Influenza A virus strains differ in sensitivity to the antiviral action of the Mx-GTPase

31 Pages·2008·1.51 MB·English

JVI Accepts, published online ahead of print on 16 January 2008
J. Virol. doi:10.1128/JVI.01753-07
Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Influenza A virus strains differ in sensitivity to the antiviral action of
2 the Mx-GTPase
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4 Jan Dittmann1#, Silke Stertz1,3#, Daniel Grimm1, John Steel2, Adolfo García-
5 Sastre2, Otto Haller1, and Georg Kochs1* E
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7 1 Department of Virology, University of Freiburg, D-79008 Freiburg, Germany;
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8 2 Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, w
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9 USA. E ad
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10 3 present address: Department of Microbiology, Mount Sinai School of Medicine, New fr
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11 York, NY 10029, USA. h
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12 # These authors contributed equally. /jv
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14 Running title: Influenza A virus sensitivity to antiviral Mx proteins g
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15 * Corresponding author: Georg Kochs A
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16 Department of Virology, University of Freiburg il 5
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17 79107 Freiburg, Germany, Hermann-Herder-Strasse 11 1
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18 Phone: [phone]
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19 Fax: [phone] st
20 e-mail: [email protected]
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22 Word count abstract: 212 words
23 Word count text: 4231 words
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1 Influenza A virus strains differ in sensitivity to the antiviral action of
2 the Mx-GTPase
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4 Jan Dittmann, Dept. Virology, Freiburg,
5 e-mail: [email protected] E
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7 Silke Stertz, Dept. Virology, Freiburg,
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8 e-mail: [email protected] w
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10 Daniel Grimm, Dept. Virology, Freiburg, fr
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11 e-mail: [email protected] h
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13 JoAhn Steel, Department of Microbiology, Mount Sinai School of Medicine, New York, m
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14 e-mail: [email protected] g
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15 A
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16 Adolfo García-Sastre, Department of Microbiology, MSSM, New York, il 5
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17 e-mail: [email protected] 1
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19 Otto Haller, Dept. Virology, Freiburg, st
20 e-mail: [email protected]
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22 Georg Kochs, Dept. Virology, Freiburg,
23 e-mail: [email protected]
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1 Abstract:
2 Interferon-mediated host responses are of great importance for controlling influenza
3 A virus infections. It is well established that the interferon-induced Mx proteins
4 possess powerful antiviral activities towards most influenza viruses. Here, we D
5 analyzed a range of influenza A virus strains for their sensitivities to murine Mx1 and
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6 human MxA proteins and found remarkable differences. Virus strains of avian origin
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7 were highly sensitive to Mx1, whereas strains of human origin showed much weaker
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8 responses. Artificial reassortments of the viral compPonents in a minireplicon system o
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9 identified the viral nucleoprotein as the main target structure of Mx1. Interestingly, the lo
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10 recently reconstructed 1918 H1N1 “Spanish Flu” virus was much less sensitive than d

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11 the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 when tested in a

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12 minireplicon syCstem. Importantly, the human 1918 virus-based minireplicon system :
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13 was almost insensitive to inhibition by human MxA, whereas the avian H5N1-derived a
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14 system was well controlled by MxA. These findings suggest that Mx proteins provide or
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15 a formidable hurdle that hinders influenza A viruses of avian origin to cross the n

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16 species barrier to humans. They further imply that the observed insensitivity of the ril
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17 1918 virus-based replicon towards the antiviral activity of human MxA is a hitherto 0
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18 unrecognized characteristic of the “Spanish Flu” virus which may contribute to the by

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19 high virulence of this unusual pandemic strain. e
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1 Introduction:
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3 Influenza A viruses pose a major health problem worldwide as thousands of people
4 suffer from seasonal influenza A virus epidemics each year. Moreover, the virus has D
5 the potential to cause devastating pandemics (29). It is therefore important to
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6 understand the factors which determine influenza A virus virulence. Among them,
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7 polymerase activity, receptor specificity, and cleavability of the hemagglutinin as well
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8 as the NS1 interferon (IFN) antagonist have previoPusly been identified as important o
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9 (27). In particular, the ability to down-regulate IFN production and/or action lo
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10 determines the pathogenic potential of some highly virulent strains to a large extent d

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11 (7, 19, 35, 42).

