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Journal of Virology, April 2008, p. 3624-3631, Vol. 82, No. 7
0022-538X/08/$08.00+0     doi:10.1128/JVI.01753-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Influenza A Virus Strains Differ in Sensitivity to the Antiviral Action of Mx-GTPase

Jan Dittmann,^1, Silke Stertz,^1,, Daniel Grimm,¹ John Steel,² Adolfo García-Sastre,² Otto Haller,¹ and Georg Kochs^1*

Department of Virology, University of Freiburg, D-79008 Freiburg, Germany,¹ Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029²

Received 10 August 2007/ Accepted 24 December 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Interferon-mediated host responses are of great importance for controlling influenza A virus infections. It is well established that the interferon-induced Mx proteins possess powerful antiviral activities toward most influenza viruses. Here we analyzed a range of influenza A virus strains for their sensitivities to murine Mx1 and human MxA proteins and found remarkable differences. Virus strains of avian origin were highly sensitive to Mx1, whereas strains of human origin showed much weaker responses. Artificial reassortments of the viral components in a minireplicon system identified the viral nucleoprotein as the main target structure of Mx1. Interestingly, the recently reconstructed 1918 H1N1 "Spanish flu" virus was much less sensitive than the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 when tested in a minireplicon system. Importantly, the human 1918 virus-based minireplicon system was almost insensitive to inhibition by human MxA, whereas the avian influenza A virus H5N1-derived system was well controlled by MxA. These findings suggest that Mx proteins provide a formidable hurdle that hinders influenza A viruses of avian origin from crossing the species barrier to humans. They further imply that the observed insensitivity of the 1918 virus-based replicon to the antiviral activity of human MxA is a hitherto unrecognized characteristic of the "Spanish flu" virus that may contribute to the high virulence of this unusual pandemic strain.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Influenza A viruses pose a major health problem worldwide: thousands of people suffer from seasonal influenza A virus epidemics each year. Moreover, the virus has the potential to cause devastating pandemics (29). It is therefore important to understand the factors that determine influenza A virus virulence. Among them, polymerase activity, receptor specificity, and the cleavability of the hemagglutinin as well as the NS1 interferon (IFN) antagonist have previously been identified as important (27). In particular, the ability to down-regulate IFN production and/or action determines the pathogenic potential of some highly virulent strains to a large extent (7, 19, 35, 42).

The IFN system is a major component in innate immunity against viruses. IFNs help to limit virus propagation by inducing an antiviral state in potential target cells and by enhancing adaptive immune responses (40). They are known to induce hundreds of cellular genes, among them the Mx gene, which codes for the Mx protein with antiviral activity against influenza A viruses (10). Mx proteins belong to the dynamin superfamily of large GTPases and are found in many species, including fish, birds, and mammals (11).

The mechanisms by which Mx proteins exert their antiviral action are still not fully understood. The mouse Mx1 protein accumulates in the cell nucleus and inhibits primary transcription of influenza A virus, which occurs in the same subcellular compartment (22, 31), indicating that the viral ribonucleoprotein complex is a likely target structure of Mx1. Interference with this early step in the viral replication cycle should result in a dramatic inhibition of virus growth. Indeed, strong inhibition of influenza A virus replication by Mx1 has been observed in tissue culture experiments (37) and in experimentally infected Mx1^+/+ mice that carry the Mx1 resistance gene (8, 9, 34, 44). IFN-regulated Mx genes are also present in humans. The human MxA protein inhibits the replication of influenza A virus and related orthomyxoviruses in cells and transgenic animals (5, 13, 31). In contrast to mouse Mx1, which resides in the cell nucleus, the cytoplasmic human MxA protein does not inhibit primary transcription of influenza A viruses but inhibits a subsequent step involved in genome amplification and secondary transcription (31). Whether primary or secondary transcription is affected depends on the subcellular localization. When MxA is moved into the nucleus by virtue of a foreign nuclear localization signal, it, like the murine Mx1 protein, blocks primary transcription (47), indicating that the Mx proteins of the two species act in a comparable way by recognizing the same or similar viral target structures.

