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. 2009 Mar;83(5):2310-20.
doi: 10.1128/JVI.00781-08. Epub 2008 Dec 24.

Inhibition of a large double-stranded DNA virus by MxA protein

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Inhibition of a large double-stranded DNA virus by MxA protein

Christopher L Netherton et al. J Virol. 2009 Mar.

Abstract

Increasing evidence points to the importance of the interferon (IFN) response in determining the host range and virulence of African swine fever virus (ASFV). Infection with attenuated strains of ASFV leads to the upregulation of genes controlled by IFN pathways, including myxovirus resistance (Mx) genes that are potent effectors of the antiviral state. Mx gene products are known to inhibit the replication of many negative-sense single-stranded RNA viruses, as well as double-stranded RNA viruses, positive-sense single-stranded RNA viruses, and the reverse-transcribing DNA virus hepatitis B virus. Here, we provide data that extend the known range of viruses inhibited by Mx to include the large double-stranded DNA viruses. Stably transfected Vero cells expressing human MxA protein did not support ASFV plaque formation, and virus replication in these cells was reduced 100-fold compared with that in control cells. In contrast, ASFV replication in cells expressing MxB protein or a mutant MxA protein was similar to that in control Vero cells. There was a drastic reduction in ASFV late protein synthesis in MxA-expressing cells, correlating with the results of previous work on the effect of IFN on viral replication. Strikingly, the inhibition of ASFV replication was linked to the recruitment of MxA protein to perinuclear viral assembly sites, where the protein surrounded the virus factories. Interactions between ASFV and MxA were similar to those seen between MxA and different RNA viruses, suggesting a common inhibitory mechanism.