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12 The IFN systeCm is a major component in innate immunity against viruses. IFNs help :
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13 to limit virus propagation by inducing an antiviral state in potential target cells and by a
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14 enhancing adaptive immune responses (40). They are known to induce hundreds of or
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15 cellular genes, among them the Mx gene which codes for the Mx protein with antiviral n

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16 activity against influenza A viruses (10). Mx proteins belong to the dynamin ril
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17 superfamily of large GTPases and are found in many species, including fish, birds 0
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18 and mammals (11). by

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19 The mechanisms by which Mx proteins exert their antiviral action are still not fully e
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20 understood. The mouse Mx1 protein accumulates in the cell nucleus and inhibits
21 primary transcription of influenza A virus which occurs in the same subcellular
22 compartment (22, 31), indicating that the viral ribonucleoprotein complex is a likely
23 target structure of Mx1. Interference with this early step in the viral replication cycle
24 should result in a dramatic inhibition of virus growth. Indeed, a strong inhibition of
25 influenza A virus replication by Mx1 has been observed in tissue culture experiments
26 (37) and in experimentally infected Mx1+/+ mice which carry the Mx1 resistance gene
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1 (8, 9, 34, 44). IFN-regulated Mx genes are also present in humans. The human MxA
2 protein inhibits the replication of influenza A virus and related orthomyxoviruses in
3 cells and transgenic animals (5, 13, 31). In contrast to mouse Mx1 residing in the cell
4 nucleus, the cytoplasmic human MxA protein does not inhibit primary transcription of D
5 influenza A viruses but rather a subsequent step involved in genome amplification
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6 and secondary transcription (31). Whether primary or secondary transcription is
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7 affected depends on the subcellular localization. When MxA is moved into the
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8 nucleus by virtue of a foreign nuclear localizaPtion signal, it blocks primary o
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9 transcription like the murine Mx1 protein (47), indicating that Mx proteins of both lo
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10 species apparently act in a comparable way by recognizing the same or similar viral d

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11 target structures.

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12 Influenza A viCrus strains markedly differ in their virulence for a given host. It is :
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13 conceivable that variations in Mx sensitivity could partly explain these differences. a
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14 We therefore compared the sensitivities of different influenza A virus strains to or
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15 inhibition by mouse Mx1 and human MxA proteins. We found that all influenza A n

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16 virus strains tested were Mx1 sensitive, albeit to varying degrees. In general, avian ril
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17 influenza viruses were found to be better inhibited than human strains. When tested 0
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18 in a minireplicon system, the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 by

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19 was more sensitive to the inhibitory action of Mx1 than the reconstructed 1918 H1N1 e
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20 “Spanish Flu” virus. Moreover, the human 1918 virus-based minireplicon system was
21 virtually insensitive to inhibition by human MxA, in stark contrast to the avian H5N1-
22 derived system. Finally, we could show that the viral nucleoprotein is an important
23 determinant of Mx1 sensitivity and may therefore represent the viral target structure.
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1 Material and Methods:
2
3 Cells and Viruses
4 Vero cells stably transfected with an Mx1-expression plasmid (Vero-Mx1) or a control D
5 plasmid (Vero-control), as well as the mouse 3T3 cell line expressing Mx1 (3T3-Mx1)
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6 and the control cells (3T3-control) were described previously (4, 31). Mouse embryo
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7 fibroblasts (MEF) were prepared as described in (21). MEF, Vero, 3T3, 293T and
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8 MDCK cells were maintained in DMEM supplementPed with 10% fetal calf serum and o
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9 antibiotics. For Mx1 induction, MEF were treated with 1000 units/ml of human hybrid lo
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10 IFN-α B/D which is active on mouse cells (14). d

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11 A mammalian cell-adapted variant of influenza A virus strain A/fowl/Dobson/27
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12 (H7N7), calledC FPV-B (17), was grown on Vero cells to a titer of 6x107 pfu/ml. :/
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13 Influenza A virus strain A/Udorn/307/72 (H3N2) (18) was also propagated on Vero as
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14 cells (4x106 pfu/ml). Strain A/WSN/33 (H1N1) was grown on MDCK cells (6x107 r
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15 pfu/ml). The strains SC35 (H7N7) (23) (provided by Dr. Juergen Stech, University of n

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16 Marburg, Germany; 3x108 pfu/ml), A/Texas/36/91 (H1N1) (5x107 pfu/ml) (1), il
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17 A/Panama/2007/99 (H3N2) (2x107 pfu/ml) and A/Wyoming/3/03 (H3N2) (2x107 0
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18 pfu/ml) (kindly provided by Dr. Terrence M. Tumpey, Centre of disease control, y