Influenza A virus strains differ markedly in their virulence for a given host. It is conceivable that variations in Mx sensitivity could partly explain these differences. We therefore compared the sensitivities of different influenza A virus strains to inhibition by mouse Mx1 and human MxA proteins. We found that all influenza A virus strains tested were Mx1 sensitive, albeit to different degrees. In general, avian influenza viruses were found to be better inhibited than human strains. When tested in a minireplicon system, the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 was more sensitive to the inhibitory action of Mx1 than the reconstructed 1918 H1N1 "Spanish flu" virus. Moreover, the human 1918 virus-based minireplicon system was virtually insensitive to inhibition by human MxA, in stark contrast to the avian H5N1 strain-derived system. Finally, we could show that the viral nucleoprotein (NP) is an important determinant of Mx1 sensitivity and may therefore represent the viral target structure.

(This work was conducted by Jan Dittmann in partial fulfillment of the requirements for an M.D. degree from the Medical Faculty of the University of Freiburg, Freiburg, Germany.)

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cells and viruses. Vero cells stably transfected with an Mx1 expression plasmid (Vero-Mx1) or a control plasmid (Vero control), as well as the mouse 3T3 cell line expressing Mx1 (3T3-Mx1) or not expressing Mx1 (3T3 control), have been described previously (4, 31). Mouse embryonic fibroblasts (MEF) were prepared as described in reference 21. MEF Vero, 3T3, 293T, and MDCK cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum and antibiotics. For Mx1 induction, MEF were treated with 1,000 U/ml of human hybrid IFN- B/D, which is active on mouse cells (14).

A mammalian-cell-adapted variant of influenza A virus strain A/Fowl/Dobson/27 (H7N7), called FPV-B (17), was grown on Vero cells to a titer of 6 x 10⁷ PFU/ml. Influenza A virus strain A/Udorn/307/72 (H3N2) (18) was also propagated on Vero cells (4 x 10⁶ PFU/ml). Strain A/WSN/33 (H1N1) was grown on MDCK cells (6 x 10⁷ PFU/ml). Strains SC35 (H7N7) (23) (3 x 10⁸ PFU/ml; provided by Juergen Stech, University of Marburg, Marburg, Germany), A/Texas/36/91 (H1N1) (5 x 10⁷ PFU/ml) (1), A/Panama/2007/99 (H3N2) (2 x 10⁷ PFU/ml), and A/Wyoming/3/03 (H3N2) (2 x 10⁷ PFU/ml; kindly provided by Terrence M. Tumpey, Centers for Disease Control and Prevention, Atlanta, GA) were grown in 8-day-old embryonated chicken eggs. Titers were determined by plaque assays on MDCK cells.

Antibodies and plasmids. Monoclonal antibody M143 was used to detect the Mx proteins (3), and a polyclonal rabbit serum was used to detect the influenza A virus nucleoprotein by Western blotting and immunofluorescence analysis. The monoclonal antibody against β-tubulin was purchased from Sigma (Munich, Germany).

Full-length cDNAs for the open reading frames encoding proteins PA, PB1, PB2, and NP of influenza virus A/Vietnam/1203/04 (H5N1) were amplified by PCR from viral RNA expression plasmids pPol1VN1203 PB2, pPol1VN1203 PB1, pPol1VN1203 PA, and pPol1VN1203 NP (30) and were cloned into pCAGGS (28) using Not1 and Xho1 restriction sites for PB2, PB1, and NP and Not1 and Nhe1 sites for PA. Expression plasmids pCAGGS-PB1, -PB2, -PA, and -NP derived from influenza A virus strain A/WSN/33 (2), as well as pCAGGS-PB1, -PB2, -PA, and -NP derived from strain A/BM/1/18 (43) (kindly provided by Chris Basler, Mount Sinai School of Medicine, New York, NY), have been described previously. Plasmids pDZ-PB1, pDZ-PB2, pDZ-PA, and pDZ-NP, derived from strain A/Texas/36/91 (43), were a kind gift from Dimitriy Zamarin and Peter Palese, Mount Sinai School of Medicine, New York, NY. Plasmids pHW2000-PB1, pHW2000-PB2, pHW2000-PA, and pHW2000-NP of strain SC35 (6) were obtained from Juergen Stech and Hans-Dieter Klenk, University of Marburg, Marburg, Germany, and plasmids pPolI/II-PB1, -PB2(E627K), -PA, and -NP, derived from strain A/Turkey/England/91, were kindly provided by Wendy Barclay, Imperial College London, London, United Kingdom (15).