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Figures

FIG. 1.
FIG. 1.
MxA protein inhibits ASFV replication. (A) Confluent monolayers of VN36 (control), VA3 (MxA), VB22 (MxB), or VA(E645R) [MxA (E645R)] cells were infected with the indicated amounts of Ba71v (ASFV), then overlaid, incubated for 7 days, and finally fixed and stained with crystal violet. (B) Monolayers from VN36 (control) and VA3 (MxA) cells infected with Ba71v were imaged at an original magnification of ×4 (0.10 normal aperture) by using a Leica DMLA microscope. A digital zoom view of the boxed VA3 cells exhibiting a CPE is shown in the bottom right corner of the image. (C) VN36 (control), VA3 (MxA), and VA(E645R) [MxA (E645R)] cells were infected with Ba71v at a MOI of 0.5 PFU/cell. Cells were incubated until a 100% CPE on the VN36 cells was observed, and then virus was harvested and quantified by plaque assay. Error bars show standard deviations for 12 replicates. (D) The indicated amounts of total protein from cell lysates prepared from VN36, VA3, or PK-15 cells incubated with or without 200 U/ml porcine IFN-α for 24 h were analyzed by immunoblotting using anti-MxA antibody M143. The position of a molecular mass marker in kilodaltons is indicated to the left of the gel. +, present; −, absent.
FIG. 2.
FIG. 2.
MxA inhibits late ASFV protein synthesis. (A) VN36 (control) or VA3 (MxA) cells were infected overnight with Ba71v, and ASFV proteins in cell lysates were analyzed by immunoblotting using antibodies against the indicated proteins. The positions of molecular mass markers in kilodaltons are indicated to the left of the gels. +, present; −, absent. (B) VN36 or VA3 cells were infected overnight with Ba71v, fixed, and stained with antibody 4H3 specific for p73. Factories in VN36 (control) and VA3 (MxA) cells were imaged and measured using Openlab 3.1.7 as described in Materials and Methods. Error bars show standard deviations in the measurements of 56 factories in VN36 cells and 50 factories in VA3 cells. (C) MxA does not inhibit viral DNA synthesis. VN36 (control) and VA3 (MxA) cells were infected overnight with Ba71v and then pulsed with [3H]thymidine for 15 min. Cytosolic fractions were collected, and incorporated radioactivity was measured with a scintillation counter. Error bars show standard deviations for three replicates.
FIG. 3.
FIG. 3.
MxA is recruited to ASFV factories in MxA-expressing cells. VN36 (control), VA3 (MxA), or VA(E645R) [MxA(E645R)] cells were infected overnight with Ba71v. Cells were fixed and processed for immunofluorescence with DAPI (A, E, and I), anti-MxA antibody (C, G, and K), and p73 antibody (4H3) (B, F, and J) and analyzed by confocal microscopy. Scale bars, 10 μm.
FIG. 4.
FIG. 4.
MxA associates with ASFV factories throughout the focal plane of the cell. VA3 cells were infected overnight with Ba71v and then fixed and processed for immunofluorescence with DAPI (A), anti-p34 antibody (B), and anti-MxA (C) and analyzed by confocal microscopy. The boxes in panel D highlight zoomed-in areas shown in panels E through H. Panels G and H show focal planes located 1.1 and 2.2 μm, respectively, from that in panel F. Scale bars, 10 μm.
FIG. 5.
FIG. 5.
The recruitment of MxA to a factory requires DNA synthesis but not microtubules or actin filaments. VA3 cells were infected for 4 h with Ba71v prior to the addition of AraC (A to D), nocodazole (E to H), or cytochalasin D (I to L). Cells were then incubated for a further 16 h, fixed, processed for immunofluorescence with DAPI (A, E, and I), anti-MxA antibody (C, G, and K), anti-p30 (B), anti-α-tubulin (F), or phalloidin-Alexa 488 (J), and analyzed by confocal microscopy. Arrows in panel B indicate areas of diminished p30 labeling, and arrowheads in panels E and I show cytoplasmic viral DNA. Scale bars, 10 μm.
FIG. 6.
FIG. 6.
MxA colocalizes with ASFV DNA binding protein pA104R. VN36 (control) and VA3 (MxA) cells were infected overnight with Ba71v, fixed, processed for immunofluorescence with DAPI (A, E, I, and M), anti-MxA antibody (C, G, K, and O), and anti-pE120R (B and F) or anti-pA104R (J and N), and then analyzed by confocal microscopy. Scale bars, 10 μm.
FIG. 7.
FIG. 7.
ASFV factories are surrounded by filaments. VN36 (A) and VA3 (B to D) cells were infected overnight with Ba71v, fixed, and processed for electron microscopy. Abbreviations: f, filaments; m, mitochondria; v, virions (mature and immature); vp, viroplasm and viral membranes; and z, zipper structures.
FIG. 8.
FIG. 8.
Porcine Mx1 is recruited to ASFV factories. Vero cells transfected with either human MxA (A to D) or porcine Mx1 (E to H) were infected overnight with Ba71v, fixed, processed for immunofluorescence with DAPI (A and E), anti-p34 antisera (B and F), and anti-MxA antibody (D and H), and then analyzed by confocal microscopy. Scale bars, 10 μm.

References

    1. Aebi, M., J. Fäh, N. Hurt, C. E. Samuel, D. Thomis, L. Bazzigher, J. Pavlovic, O. Haller, and P. Staeheli. 1989. cDNA structures and regulation of two interferon-induced human Mx proteins. Mol. Cell. Biol. 95062-5072. - PMC - PubMed
    1. Afonso, C. L., L. Zsak, C. Carrillo, M. V. Borca, and D. L. Rock. 1998. African swine fever virus NL gene is not required for virus virulence. J. Gen. Virol. 792543-2547. - PubMed
    1. Afonso, C. L., M. E. Piccone, K. M. Zaffuto, J. Neilan, G. F. Kutish, Z. Lu, C. A. Balinsky, T. R. Gibb, T. J. Bean, L. Zsak, and D. L. Rock. 2004. African swine fever virus multigene family 360 and 530 genes affect host interferon response. J. Virol. 781858-1864. - PMC - PubMed
    1. Andersson, I., L. Bladh, M. Mousavi-Jazi, K. E. Magnusson, A. Lundkvist, O. Haller, and A. Mirazimi. 2004. Human MxA protein inhibits the replication of Crimean-Congo hemorrhagic fever virus. J. Virol. 784323-4329. - PMC - PubMed
    1. Andrés, G., C. Simón-Mateo, and E. Viñuela. 1997. Assembly of African swine fever virus: role of polyprotein pp220. J. Virol. 712331-2341. - PMC - PubMed

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