| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
Institute of Molecular Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany
Correspondence
Egbert Mundt
emundt{at}uga.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Present address: Poultry Diagnostic and Research Center, College of Veterinary Medicine, The University of Georgia, 953 College Station Rd, Athens, GA 30602, USA. 
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). The antiviral activity of MxA was shown first for Influenza A virus and vesicular stomatitis virus (VSV; Pavlovic et al., 1990). MxA accumulated in the cytoplasm of IFN-activated cells and showed antiviral activity against members of several virus families with negative-stranded RNA genomes (Lee & Vidal, 2002), positive-stranded RNA genomes (Chieux et al., 2001) and a virus with a DNA genome (Gordien et al., 2001). The mechanism by which human MxA protein is able to inhibit virus replication is not fully understood. It has experimentally been shown that MxA targeted the viral nucleocapsids of Thogoto virus (Kochs & Haller, 1999a, b) and La Crosse virus (Weber et al., 2000; Kochs et al., 2002; Reichelt et al., 2004) or interfered with amplification of viral RNA of Semliki Forest virus (Landis et al., 1998). In addition, in its purified form, MxA was able to inhibit transcription of VSV RNA (Schwemmle et al., 1995). Furthermore, the inhibitory effect of MxA seemed to be dependent on the cellular model used, as described for Measles virus (Schnorr et al., 1993; Schneider-Schaulies et al., 1994). This indicated that suppression of viral infection by human MxA did not occur by one single mechanism.
In order to assay whether MxA is able to influence replication of double-stranded RNA (dsRNA) viruses, two viral systems were investigated, the Infectious bursal disease virus (IBDV) and a human reovirus. IBDV is member of the family Birnaviridae (Delmas et al., 2004) and the cause of a worldwide economically important immunosuppressive disease in chicken livestock. The genome of birnaviruses consists of two segments, A and B, which are localized within a single-shelled icosahedral capsid of 60 nm diameter. Few reports have been published regarding Mx protein of poultry. For duck Mx protein, no enhanced antiviral activity was observed after influenza virus infection (Bazzigher et al., 1993). This is an interesting finding since Ko et al. (2002) described that cloned chicken Mx showed antiviral activity against influenza virus and VSV. However, the antiviral effect was dependent on cells obtained from different chicken breeds. In subsequent investigations, they showed that aa 631, located at the carboxy terminus of the protein, corresponded with the positive antiviral phenotype against VSV (Ko et al., 2004). In fish, it was shown that expression of Mx1 protein of Atlantic salmon inhibited the replication of Infectious pancreatic necrosis virus, another member of the family Birnaviridae (Larsen et al., 2004).
The second dsRNA virus used was the mammalian reovirus which belongs to genus Orthoreovirus, one of the six genera of the family Reoviridae. Mammalian reoviruses are non-enveloped viruses that replicate in the cytoplasm of infected cells and contain 10 dsRNA genome segments surrounded by two protein shells termed the outer capsid and the core of 70–80 nm in diameter (Tyler, 2001). It is known that two IFN-induced proteins contribute to the innate immune response due to reovirus-induced host cell shutoff, the dsRNA-activated protein kinase (PKR) and RNase L (Smith et al., 2005). This study shows that these cellular factors are not the sole determinants of the cellular innate antiviral activity against reovirus and it contrasts with reports showing well-documented antiviral activities of PKR and RNase L (De Benedetti et al., 1985; Li et al., 1998; Balachandran et al., 2000). Smith et al. (2005) assumed an additional cellular factor responsible for the antiviral activity against mammalian reovirus. Studies with reovirus including MxA have so far not been described.