The pPOLI-Luc-RT reporter construct, encoding firefly luciferase in the negative-sense orientation flanked by the noncoding regions of segment 8 of strain A/WSN/33, has been described recently (38). The second reporter construct, pPOLI-SP-Luc-RT, coding for firefly luciferase in the negative-sense orientation flanked by modified noncoding regions of segment 4 of strain A/WSN/33, as described by Neumann and Hobom for pHL1104 (26), was constructed by a PCR-mediated approach using plasmid pGL3-FF-Luc (Promega, Madison, WI) as a template and primers 5'-GACACGTCTCGTATTAGTAGAAACAAGGGTGTTTTTTCTTACACGGCGATCTTTCCGCC-3' and 5'-GACACGTCTCCGGGAGTAGAAACAGGGGAAAATAAAAACAACCATGGAAGACGCCAAAAACATAAAG-3'. The resulting PCR product and the pHH21 vector were digested with the restriction enzyme Esp3I (Fermentas, St. Leon-Rot, Germany), and subsequently the insert was ligated into the vector. Expression plasmids pcDNA3-Mx1 and pcDNA3-MxA and the plasmids encoding the inactive mutants, pcDNA3-Mx1(K49A) and pcDNA3-MxA(T103A), have been described previously (38, 39).

Tissue culture infection experiments. Cells were incubated with the appropriate dilution of virus stock in phosphate-buffered saline with 0.3% bovine serum albumin for 1 h. Subsequently, the inoculum was washed off, and cells were incubated with DMEM containing 10% fetal calf serum. At the time points indicated, cells were incubated with lysis buffer (50 mM Tris [pH 7.5], 250 mM NaCl, 20% glycerol, 0.5% NP-40, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 2 U/ml benzonase, protease inhibitor [Complete Mini; Roche Diagnostics, Mannheim, Germany]) for 10 min on ice. The lysates were centrifuged for 1 min at 13,000 rpm, and the supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.

For immunofluorescence analysis, the cells were seeded onto glass coverslips and infected with 2 PFU per cell. At 8 h postinfection (p.i.), the cells were fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were then stained for Mx1 and viral nucleoprotein by using specific antibodies and fluorophore (Cy2, Cy3)-conjugated secondary donkey antibodies (Dianova, Hamburg, Germany). The cells were analyzed with a Leica TCS^SP2 confocal laser scanning microscope.

For plaque assays, cells were incubated with serial dilutions of virus in phosphate-buffered saline containing 0.3% bovine serum albumin for 90 min. After removal of the inoculum, cells were overlaid with DMEM containing 0.6% agar (Oxoid Ltd., Basingstoke, Hampshire, England) and trypsin (1 µg/ml). Two to 3 days later, cells were fixed with 3.7% formaldehyde and stained with 1% crystal violet in 20% ethanol. Virus titers are expressed as PFU.

Influenza A virus minireplicon system. Transfection assays were carried out with human embryonic kidney cells (HEK 293T cells) seeded into 6-well plates. Cells were transfected using Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The indicated amounts of the three plasmids encoding the subunits of the viral RNA polymerase and the plasmid coding for the NP were cotransfected with 0.05 µg of plasmid pPOLI-Luc-RT or 0.5 µg of pPOLI-SP-Luc-RT carrying the firefly luciferase reporter gene. To provide a measure of transfection efficiency, 0.1 µg of the Renilla luciferase-encoding plasmid pRL-SV40-Rluc (Promega, Madison, WI) was cotransfected. To analyze the antiviral potential of Mx, increasing amounts of the Mx-encoding plasmids were cotransfected. As a positive control, plasmids encoding the antivirally inactive Mx1(K49A) and MxA(T103A) mutants were used. The negative control lacked the plasmid encoding NP. Equal amounts of DNA in the transfection mixtures were achieved by adding an empty pcDNA3 vector. Cells were harvested and lysed 24 h posttransfection. Firefly and Renilla luciferase activities were determined using the Dual Luciferase assay (Promega, Madison, WI) according to the manufacturer's instructions. Luminescence intensities were measured with a luminometer for 10 s each.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Influenza A virus strains differ in their sensitivity to Mx1. Initially, we compared influenza A virus strain FPV-B, a mammalian cell culture-adapted variant of the avian isolate A/Fowl/Dobson/27 (17), and the human isolate A/Udorn/72 with regard to their sensitivities to the antiviral effect of Mx1. For this purpose, we performed plaque assays on Vero cells constitutively expressing the mouse Mx1 gene. FPV-B was completely inhibited in Vero-Mx1 cells compared to Vero control cells (Fig. 1, upper panel), as expected from previous studies (37). However, much to our surprise, isolate A/Udorn/72 was able to form plaques on Mx1-expressing cells (Fig. 1, lower panel), although the number and size of plaques were lower than those in control cells (Fig. 1, lower panel).

