Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes - PubMed (original) (raw)

Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes

Georg Kochs et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Bunyaviruses replicate in the cytoplasm of infected cells. New viral particles are formed by budding of nucleocapsids into the Golgi apparatus. We have previously shown that the IFN-induced human MxA protein inhibits bunyavirus replication by an unknown mechanism. Here we demonstrate that MxA binds to the nucleocapsid protein of La Crosse virus (LACV) and colocalizes with the viral protein in cytoplasmic complexes. Electron microscopy revealed that these complexes accumulated in the perinuclear area and consisted of highly ordered fibrillary structures. A similar MxA-mediated redistribution of viral nucleocapsid proteins was detected with other bunyaviruses, such as Bunyamwera virus and Rift Valley fever virus. MxA(E645R), a carboxy-terminal mutant of MxA without antiviral activity against LACV, did not lead to complex formation. Wild-type MxA, but not MxA(E645R), was able to bind to LACV nucleocapsid protein in coimmunoprecipitation assays, demonstrating the importance of the carboxy-terminal effector domain of MxA. These results illustrate an efficient mechanism of IFN action whereby an essential virus component is trapped in cytoplasmic inclusions and becomes unavailable for the generation of new virus particles.

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Figures

Figure 1

Figure 1

MxA binds to the N protein of bunyaviruses and colocalizes with N in large perinuclear complexes. (A) MxA-expressing Vero cells were either infected with 10 pfu of LACV per cell (a_–_f) or were transfected with plasmid pSC-N coding for the N protein of LACV (g–i, note that the transfected cell is surrounded by nontransfected cells expressing only MxA) or were left untreated (j). Cells were fixed 24 h later and analyzed by immunofluorescence for the presence of viral N or G protein (red) and MxA (green), using specific Abs. (B) MxA-expressing Vero cells were infected with 20 pfu per cell of either BUNV or RVFV for 20 h, fixed, and analyzed for MxA and N expression. The cells were stained with Abs directed against the N proteins of either BUNV (red; a) or RVFV (d) together with an MxA-specific Ab (green; b and e). Panels at the right show the superimposition of the two images. Bars, 20 μm. (C) MxA-expressing Vero cells were infected with 10 pfu of LACV per cell for 20 h or were left untreated. Cell lysates were used for immunoprecipitation in the presence or absence of GTPγS by using the N-specific antiserum. The coprecipitated proteins were detected by Western-blotting with the MxA-specific mAb M143. Positions of _M_r markers (in kDa) are indicated at the left.

Figure 2

Figure 2

Ultrastructure of MxA/N complexes. MxA-expressing Vero cells (a) or control cells (b) were infected with 10 pfu of LACV per cell for 20 h. Cells were prepared for transmission EM by cellulose capillary technique. The nucleus is marked by Nu. For immuno-EM, MxA-expressing cells infected with LACV were prepared by using the progressive lowering of temperature method. Thin sections of the cells were labeled with the N-specific Ab (c) or with mAb M143 directed against MxA (d). Primary Abs were detected with gold-labeled protein A. The magnification is indicated by the bars. The diameter of the gold particles is 10 nm.

Figure 3

Figure 3

MxA(E645R) lacks antiviral activity against LACV and does not interact with the viral N protein. (A) Plaque formation of LACV is not inhibited by MxA(E645R). Vero cells expressing wild-type MxA, MxA(E645R), or control cells were infected with 200 pfu of LACV and stained with crystal violet 5 days later. (B) MxA(E645R) does not coimmunoprecipitate with N of LACV. Vero cells expressing MxA or MxA(E645R) and control cells were infected with 10 pfu per cell of LACV or were left untreated. Cell lysates were prepared 20 h later and immunoprecipitated with the N-specific antiserum in the presence of GTPγS. The immunoprecipitate was analyzed for the presence of MxA with mAb M143 by immunoblot (Upper). The coprecipitated background bands visible in all lanes are of unknown origin. The expression levels of MxA, MxA(E645R), and N were assessed by immunoblot analysis of the crude cell lysates, using the same Abs (Lower).

Figure 4

Figure 4

The antiviral activity of MxA correlates with the formation of MxA/N complexes. Control cells (a_–_c) or Vero cells constitutively expressing MxA (d–f) or MxA(E645R) (g–i) were used for infection. In parallel, Vero cells that transiently expressed MxA(L612K) (j–l), MxA(T103A) (m–o), or TMxA (p–r) were used. All cells were infected with 10 pfu/cell of LACV. Twenty hours later, the cells were fixed and analyzed for the viral N (red) and the various MxA proteins (green) by immunofluorescence. Panels at the right show the superimposition of the two images. Bar, 20 μm.

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