In order to assay whether human MxA has antiviral activity against dsRNA viruses, recombinant African green monkey kidney (Vero) cells were used, which constitutively express wild-type MxA (clones VA9, VA3) or the mutant MxA(E645R) [VA(E645R)], and control cells without MxA (VN36) which contain only the cloning vector (Frese et al., 1996). Cells were incubated with Dulbecco’s minimal essential medium (DMEM) containing 10 % fetal calf serum and 2 mg G418 ml–1 (Invitrogen). Cells were seeded into 24-well plates, grown to confluence and infected at an m.o.i. of 1 with the IBDV strain D78 (Mundt, 1999). After incubation for 60 min at 37 °C supernatants were removed, cells were rinsed once with serum-free medium and 1 ml G418-containing medium was added. At 24 and 48 h supernatants were removed and stored at –70 °C. The TCID50 was measured on chicken embryonic cells (CEC) as described before (Mundt et al., 2003). As shown in Fig. 1(a), virus yield in the supernatant was reduced about 100- (VA3) to 500-fold (VA9) in comparison to cells expressing the mutated MxA(E645R) [VA(E645R)] or the control cells (VN36). These results indicated that human MxA protein had a strong antiviral effect on IBDV replication. In order to exclude that this finding was due to different amounts of MxA proteins, Western blot analysis was performed using lysates of simultaneously infected cells at 48 h post-infection (p.i.; Fig. 1b). After separation by SDS-PAGE (10 % gel), proteins were blotted on nitrocellulose membranes, blocked with 5 % skim milk solution in TBST [10 mM Tris/HCL, 150 mM NaCl (pH 8.0)] containing 0.05 % (v/v) Tween 20 and simultaneously incubated with mAb raised against VP3 of IBDV (Granzow et al., 1997), MxA (Flohr et al., 1999) and cellular 
-tubulin (Sigma). After incubation with anti-mouse horseradish peroxidase-conjugated goat antibodies (Sigma) and application of SuperSignal West Pico chemiluminescent substrate (Perbio Science), it was observed that the amount of MxA protein was very similar in lysates of the different cell lines. VP3 of IBDV was detectable only in lysates of those cells where either the mutated MxA(E645R) or no human MxA protein was expressed (VN36). That equal amounts of proteins were analysed was proven by the presence of similar amounts of -tubulin. The simultaneous incubation with three different antibodies was possible due to the different molecular masses of the proteins analysed. In order to analyse to what extent the replication was inhibited, recombinant cell lines were simultaneously infected and immunofluorescence analysis was performed (Fig. 1c) at 48 h p.i. by confocal laser scanning microscopy using an LSM 510 (Zeiss). To this end, cells were fixed with ice-cold ethanol for 10 min, rinsed with PBS and incubated with a polyclonal anti-IBDV serum obtained from a rabbit (Granzow et al., 1997) and the anti-MxA mAb, followed by incubation with anti-mouse DTAF-conjugated swine antibodies and anti-rabbit Cy3-conjugated goat antibodies (Dianova). Specific fluorescence was observed in a very low number of VA3 or VA9 cells infected with IBDV, whereas in the other cell lines used [VN36, VA(E645R)] a large number of cells showed IBDV antigen. This finding confirmed the results from Western blot analysis where VP3 was not detectable in lysates of infected VA3 and VA9 cells, probably due to the low amount of viral protein present. The observation that in principle the MxA-expressing cells are permissive for IBDV infection and that the virus was amplified in the cells, although to a lower number as shown by the growth kinetics, raised the question about the mechanism of the antiviral effect. In order to assay whether there is a protein–protein interaction as described for Thogoto virus (Kochs & Haller, 1999a), immunoprecipitation assays were performed. IBDV and MxA proteins couldn’t be co-precipitated with appropriate antibodies, whether GTP
S was present or not (data not shown). In addition, during immunofluorescence studies using IBDV protein-specific mAbs raised against VP2, VP3, VP4 (Granzow et al., 1997) and VP5 (Mundt et al., 1997) and a polyclonal anti-MxA serum from a rabbit (Ponten et al., 1997), no co-localization was observed by confocal laser scanning microscopy. No co-localization was observed by immunofluorescence using anti-VP1 antiserum (Birghan et al., 2000) and the anti-MxA mAb either. In conclusion, the mechanism by which MxA inhibited IBDV replication remains unknown.
|
). No significant differences in virus titres were detected at 48 h p.i. At later time points, viral multiplication was reduced 50-fold (VA3, VA9) at 96 h p.i. and 10- (VA3) to 100-fold (VA9) at 144 h p.i. in supernatants of infected wild-type MxA-expressing cells in comparison to cells expressing either the mutant protein [VA(E645R)] or cells lacking MxA (VN36). Western blot analysis of cellular lysates at 96 h p.i. using a rabbit polyclonal anti-reovirus serum (a generous gift of Terence Dermody, Vanderbilt University, USA) and anti-rabbit horseradish peroxidase-conjugated goat antibodies substantiated this finding, since the amount of viral proteins was reduced in cells expressing the wild-type MxA protein (Fig. 3a). In addition, these data were confirmed after analysis of infected cells by immunofluorescence (Fig. 3c), since the number of infected cells was reduced in MxA-expressing cell cultures. This showed that the presence of human MxA protein was also able to reduce replication of human orthoreovirus type 3 strain Dearing. Whether MxA could be one of the additional cellular factors assumed to be responsible for the antiviral activity against mammalian reovirus (Smith et al., 2005) needs to be further elucidated. Reovirus was a strong inducer of IFN expression (Tytell et al., 1967), but the results obtained were very likely not the result of IFN-induced genes since Vero cells contain a genetic defect for expression of IFN (Emeny & Morgan, 1979). The effect observed was probably due to the action of human MxA on reovirus replication by an unknown mechanism.