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FIG. 1. Effect of Mx1 on viral plaque formation. Vero-control cells and Vero-Mx1 cells were infected with 200 PFU of either influenza A virus strain FPV-B (upper wells) or A/Udorn/72 (lower wells) and were subsequently overlaid with medium containing agar. At 3 days p.i., cells were fixed and plaques were stained with crystal violet. Arrows mark plaques that have been formed on Mx1-expressing cells.

We then extended our studies to a wider range of virus strains. Because viral replication does not necessarily lead to plaque formation and because most influenza A virus strains do not form plaques on Vero and mouse 3T3 cell lines, we determined the level of accumulation of the viral NP as a measure of virus growth in constitutively Mx1-expressing 3T3 cells by using Western blot analysis. Comparison of Mx1 expression levels in 3T3-Mx1 cells with those in IFN-treated primary MEF derived from congenic B6.A2G-Mx1 mice (21) revealed comparable amounts of Mx1 protein accumulation in the two cell cultures (Fig. 2B). In addition to strains FPV-B and A/Udorn/72, we used SC35 (an influenza virus strain that was derived from A/Seal/Massachussetts/1/80 by serial passages in chicken embryo cells and is pathogenic for chickens [23]) as well as strains A/WSN/33, A/Texas/36/91, A/Panama/2007/99, and A/Wyoming/3/03, which are derived from human isolates. At 6 h p.i., 3T3-Mx1 and 3T3 control cells were lysed, and Western blot analysis using a polyclonal rabbit serum against the viral NP was performed. Analysis of the viral NP revealed differences in signal intensities between the various strains (Fig. 2A). The intensities of the NP signals were strongly reduced in Mx1-expressing cells from those in control cells for the avian strains FPV-B and SC35 and for A/WSN/33, whereas only slight reductions in intensity were observed for the four human strains A/Texas/36/91, A/Udorn/72, A/Panama/2007/99, and A/Wyoming/3/03 (Fig. 2A). In order to exclude the possibility that the expression levels or the nuclear localization of the Mx1 protein was affected by infection with these less-sensitive viruses, we determined the levels of Mx1 by Western blotting and the subcellular localization of Mx1 by immunofluorescence analysis. Figure 2B shows that infection of 3T3-Mx1 cells by strain A/WSN/33, A/Texas/36/91, or SC35 did not alter Mx1 protein levels in the cells. Parallel detection of β-tubulin confirmed the loading of equal amounts of cell lysates. In addition, the nuclear localization of Mx1 was not affected by infection with the viruses (Fig. 2C and data not shown).

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FIG. 2. Effect of Mx1 on viral protein synthesis. Swiss 3T3 control cells and 3T3 Mx1-expressing cells were infected with 2 PFU per cell of different influenza A virus strains. At 6 h p.i., cells were lysed and proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (A) The viral NP was detected by Western blotting using a polyclonal rabbit serum. Detection of β-tubulin served as a loading control. (B) Expression of Mx1 in IFN--treated MEF and virus-infected 3T3-Mx1 cells. MEF were treated for 20 h with 1,000 U/ml of IFN- B/D. 3T3-Mx1 cells were infected as described for panel A. Mx1 expression was monitored using a mouse monoclonal antibody. Detection of β-tubulin reveals loading of equal amounts of cell lysates. (C) Localization of Mx1 in infected cells. 3T3-Mx1 cells were either infected with 2 PFU per cell of A/Texas/36/91 or mock treated. At 8 h p.i., cells were fixed, permeabilized, and stained for Mx1 and viral NP using specific antibodies and Cy2 or Cy3 fluorophore-conjugated secondary antibodies, respectively.

