Mx1T69A, SMx1, SMx1T69A, and the GTPase domain of hybrid Mx1 proteins were produced in E. coli and purified through an N-terminal His6 tag with Ni-NTA agarose beads. The purity and relative Mx1 amounts used in the assay whose results are presented in panel C were visualized on a Coomassie brilliant blue-stained SDS-polyacrylamide gel. (C) The GTPase activity of the different recombinant Mx1 proteins was determined with a Transcreener GDP FI assay kit. Bars represent average fluorescence values from three independent experiments, each of which was performed in triplicate, and the error bars depict the standard errors. The GTPase activities of the various Mx1 proteins were compared using a one-way ANOVA, with the replicates of the experiment being set as blocks. Significance was assessed by an F test. ***, P ⬍ 0.001.

spretus. M. spretus mice carrying Mx1 are better protected against PR8 (H1N1) or A/X-47 (X47; H3N2) virus infection than M. musculus A2G mice carrying Mx1 (32), which could indicate that SMx1 has a higher intrinsic antiviral activity. However, when we

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FIG 7 SMx1 reduces maSwO virus infection. HEK293T cells were transfected in triplicate with 100 ng of pCAXL-SMx1, -SMx1E540G, -Mx1, or -A2G Mx1dL4. After 24 h, the cells were infected with maSwO (H3N3) at an MOI of 1. After 8 h, the cells were collected, fixed, permeabilized, and stained for Mx1 (anti-Mx1) and NP (anti-RNP). The number of infected (RNP-positive) cells in the Mx1-positive population was determined on an LSR-II flow cytometer, and the data were analyzed with FACSDiva and FlowJo software. The graph represents the data from two independent experiments, each of which was performed in triplicate. Infected cell proportions were analyzed by logistic regression (logit was used as the link function). Significance levels of pairwise comparisons were assessed by a Fisher’s protected least significant difference test. **, P ⬍ 0.01. Western blot detection of Mx1 expression in lysates of transfected cells from one of the two experiments is shown.

FIG 6 Loop L4 in Mx1 is important for influenza A virus NP binding and for

antiviral activity. (A) HEK293T cells were transfected with 0.5 ␮g of pCAXLPB1, -PB2, -PA, and -NP and 0.5 ␮g of pHW-NSLuc. In addition, 1 ␮g of pCAXL-SMx1, -Mx1, or -A2G Mx1dL4 was cotransfected. After 24 h, total lysates were made in the presence of 25 mM N-ethylmaleimide, and NP was immunoprecipitated with anti-NP. Proteins were visualized by Western blotting with antibodies recognizing NP (anti-RNP) and Mx1. Ab, antibody. (B) HEK293T cells were transfected in triplicate with PB1, PB2, PA, and NP expression plasmids (25 ng each), pHW-NSLuc (100 ng), and pRL-CMV (25 ng). An empty control vector or an expression vector for one of the Mx1 mutants was cotransfected (125 ng of pCAXL or pCAXL-SMx1, -SMx1V516A, -SMx1E540G, -SMx1V516AE540G, -Mx1, or -A2G Mx1dL4). After 48 h, the relative luciferase activity in the lysates was determined. Bars represent the averages from triplicate transfections, and the error bars depict the standard errors. This graph is a representative of the graphs from at least two independent experiments. (C) HEK293T cells were transfected in triplicate with 100 ng of pCAXL-SMx1, -SMx1E540G, -Mx1, or -A2G Mx1dL4. After 30 h, the cells


compared the antiviral activity of the isolated SMx1 protein in a PR8 minireplicon or infection assay, we found that its antiviral activity was actually lower than that of M. musculus A2G Mx1. The sequences of the SMx1 and A2G Mx1 proteins differ at 25 positions, which are scattered over the entire protein. Approximately half (n ⫽ 13) of these substitutions are present in the GTPase domain. As GTPase activity is important for antiviral activity (23, 39), we reasoned that one or more of these substitutions could be responsible for the reduced antiviral activity of SMx1. However, we did not detect a difference in GTPase activity between A2G Mx1 and SMx1. This suggests that the reduced antiviral activity of SMx1 is not the result of a reduced GTPase activity. The GTPase activity is a combination of the intrinsic GTPase activity of the GTPase domain and the stimulated GTPase activity caused by the nucleotide-dependent dimerization of the GTPase domains at the G interface (40). Recently, it has been described that mutations in this G interface can also influence antiviral activity (40). For example, the natural human MxA variants G255E (G221 in mouse Mx1) and V268M (V234 in mouse Mx1) show

