Human MxA protein inhibits the replication of Crimean-Congo hemorrhagic fever virus

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Nairovirus within the family Bunyaviridae and is the causative agent of severe hemorrhagic fever. Despite increasing knowledge about hemorrhagic fever viruses, the factors determining their pathogenicity are still poorly understood. The interferon-induced MxA protein has been shown to have an inhibitory effect on several members of the Bunyaviridae family, but the effect of MxA against CCHFV has not previously been studied. Here, we report that human MxA has antiviral activity against CCHFV. The yield of progeny virus in cells constitutively expressing MxA was reduced up to 1,000-fold compared with control cells, and accumulation of viral genomes was blocked. Confocal microscopy revealed that MxA colocalizes with the nucleocapsid protein (NP) of CCHFV in the perinuclear regions of infected cells. Furthermore, we found that MxA interacted with NP by using a coimmunoprecipitation assay. We also found that an amino acid substitution (E645R) within the C-terminal domain of MxA resulted in a loss of MxA antiviral activity and, concomitantly, in the capacity to interact with CCHFV NP. These results suggest that MxA, by interacting with a component of the nucleocapsid, prevents replication of CCHFV viral RNA and thereby inhibits the production of new infectious virus particles.

FIG. 1.

Antiviral activity of human MxA protein against CCHFV. Vero cells constitutively expressing wild-type MxA (VA9, VA12, and VA3), nontransfected Vero cells, cells expressing only the neomycin resistance gene (VN36 and VN41) and cells expressing a mutant form of MxA [VA(E645R)] were infected with CCHFV at an MOI of 0.01. At 24 and 48 hpi, the cells and supernatants were harvested and the amount of infectious virus was determined as described in Materials and Methods. The mean log titers given are the average of three independent experiments. Error bars indicate standard deviations.

FIG. 2.

(A) Formation of foci was inhibited in cells expressing MxA protein. Nontransfected Vero cells, Vero cells expressing only the neomycin resistance gene (VN36), Vero cells expressing MxA(E645R) [VA(E645R)] or cells expressing wild-type MxA (VA9) were grown in chamber slides and infected with CCHFV at MOI of 0.01. At 12 or 24 hpi, the cells were fixed and analyzed for CCHFV NP expression by indirect immunofluorescence. (B) Monolayers of control cells (Vero E6 cells and VN36) and cells constitutively expressing MxA (VA9) were infected with CCHFV at MOI of 0.1. At 12 (a) or 24 (b) hpi, the cells were harvested and analyzed by Western blot using an anti-CCHFV NP antibody. Molecular markers are shown at the right side of the panel.

FIG. 3.

Accumulation of the CCHFV genome is blocked in cells expressing wild-type MxA. VA9 and VN36 monolayers were infected with CCHFV at an MOI of 0.01. After 12 and 24 hpi, the cells were lysed in TRIZOL reagent. Total RNA was extracted and the amounts of vRNA for the S (a) and M (b) segments were determined by a quantitative real-time PCR as described in Materials and Methods. Values given are the average of three independent experiments. Error bars indicate standard deviations.

FIG. 4.

(Color panels) Wild-type MxA colocalizes with NP of CCHFV in perinuclear regions of infected cells. VN36, VA(E645R), and VA9, seeded in chamber slides, were infected with CCHFV at an MOI of 0.01. At 24 hpi, the cells were fixed and analyzed for CCHFV NP (green) and MxA (red) under the confocal microscope. Panels at the right show the superimposition of the two images. (Black-and-white panels) MxA coimmunoprecipitated with CCHFV NP. Wild-type MxA-expressing Vero cells (VA9), a mutant form of MxA [VA(E645R)], and cells expressing only the neomycin gene were infected with CCHFV at an MOI of 0.1. (a) At 24 hpi, the cell monolayers were lysed and immunoprecipitated with an anti-CCHFV NP-specific antibody. The immunoprecipitate was analyzed for the presence of MxA by Western blot analysis using specific MxA antibodies. Molecular markers are shown on the left side of the panel. (b) Cell lysates from panel a were analyzed for the expression of MxA and CCHFV NP by Western blot analysis.