|
|
; Frese et al., 1995). Therefore, the GTPase activity which is retained in the mutant cannot be the sole determinant for the inhibitory effect.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Bazzigher, L., Schwarz, A. & Staeheli, P. (1993). No enhanced influenza virus resistance of murine and avian cells expressing cloned duck Mx protein. Virology 195, 100–112.[CrossRef][Medline]
Birghan, C., Mundt, E. & Gorbalenya, A. E. (2000). A non-canonical Lon proteinase deficient of the ATPase domain employs the Ser-Lys catalytic dyad to impose broad control over the life cycle of a double-stranded RNA virus. EMBO J 19, 114–123.[CrossRef][Medline]
Chieux, V., Chehadeh, W., Harvey, J., Haller, O., Wattre, P. & Hober, D. (2001). Inhibition of coxsackievirus B4 replication in stably transfected cells expressing human MxA protein. Virology 283, 84–92.[CrossRef][Medline]
De Benedetti, A., Williams, G. J., Comeau, L. & Baglioni, C. (1985). Inhibition of viral mRNA translation in interferon-treated L cells infected with reovirus. J Virol 55, 588–593.
Delmas, B., Kibenge, F. S. B., Leong, J. C., Mundt, E., Vakharia, V. N. & Wu, J. L. (2004). Birnaviridae. In Virus Taxonomy, Eighth Report of the International Committee on Taxonomy of Viruses, pp. 561–569. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier/Academic Press.
Emeny, J. M. & Morgan, M. J. (1979). Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production. J Gen Virol 43, 247–252.
Flohr, F., Schneider-Schaulies, S., Haller, O. & Kochs, G. (1999). The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures. FEBS Lett 463, 24–28.[CrossRef][Medline]
Frese, M., Kochs, G., Meier-Dieter, U., Sielber, J. & Haller, O. (1995). Human MxA protein inhibits tick-borne Thogoto virus but not Dhori virus. J Virol 69, 3904–3909.[Abstract]
Frese, M., Kochs, G., Feldmann, H., Hertkorn, C. & Haller, O. (1996). Inhibition of bunyaviruses, phleboviruses, and hantaviruses by human MxA protein. J Virol 70, 915–923.[Abstract]
Gordien, E., Rosmorduc, O., Peltekian, C., Garreau, F., Brechot, C. & Kremsdorf, D. (2001). Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein. J Virol 75, 2684–2691.
Granzow, H., Birghan, Ch., Mettenleiter, T. C., Beyer, J., Köllner, B. & Mundt, E. (1997). A second form of infectious bursal disease virus-associated tubules contains VP4. J Virol 71, 8879–8885.[Abstract]
Horisberger, M. A. (1992). Interferon-induced human protein MxA is a GTPase which binds transiently to cellular proteins. J Virol 66, 4705–4709.
Kaerber, G. (1931). Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Arch Exp Pathol Pharmakol 162, 480–483(in German).
Ko, J. H., Jin, H. K., Asano, A., Takada, A., Ninomiya, A., Kida, H., Hokiyama, H., Ohara, M., Tsuzuki, M. & other authors (2002). Polymorphisms and the differential antiviral activity of the chicken Mx gene. Genome Res 12, 595–601.
Ko, J. H., Takada, A., Mitsuhashi, T., Agui, T. & Watanabe, T. (2004). Native antiviral specificity of chicken Mx protein depends on amino acid variation at position 631. Anim Genet 35, 119–122.[CrossRef][Medline]
Kochs, G. & Haller, O. (1999a). GTP-bound human MxA protein interacts with the nucleocapsids of Thogoto virus (Orthomyxoviridae). J Biol Chem 274, 4370–4376.
Kochs, G. & Haller, O. (1999b). Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids. Proc Natl Acad Sci U S A 96, 2082–2086.
Kochs, G., Janzen, C., Hohenberg, H. & Haller, O. (2002). Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes. Proc Natl Acad Sci U S A 99, 3153–3158.