Inhibition of influenza A virus minireplicon systems derived from different strains by Mx1. Previous publications have shown that Mx1 affects primary transcription of influenza A virus (22, 31). Therefore, we reasoned that the effect of Mx1 should be visible in minireplicon systems derived from influenza A viruses. It was shown previously that expression of reporter gene activity from a minigenome containing the noncoding regions of influenza A virus segments quantitatively reflects the activity of the viral polymerase complex in virus-infected as well as cDNA-transfected cells (24). To investigate differences in sensitivity to Mx1, we used minireplicon systems derived from strains A/WSN/33, A/Texas/36/91, SC35, and A/Turkey/England/91. First, we defined settings that allowed us to directly compare the viral polymerase activities of the different systems. For this purpose, we titrated the amounts of the polymerase- and NP-encoding plasmids to determine the conditions for each system that lead to 50% of the system's maximum activity (data not shown). We then used these settings to analyze the inhibitory effect of Mx1. By cotransfection of increasing amounts of an Mx1-encoding plasmid, we determined the amount of Mx1 plasmid necessary for 50% inhibition. The resulting curves are shown for the A/WSN/33 and A/Texas/36/91 systems (Fig. 3A). The largest amount of plasmid used for Mx1 (750 ng) was also used for an expression plasmid encoding the inactive mutant Mx1(K49A) (32). As expected, Mx1(K49A) did not have an inhibitory effect, and the respective luciferase activities were set to 100%. For both A/WSN/33 and A/Texas/36/91, increasing amounts of the Mx1-encoding plasmid led to decreasing luciferase activities. However, the expression of the reporter gene was much more sensitive to Mx1 in the system derived from strain A/WSN/33 than in that derived from A/Texas/36/91 (Fig. 3A).

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FIG. 3. Effect of Mx1 on viral polymerase activity. 293T cells were transfected with the following amounts of expression plasmids encoding PA, PB1, and PB2: 90 ng (A and B) or 5 ng (C) for A/WSN/33, 30 ng for A/Texas/36/91 (A and B), 5 ng for A/Turkey/England/91 (C), and 25 ng for SC35 (C). In addition, 100 ng of pRL-SV40-Rluc, 50 ng of pPOLI-Luc-RT, and 300 ng of the respective NP-encoding plasmid for strains A/WSN/33 and A/Texas/36/91 (A and B) or 500 ng of pPOLI-SP-Luc-RT and 500 ng of the respective NP-encoding plasmid for A/WSN/33, A/Turkey/England/91, and SC35 (C) were cotransfected. These amounts of expression plasmids had been determined before to lead to 50% of the maximum activity for each system. (A) To investigate the inhibitory potential of Mx1, increasing amounts of the Mx1-encoding plasmid were cotransfected. At 24 h after transfection, cells were lysed and luciferase activities were measured. Cotransfection of the inactive mutant Mx1(K49A) was used as a positive control, and activity in the presence of Mx1(K49A) was set to 100%. (B and C) Amount of Mx1 expression plasmid necessary for 50% inhibition of the different minireplicon systems, determined as described for panel A. Error bars, standard deviations calculated from three experiments.

The amount of Mx1 plasmid needed for 50% inhibition was then fine-titrated within the range determined in the first series of transfections (10 to 100 ng of Mx1-encoding plasmid for A/WSN/33 and 300 to 400 ng for A/Texas/36/91). The exact amounts were determined to be 37.5 ng for the A/WSN/33 system and 375 ng for the A/Texas/36/91 system (Fig. 3B).

Using this strategy, we also determined the amount of Mx1-encoding plasmid necessary to inhibit two avian systems derived from strains SC35 and A/Turkey/England/91, respectively. Because the assay did not work efficiently with the conventional reporter construct, pPOLI.Luc-RT, which is based on the wild-type noncoding regions of segment 8, we used pPOLI-SP-Luc-RT, a reporter construct with enhanced expression properties based on the modified noncoding regions of segment 4 of A/WSN/33, which has been described by Neumann and Hobom (26). To enable comparison of the results with those of our previous assays, we also included the A/WSN/33 expression plasmids in this setting. We titrated plasmid amounts as described above and found that 200 ng of the Mx1-encoding plasmid was necessary to reduce the activity of the system derived from A/WSN/33 to 50% relative to the activity observed when the mutant promoter construct was used (Fig. 3C). In contrast, cotransfection with only 5 or 6 ng of Mx1-encoding plasmid resulted in 50% inhibition of the polymerase activities of SC35 and A/Turkey/England/91, respectively (Fig. 3C). These data clearly demonstrate that there are large differences in sensitivity to Mx1 between the strains. The avian strains A/Turkey/England/91 and SC35 are the most Mx1 sensitive. The human strain A/Texas/36/91 is the most resistant, whereas A/WSN/33 displays an intermediate phenotype.

