Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids

Abstract

Interferon-induced human MxA protein belongs to the dynamin superfamily of large GTPases. It exhibits antiviral activity against a variety of RNA viruses, including Thogoto virus, an influenza virus-like orthomyxovirus transmitted by ticks. Here, we report that MxA blocks the transport of Thogoto virus nucleocapsids into the nucleus, thereby preventing transcription of the viral genome. This interaction can be abolished by a mAb that neutralizes the antiviral activity of MxA. Our results reveal an antiviral mechanism whereby an interferon-induced protein traps the incoming virus and interferes with proper transport of the viral genome to its ultimate target compartment within the infected cell.

Figure 1

Detection of primary THOV transcripts by Northern blot analysis. Parallel cultures of MxA-expressing 3T3 cells (MxA, lane 2) or control cells (neo, lane 1) (14) were infected with 20 plaque forming units of THOV (31) per cell in the presence of 50 μg/ml CHX. Total RNA was prepared 7 h after infection, and samples of 20 μg of RNA were loaded into each lane. Northern blots were hybridized with radiolabeled, negative-sense RNA probes derived from the PB2, NP, and M genes of THOV (34), as indicated. For the detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts, blots were analyzed with a specific, nick-translated cDNA probe. Blots were exposed to x-ray films to detect the radioactive signals. The position of the cross-hybridizing 18S rRNA is indicated.

Figure 2

MxA inhibits nuclear import of viral nucleocapsids. MxA-expressing (MxA) and control (neo) 3T3 cells were microinjected with purified viral nucleocapsids and incubated for 2 h in the presence of 50 μg/ml CHX. Cells were then analyzed by indirect double-immunofluorescence with anti-MxA (a and d; green) and anti-NP (b and e; red) antibodies. Panels at the right (c and f) show the superimposition of two images. Colocalization of NP (red) and MxA (green) results in yellow staining.

Figure 3

mAb 2C12 abrogates import block. (A) MxA-expressing 3T3 cells were microinjected with a mixture of viral nucleocapsids and antibody 2C12 (9). Microinjected cells were kept for 2 h in the presence of CHX and then analyzed for the localization of the antibody (a; green) or the viral NP protein (b; red). (B) MxA-expressing 3T3 cells were microinjected with either antibody 2C12 (a and c) or an unrelated control antibody (b and d), and were infected 8 h later with THOV at a multiplicity of 50 per cell. Cells were fixed 16 h later and analyzed for viral protein synthesis by double-immunofluorescence labeling using a hyperimmune anti-THOV antiserum (c and d). The microinjected antibodies were detected with the help of a fluorescein-conjugated goat anti-mouse IgG antibody (a and b).

Figure 4

MxA-mediated antiviral effect of IFN. MxA binds to THOV nucleocapsids upon entry into the cytosol and prevents their transport to the nucleus. As a consequence, primary transcription of the viral genome cannot take place.