Landis, H., Simon-Jodicke, A., Kloti, A., Di Paolo, C., Schnorr, J. J., Schneider-Schaulies, S., Hefti, H. P. & Pavlovic, J. (1998). Human MxA protein confers resistance to Semliki Forest virus and inhibits the amplification of a Semliki Forest virus-based replicon in the absence of viral structural proteins. J Virol 72, 1516–1522.
Larsen, R., Rokenes, T. P. & Robertsen, B. (2004). Inhibition of infectious pancreatic necrosis virus replication by Atlantic salmon Mx1 protein. J Virol 78, 7938–7944.
Lee, S.-H. & Vidal, S. M. (2002). Functional diversity of Mx proteins: variations on a theme of host resistance to infection. Genome Res 12, 527–530.
Li, X. L., Blackford, J. A. & Hassel, B. A. (1998). RNase L mediates the antiviral effect of interferon through a selective reduction in viral RNA during encephalomyocarditis virus infection. J Virol 72, 2752–2759.
Mundt, E. (1999). Tissue culture infectivity of different strains of infectious bursal disease virus is determined by distinct amino acids in VP2. J Gen Virol 80, 2067–2076.
Mundt, E., Kollner, B. & Kretzschmar, D. (1997). VP5 of infectious bursal disease virus is not essential for viral replication in cell culture. J Virol 71, 5647–5651.[Abstract]
Mundt, E., de Haas, N. & van Loon, A. A. (2003). Development of a vaccine for immunization against classical as well as variant strains of infectious bursal disease virus using reverse genetics. Vaccine 21, 4616–4624.[CrossRef][Medline]
Pavlovic, J., Züricher, T., Haller, O. & Staeheli, P. (1990). Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein. J Virol 64, 3370–3375.
Ponten, A., Sick, C., Weeber, M., Haller, O. & Kochs, G. (1997). Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity. J Virol 71, 2591–2599.[Abstract]
Reichelt, M., Stertz, S., Krijnse-Locker, J., Haller, O. & Kochs, G. (2004). Missorting of La Crosse virus nucleocapsid protein by the interferon-induced MxA GTPase involves smooth ER membranes. Traffic 5, 772–784.[CrossRef][Medline]
Schneider-Schaulies, S., Schneider-Schaulies, J., Schuster, A., Bayer, M., Pavlovic, J. & ter Meulen, V. (1994). Cell type-specific MxA-mediated inhibition of measles virus transcription in human brain cells. J Virol 68, 6910–6917.
Schnorr, J. J., Schneider-Schaulies, S., Simon-Jodicke, A., Pavlovic, J., Horisberger, M. A. & ter Meulen, V. (1993). MxA-dependent inhibition of measles virus glycoprotein synthesis in a stably transfected human monocytic cell line. J Virol 67, 4760–4768.
Schwemmle, M., Weining, K. C., Richter, M. F., Schumacher, B. & Staehli, P. (1995). Vesicular stomatitis virus transcription inhibited by purified MxA protein. Virology 206, 545–554.[CrossRef][Medline]
Smith, J. A., Schmechel, S. C., Williams, B. R. G., Silverman, R. H. & Schiff, L. A. (2005). Involvement of the interferon-regulated antiviral proteins PKR and RNase L in reovirus-induced shutoff of cellular translation. J Virol 79, 2240–2250.
Tyler, K. L. (2001). Mammalian reoviruses. In Fields Virology, 4th edn, pp. 1729–1945. Edited by D. M. Knipe & P. M. Howley. Philadelphia, USA: Lippincott Williams & Wilkins.
Tytell, A. A., Lampson, G. P., Field, A. K. & Hilleman, M. R. (1967). Inducers of interferon and host resistence. III. Double-stranded RNA from Reovirus type 3 virions (reo-3-RNA). Proc Natl Acad Sci U S A 58, 1719–1722.
Weber, F., Haller, O. & Kochs, G. (2000). MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes. J Virol 74, 560–563.
Zürcher, T., Pavlovic, J. & Staehli, P. (1992). Mechanism of human MxA protein action: variants with changed antiviral properties. EMBO J 11, 1657–1661.[Medline]
Received 1 September 2006;
accepted 1 December 2006.
This article has been cited by other articles:
|
|
 |
|
  A. Hoenen, W. Liu, G. Kochs, A. A. Khromykh, and J. M. Mackenzie West Nile virus-induced cytoplasmic membrane structures provide partial protection against the interferon-induced antiviral MxA protein J. Gen. Virol., November 1, 2007; 88(11): 3013 - 3017. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