The viral nucleoprotein as a possible target of Mx1 action. Next, we used the minireplicon system to identify the Mx1 target protein. For this purpose, we exchanged expression plasmids between the different strains to determine whether an Mx1-sensitive minireplicon system could be converted into a more resistant system and vice versa. Unfortunately, the exchange of plasmids encoding PB1, PB2, or PA led to a strong decrease in the activities of the mixed polymerase complexes, making a comparison with the original minireplicon systems very difficult. However, exchanging the NP-encoding plasmids was compatible for the systems derived from strains A/WSN/33, A/Texas/36/91, and SC35, resulting in approximately the same luciferase activities as those measured with the original systems. We used the minireplicon systems at 50% of maximum activity and cotransfected the amount of Mx1-encoding plasmid leading to 50% inhibition. The inactive mutant Mx1(K49A) was used as a control, and activity with this mutant was set to 100%. In this context, the effect of exchanging the NP-encoding plasmid was determined. We observed approximately 50% inhibition when we used 37.5 ng of the Mx1-encoding plasmid and the NP from strain A/WSN/33 in the A/WSN/33 system, as expected (Fig. 4A). When we used the same amount of the NP-encoding plasmid of A/Texas/36/91 in the A/WSN/33 system, luciferase activity in the presence of Mx1 increased to 70% of that in the presence of Mx1(K49A). In contrast, the use of the NP of SC35 resulted in a reduction of luciferase activity to 6% by Mx1 (Fig. 4A). For the system derived from strain A/Texas/36/91, the use of the original NP resulted in an inhibition of approximately 50% by transfection of 375 ng of the Mx1 expression plasmid (Fig. 4B). Use of the NP of A/WSN/33 or SC35 strongly increased the inhibitory potential of Mx1, leading to a reduction of polymerase activity to 13% or 3%, respectively (Fig. 4B). When we used the avian system derived from SC35, approximately 50% inhibition of polymerase activity was achieved by cotransfection of 6 ng of the Mx1 expression plasmid. Exchange of the NP resulted in a partial offset of inhibition by Mx1, bringing the level of inhibition to only about 25% with the NP-encoding plasmid of A/WSN/33 or A/Texas/36/91 (Fig. 4C). Taken together, these data reveal that NP has a great impact on the sensitivity of the minireplicon system to Mx1, since it was possible to convert a sensitive minireplicon system into a more resistant system and vice versa by changing the NP-encoding plasmid. Therefore, our approach identified the influenza A virus nucleoprotein as a possible target structure of Mx1.

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FIG. 4. Role of the viral NP in Mx1 sensitivity. 293T cells were transfected with the respective amounts of plasmids for minireplicon assays leading to 50% of the maximum activity of each system. The specific amount of Mx1-encoding plasmid causing 50% inhibition of luciferase activity or the same amount of a plasmid encoding the antivirally inactive mutant Mx1(K49A) was cotransfected: 37.5 ng for A/WSN/33 (A), 375 ng for A/Texas/36/91 (B), and 6 ng for SC35 (C). For each system, the NP-encoding plasmids of the three virus strains were cotransfected with the respective polymerase-encoding plasmids. Luciferase activity was determined 24 h after transfection. The values for the inactive mutant were set to 100%, and the values for Mx1 are expressed as percentages. Error bars, standard deviations calculated from three experiments. Abbreviations: wt, wild-type Mx1; K49A, mutant Mx1(K49A); WSN, A/WSN/33; Tx, A/Texas/36/91.