were infected with maPR8 at an MOI of 0.5. After 16 h, the cells were collected, fixed, permeabilized, and stained for Mx1 (anti-Mx1) and NP (anti-RNP). The number of infected (RNP-positive) cells in the Mx1-positive population was determined on an LSR-II flow cytometer, and the data were analyzed with FACSDiva and FlowJo software. The graph presents the data from three independent experiments, each of which was performed in triplicate. Infected cell proportions were analyzed by logistic regression (logit was used as the link function). Significance levels of pairwise comparisons were assessed by a Fisher’s protected least significant difference test. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001. A Western blot showing detection of Mx1 expression in lysates of transfected cells from one of three experiments is shown.

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FIG 8 SMx1 has a lower antiviral activity against PR8 and maSwO than A2G Mx1. (A and C) HEK293T cells were transfected in triplicate with 80 ng of pHW192-PB1, pHW191-PB2, pHW193-PA, or pHW195-NP (A) or pHWS-maSwO-PB1, pHWS-maSwO-PB2, pHWS-maSwO-PA, or pHWS-maSwO-NP (C). In addition, pHW-NSLuc (80 ng), pRL-CMV (25 ng), and an empty vector control or an increasing concentration of the expression vector for one of the Mx1 mutants was cotransfected (5 ng, 10 ng, 20 ng, or 40 ng of pCAXL-SMx1, -SMx1E540G, -Mx1, -Mx1G540E, or -A2G Mx1dL4). After 24 h, the relative luciferase activity in the lysates was determined. Bars represent the average from 5 (PR8) or 4 (maSwO) independent experiments, each of which was performed in triplicate, except for experiments with A2G Mx1dL4, for which the results are the average from two independent experiments. The error bars depict the standard errors. Analysis of firefly luciferase was performed by a hierarchical generalized linear mixed model regression. The significances of pairwise comparisons of the effects if the Mx1 protein at a fixed concentration were assessed by a Fisher’s protected least significant difference test. *, P ⬍ 0.05; **, P ⬍ 0.01. Mx1 and NP expression was determined by Western blotting, and a representative blot is shown.

reduced antiviral activity as a result of decreased GTPase activity (40, 41). Comparison of the G interface of SMx1 and A2G Mx1 revealed 5 differences (at positions 152, 227, 230, 231, and 236) which might influence antiviral activity through an altered dimerization at the G interface. However, if these mutations affect the stimulated GTPase activity, this should have been revealed in our in vitro assay that determined the total GTPase activity. Unexpectedly, the hybrid Mx1 constructs with swapped GTPase domains had poor or no anti-influenza virus activity, although these hybrids showed GTPase activity similar to that of the wild-type Mx1 proteins. This suggests that other residues in the Mx1 protein cooperate with the GTPase domain and that both Mx1 proteins have evolved separately to perform optimally with their respective GTPase domains. Although GTPase activity is essential for antiviral activity, there is no strict direct correlation between GTPase activity and antiviral activity. It was shown for MxA that mutations in the dimer interface, e.g., M527D, or in the hinge 1 region, e.g., R640E, abolish antiviral activity but actually increase GTPase activity (24, 25). In addition, mutations in the intermolecular BSE-stalk interface of MxA, e.g., Q358A or K503A, also increased GTPase activity and at the same time abolished antiviral activity (25). The findings of these experiments by Gao et al. indicate that the antiviral activities of Mx proteins require an optimal GTPase