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19 Atlanta) were grown in 8 days old embryonated chicken eggs. Titers were es
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20 determined by plaque assay on MDCK cells.
21 Antibodies and Plasmids
22 A monoclonal antibody (mAb) M143 was used to detect the Mx proteins (3) and a
23 polyclonal rabbit serum to detect the influenza A virus nucleoprotein by Western blot
24 and immunofluorescence analysis. The monoclonal β-tubulin-antibody was
25 purchased from Sigma (Munich, Germany).
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1 Full length cDNAs for the open reading frames encoding proteins PA, PB1, PB2 and
2 NP of influenza A/Vietnam/1203/04 (H5N1) were amplified by PCR from vRNA
3 expression plasmids pPol1VN1203 PB2, pPol1VN1203 PB1, pPol1VN1203 PA, and
4 pPol1VN1203 NP (30) and cloned into pCAGGS (28) using Not1 and Xho1 restriction D
5 sites for PB2, PB1 and NP and Not1 and Nhe1 sites for PA. The expression plasmids
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6 pCAGGS-PB1, -PB2, -PA and -NP derived from influenza A virus strain A/WSN/33
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7 (2) as well as pCAGGS-PB1, -PB2, -PA, -NP derived from strain A/BM/1/18 (43)
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8 (kindly provided by Dr. Chris Basler, Mount Sinai PSchool of Medicine, New York, o
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9 U.S.) have been previously described. The plasmids pDZ-PB1, pDZ-PB2, pDZ-PA lo
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10 and pDZ-NP derived from strain A/Texas/36/91 (43) were a kind gift of Drs. Zamarin d

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11 and Palese, Mount Sinai School of Medicine, New York. The plasmids pHW2000-

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12 PB1, pHW200C0-PB2, pHW2000-PA and pHW2000-NP of strain SC35 (6) were :
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13 obtained from Drs. Juergen Stech and Hans-Dieter Klenk, University of Marburg, a
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14 Germany, and the plasmids pPolI/II-PB1, -PB2(E627K), -PA and –NP derived from or
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15 strain A/Turkey/England/91 were kindly provided by Dr. Wendy Barclay, Imperial n

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16 College London, U.K. (15). ril
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17 The reporter construct pPOLI-Luc-RT encoding Firefly luciferase in negative sense 0
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18 orientation flanked by the noncoding regions of segment 8 of strain A/WSN/33 has by

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19 been described recently (38). The second reporter construct pPOLI-SP-Luc-RT e
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20 coding for Firefly luciferase in negative sense orientation flanked by modified
21 noncoding regions of segment 4 of strain A/WSN/33 as described by Neumann et al.
22 for pHL1104 (26) was constructed by a PCR-mediated approach using the plasmid
23 pGL3-FF-Luc (Promega, Madison, WI 53711, USA) as template and 5’-
24 gacacgtctcgtattagtagaaacaagggtgttttttcttacacggcgatctttccgcc-3’ and 5’-
25 gacacgtctccgggagtagaaacaggggaaaataaaaacaaccatggaagacgccaaaaacataaag-3’ as
26 primers. The resulting PCR product and the vector pHH21 were digested with the
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1 restriction enzyme Esp3I (Fermentas, St. Leon-Rot, Germany) and subsequently, the
2 insert was ligated into the vector. The expression plasmids pcDNA3-Mx1, pcDNA3-
3 MxA and the plasmids encoding the inactive mutants, pcDNA3-Mx1(K49A) and
4 pcDNA3-MxA(T103A) have been described previously (38, 39). D
5 Tissue culture infection experiments
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6 Cells were incubated with the appropriate dilution of virus stock in PBS with 0.3%
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7 bovine serum albumin for 1h. Subsequently, the inoculum was washed off and cells
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8 were incubated with DMEM containing 10% fetalP calf serum. At the time points o
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9 indicated cells were incubated with lysis buffer (50 mM Tris (pH 7,5), 250 mM NaCl, lo
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10 20% Glycerol, 0.5% NP-40, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 U/ml d

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11 benzonase, protease inhibitor (Complete Mini, Roche Diagnostics, Mannheim,

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12 Germany) for 1C0 min on ice. The lysates were centrifuged for 1 min at 13000 rpm and :
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13 the supernatants were subjected to SDS-PAGE and Western blot analysis. a
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14 For immunofluorescence analysis, the cells were seeded onto glass coverslips and or
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15 infected with 2 pfu per cell. At eight hours p.i. the cells were fixed with 3% n

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16 paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were then ril
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17 stained for Mx1 and viral nucleoprotein using specific antibodies and fluorophore 0
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18 (Cy2, Cy3)-conjugated secondary donkey antibodies (Dianova, Hamburg, Germany). by