Antiviral potential of mouse Mx1 and human MxA against highly virulent 2004 H5N1 and 1918 H1N1 strains. So far, our results have shown that although there were differences between the influenza A virus strains, all were sensitive to the antiviral action of Mx1. We therefore considered whether highly virulent strains, such as the pandemic virus of 1918, A/BM/1/18 (43), or a recent H5N1 isolate from a human patient with a fatal case, A/Vietnam/1203/04 (25), are also sensitive to Mx1 action. To answer this question, we transfected 293T cells with the appropriate expression plasmids for minireplicon assays. We titrated the plasmid amounts to achieve 50% of maximum activity: 25 ng of each polymerase-encoding plasmid of A/BM/1/18 or 150 ng for A/Vietnam/1203/04. Using these conditions, we cotransfected increasing amounts of the Mx1-encoding plasmid or a plasmid encoding the inactive mutant Mx1(K49A) and determined the amount of plasmid needed for 50% inhibition (Fig. 5A). Exact titration of the amount of Mx1 expression plasmid required for 50% inhibition revealed it to be 125 ng for the system derived from A/BM/1/18 and 25 ng for the A/Vietnam/1203/04-derived system (Fig. 5B). This suggests that the sensitivities of the highly pathogenic viruses follow the same pattern as those of their moderately pathogenic counterparts.

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FIG. 5. Inhibition of the polymerase activities of the highly virulent strains A/BM/1/18 and A/Vietnam/1203/04 by mouse Mx1 and human MxA. 293T cells were transfected with expression plasmids coding for the polymerase subunits (25 ng of each plasmid for A/BM/1/18 or 150 ng for A/Vietnam/1203/04), 300 ng of the plasmid encoding the NP of A/BM/1/18 or 500 ng of the NP-encoding plasmid for A/Vietnam/1203/04, 50 ng of pPOLI-Luc-RT, and 100 ng of pRL-SV40-Rluc. (A) Increasing amounts of pcDNA3-Mx1 were cotransfected. Twenty-four hours posttransfection, cells were lysed and luciferase activities were measured. The luciferase activities obtained by cotransfection of the inactive mutant Mx1(K49A) were set to 100%. (B and C) Titration of the amount of Mx1-encoding plasmid (B) or MxA-encoding plasmid (C) needed for 50% inhibition of the polymerase activities of strains A/BM/1/18 (BM) and A/Vietnam/1203/04 (VN). Error bars, standard deviations calculated from three experiments.

Since both viruses were isolated from humans with fatal influenza A virus infections, we also tested the effect of human MxA on the minireplicon system. We used an MxA-encoding plasmid or a plasmid coding for the inactive mutant MxA(T103A) (33) and performed titration as described above. We found that MxA coexpression had a strong inhibitory effect on the avian system and that only 110 ng of the MxA-encoding plasmid was needed for 50% inhibition of the A/Vietnam/1203/04 system (Fig. 5C). However, with 1,500 ng of the MxA-encoding plasmid, no inhibitory effect on the polymerase activity of A/BM/1/18 was detectable (Fig. 5C). We also studied the sensitivity of the polymerase of A/Texas/36/91 to MxA expression and determined a similar lack of inhibition by 1,500 ng of the MxA-encoding expression plasmid (data not shown), suggesting an unexpected insensitivity of the polymerase complex of human influenza viruses to the expression of MxA.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Here we demonstrate that influenza A virus strains differ markedly in their sensitivities to the antiviral action of Mx proteins. The amounts of Mx needed to inhibit different influenza virus replicon systems differed greatly. We further show that the viral NP is a determinant of viral sensitivity to Mx. NP is a major component of the viral ribonucleoprotein complex and is essential for viral transcription and replication. Our findings indicate that NP is a target of Mx.

We found that influenza A virus strain FPV-B was highly susceptible to Mx1, confirming earlier studies (37). By screening a panel of influenza A virus strains, we could identify viruses that were able to replicate reasonably well in the presence of Mx1. Interestingly, influenza A viruses with heightened Mx resistance represented human isolates, whereas the more sensitive viruses were of avian origin. Importantly, virus sensitivities were reflected in the Mx sensitivities of minireplicon systems. Thus, the minireplicon system based on the avian H5N1 virus currently circulating in Asia (25) was sensitive to the antiviral action of Mx1. It is therefore tempting to speculate that avian influenza A viruses have only limited opportunity to adapt to the mammalian host, partly because of the mammalian Mx system. Successful human strains were presumably selected for low sensitivity to mammalian Mx proteins during the course of their adaptation. The intermediate sensitivity of strain A/WSN/33 was remarkable and is not easily explained. The virus was extensively passaged in laboratory mice that are negative for a functional Mx1 gene (36) and may have lost some Mx resistance. Further studies analyzing primary human and avian isolates with respect to their sensitivities to mammalian Mx proteins are under way to test this hypothesis.