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activity, which is influenced by many intra- and intermolecular interactions of Mx monomers and between Mx oligomers (24, 25). A recent evolution-guided comparison of 25 primate MX1 sequences revealed that the unstructured loop L4 has been particularly subjected to positive selection (26). Loop exchange and mutational studies confirmed the functional importance of loop L4 for the antiviral activity of primate MxA (26, 27). We hypothesized that loop L4 might also be important for the antiviral function of mouse Mx1, which is a more distant homolog of human MxA. In an influenza A virus minireplicon assay and in an infection experiment, deletion of loop L4 abolished the antiviral activity of Mx1. Furthermore, we demonstrated that this loop is essential for the interaction between Mx1 and influenza virus NP, indicating that this region in Mx1 is involved in viral target recognition. Interestingly, comparison of the protein sequences of SMx1 and A2G Mx1 revealed two differences in loop L4, at positions 516 and 540. As we showed that this loop was crucial for antiviral activity, these differences represent interesting candidates that could help explain the difference in antiviral activity between the two Mx1 proteins against PR8 virus. Indeed, we found that changing glutamic acid at position 540 in SMx1 to a glycine residue increased its antiviral activity against PR8 and

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maSwO. Vice versa, the change of this position in A2G Mx1 to its counterpart in SMx1 reduced antiviral activity, confirming the importance of position 540 for the anti-influenza virus activity of Mx1. Loop L4 mediates antiviral activity in multiple ways, as it is required for NP binding and for oligomerization (24). Therefore, position 540 in Mx1 could modulate the antiviral activity against influenza virus through either one of these functions. Nevertheless, SMx1E540G still had reduced anti-influenza A virus activity compared to that of A2G Mx1, indicating that another position(s) in Mx1 also controls antiviral activity. The difference in antiviral protection against PR8 infection between SMx1 and A2G Mx1 seems at odds with the report by Vanlaere et al. (32), who showed that congenic mice carrying SMx1 were even slightly better protected than A2G mice carrying Mx1. One of their hypotheses for this increased protection was a slightly higher level of SMx1 expression than A2G Mx1 expression. Differences in Mx1 expression levels could be caused by differences in the interferon responsiveness of their promoters. It is also possible that one or more other IFN-induced proteins are involved in the in vivo protection. Another possibility for the difference between the in vivo and in vitro protection provided by SMx1 is the involvement of Mx2. Feral mouse strains such as M. spretus have a functional Mx2, whereas this protein is absent in M. musculus A2G mice (8, 9). Mouse Mx2 has no anti-influenza virus activity on its own (42, 43), but it is possible that Mx2 increases the antiviral activity of Mx1 against influenza viruses, e.g., by forming hetero-oligomers. Therefore, we also isolated the Mx2 cDNA of congenic BL6.spretus-Mx1 mice but noticed that the genomes of these mice did not encode a functional Mx2 protein due to a nonsense mutation at position 422 (unpublished results). This excludes the possibility of a role of SMx2 in the in vivo protection of BL6.spretus-Mx1 mice against influenza A viruses. To conclude, our results show that loop L4 is crucial for the antiviral activity of mouse Mx1 against influenza A viruses and that deletion of this loop is detrimental for NP recognition. Furthermore, in comparison to the antiviral activity of A2G Mx1, SMx1 has a reduced antiviral activity against different influenza viruses, which is partially attributed to a difference at position 540 in loop L4 and is not caused by a reduction in GTPase activity. ACKNOWLEDGMENTS We thank Anouk Smet for excellent technical support and Gert Van Isterdael for flow cytometry support. We thank Gnomixx for statistical analysis and Amin Bredan for critically reading the manuscript. We thank St. Jude Children’s Research Hospital for providing us with the A/Puerto Rico/8/34-based eight-plasmid system for generating recombinant influenza A viruses and the Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH, for the anti-NP and anti-RNP antibodies. We thank Suzan Carman (University of Guelph, Guelph, Ontario, Canada) for the A/Swine/Ontario/42729A/01 strain. The BL6.spretusMx1 mice were a kind gift from Xavier Montagutelli (Institut Pasteur, Paris, France). This work was supported by FWO Research Project G052412N and Ghent University special research grant BOF12/GOA/014. J.S. and D.D.V. are supported by the Agentschap voor Innovatie door Wetenschap en Techniek (IWT). C.N. was supported by project STA 338/13-1 from the Deutsche Forschungsgemeinschaft (DFG). The Flow Cytometer Core Facility at IRC is supported by a Methusalem grant (BOF09/01M00709) from Ghent University.


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