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19 The cells were analyzed with a Leica TCSSP2 confocal laser scanning microscope. es
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20 For plaque assay, cells were incubated with serial dilutions of virus in PBS containing
21 0.3% BSA for 90 min. After removal of the inoculum, cells were overlaid with DMEM
22 medium containing 0.6% agar (Oxoid LTD., Basingstoke, Hampshire, England) and
23 trypsin (1 µg/ml). 2-3 days later, cells were fixed with 3.7% formaldehyde and stained
24 with 1% crystal violet in 20% ethanol. Virus titers are expressed as plaque forming
25 units (pfu).
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1 Influenza A virus minireplicon system
2 Transfection assays were carried out with human embryonic kidney cells (HEK 293T)
3 seeded into 6-well plates. Cells were transfected using Lipofectamine transfection
4 reagent (Invitrogen, Carlsbad, California 92008, U.S.) according to the D
5 manufacturer's protocol. The amounts indicated of the three plasmids encoding the
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6 subunits of the viral RNA polymerase and the plasmid coding for the nucleoprotein
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7 NP were cotransfected with 0.05 µg of the plasmid pPOLI-Luc-RT or 0.5 µg of
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8 pPOLI-SP-Luc-RT encoding the Firefly luciferaseP reporter gene. To provide a o
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9 measure of transfection efficiency 0.1 µg of the Renilla luciferase encoding plasmid lo
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10 pRL-SV40-Rluc (Promega, Madison, WI 53711, USA) were cotransfected. To d

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11 analyze the antiviral potential of Mx increasing amounts of the Mx-encoding plasmids

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12 were cotransfCected. As a positive control the plasmids encoding the antivirally :
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13 inactive mutants Mx1(K49A) and MxA(T103A) were used. The negative control was a
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14 lacking the plasmid encoding NP. Equal amounts of DNA in the transfection mixtures or
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15 were achieved by adding empty pcDNA3-vector. Cells were harvested and lysed 24 n

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16 h post transfection. Firefly and Renilla luciferase activity were determined using the ril
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17 Dual Luciferase Assay (Promega, Madison, WI 53711, USA) according to the 0
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18 manufacturer's instructions. Luminescence intensities were measured with a by

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19 luminometer for 10 s each. e
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1 Results:
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3 Influenza A virus strains differ in their sensitivity to Mx1. Initially, we compared
4 influenza A virus strain FPV-B, a mammalian cell culture-adapted variant of the avian D
5 isolate A/fowl/Dobson/27 (17), and the human isolate A/Udorn/72 with regard to their
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6 sensitivities towards the antiviral effect of Mx1. To this aim, we performed plaque
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7 assays on Vero cells constitutively expressing the mouse Mx1 gene. FPV-B was
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8 completely inhibited in Vero-Mx1 cells compared tPo the Vero-control cells (Fig. 1, o
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9 upper panel) as expected from previous studies (37). However and much to our lo
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10 surprise, isolate A/Udorn/72 was able to form plaques on Mx1-expressing cells (Fig. d

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11 1, lower panel) although there was a reduction in the number and size of plaques

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12 compared to thCe control cells (Fig. 1, lower panel). :
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13 We then extended our studies to a wider range of virus strains. As viral replication a
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14 does not necessarily lead to plaque formation and since most influenza A virus or
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15 strains do not form plaques on Vero and mouse 3T3 cell lines, we determined the n

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16 accumulation of the viral nucleoprotein (NP) as a measure of virus growth in ril
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17 constitutively Mx1-expressing 3T3-cells, using Western blot analysis. A comparison 0
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18 of the Mx1 expression level in the 3T3-Mx1 cells with that in IFN-treated primary by

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19 mouse embryonic fibroblasts derived from congenic B6.A2G-Mx1 mice (21) revealed e
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20 comparable amounts of Mx1 protein accumulation in both cell cultures (Fig. 2B). In
21 addition to strains FPV-B and A/Udorn/72, we used SC35 (an influenza virus strain
22 which was derived from A/Seal/Massachussetts/1/80 by serial passages in chicken
23 embryo cells and is pathogenic for chickens (23)), as well as strains A/WSN/33,
24 A/Texas/36/91, A/Panama/2007/99, and A/Wyoming/3/03 which are derived from
25 human isolates. At 6 h p.i., 3T3-Mx1 and 3T3 control cells were lysed and a Western
26 blot analysis using a polyclonal rabbit serum against the viral nucleoprotein (NP) was
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