Interestingly, we found that minireplicons based on the pandemic strain A/BM/1/1918 as well as minireplicons of strain A/Vietnam/1203/04 (isolated from a human patient with a fatal case) were inhibited by Mx1. These results fit very well with recent in vivo experiments showing that Mx1^+/+ mice survive infection by both viruses with doses that are lethal for genetically defective Mx^–/– mice (34, 44). Nevertheless, the polymerase activity of A/BM/1/1918 was inhibited less strongly by mouse Mx1 than that of A/Vietnam/1203/04. The difference in sensitivity between the two strains was even more pronounced with human MxA. MxA inhibited the polymerase activity of A/Vietnam/1203/04 more efficiently than those of A/BM/1/1918 and A/Texas/36/91, which were both virtually insensitive. It is therefore likely that human MxA provides an efficient block to zoonotic transmission of avian influenza A viruses, including H5N1 viruses that actually circulate in Southeast Asia. Successful viruses that adapt to and spread from humans to humans may have evolved a relative insensitivity to the antiviral activity of MxA.

Furthermore, there may be an inverse relationship between the capacity of viruses to antagonize IFN production and their sensitivity to Mx. It was shown by Hayman et al. that viruses that are less potent at inhibiting IFN induction are more resistant to IFN treatment than viruses that are strong suppressors of IFN (12).

The strain-specific differences in Mx sensitivity were used to analyze the viral target structure of Mx1. By exchange of the NP-encoding plasmids, we could convert a sensitive polymerase complex into a more resistant one, and vice versa. This shows that NP is an important viral determinant of Mx1 sensitivity and therefore a possible target structure of Mx proteins. This role of NP is not without precedent: Turan et al. were able to show an interaction of NP with a variant of human MxA carrying a foreign nuclear localization signal (45). Furthermore, MxA interacts with the NP of Thogoto virus, a member of the orthomyxovirus family (20, 46). In addition, the PB2 subunit of the influenza virus polymerase complex has been proposed as a target structure of mouse Mx1 (16, 41). It is therefore conceivable that the viral ribonucleoprotein complex, consisting of genomic RNA, NP, and viral polymerase, is the target structure of Mx proteins.

In summary, our results show that there are large differences in the sensitivities of influenza A virus strains to inhibition by the IFN-induced Mx proteins. The avian influenza virus strains are very sensitive, whereas strains of human origin are more resistant. Most remarkably, the polymerase complex of the 1918 pandemic influenza A virus was not affected by the highest MxA expression levels used in our test system, suggesting a remarkable adaptation to human cells. The viral NP proved to be a determinant of Mx sensitivity, supporting the hypothesis that viral ribonucleoprotein complexes are target structures of Mx proteins.

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ko1579/1-8) and the German-Israeli Research Foundation (GIF-841/04) to G.K. and O.H. and by grants from the National Institutes of Health (R01AI46954 and P01AI58113) to A.G.-S.

We thank Peter Staeheli and Friedemann Weber for discussions and critical comments on the manuscript. We are grateful to Wendy Barclay for A/Turkey/England/91 expression plasmids, to Jürgen Stech and Hans-Dieter Klenk for virus isolates and SC35 expression plasmids, to Christopher Basler for 1918 expression plasmids, to Terrence M. Tumpey for influenza virus strains A/Panama/2007/99 and A/Wyoming/3/03, and to Dimitriy Zamarin and Peter Palese for virus isolates and A/Texas/36/91 expression plasmids.

 
* Corresponding author. Mailing address: Department of Virology, University of Freiburg, Hermann-Herder-Strasse 11, 79107 Freiburg, Germany. Phone: 49-761-2036623. Fax: 49-761-2036562. E-mail: georg.kochs{at}uniklinik-freiburg.de

Published ahead of print on 16 January 2008.

J.D. and S.S. contributed equally to this work.

Present address: Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Virology, April 2008, p. 3624-3631, Vol. 82, No. 7
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