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

Abstract

Mx proteins form a small family of interferon (IFN)-induced GTPases with potent antiviral activity against various negative-strand RNA viruses. To examine the antiviral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiviral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of viral structural proteins, we took advantage of a recombinant vector based on the SFV replicon. The vector contains only the coding sequence for the viral nonstructural proteins and the bacterial LacZ gene, which was cloned in place of the viral structural genes. Upon transfection of vector-derived recombinant RNA, expression of the beta-galactosidase reporter gene was strongly reduced in the presence of MxA. This finding indicates that viral components other than the structural proteins are the target of MxA action.

FIG. 1

Inhibition of virus plaque formation by MxA protein expressed in stably transfected human cell lines. Confluent monolayers of HEp-2-neo control cells and the clonal lines HEp-2-MxA 44.12 and 71.2 were infected with several hundred PFU of either influenza A virus (FPV-B), VSV, or SFV. The viruses were allowed to form plaques under soft agar for 46 h (VSV infection) or 70 h (influenza virus and SFV infection).

FIG. 2

Expression of MxA protein in stably transfected human HEp-2-MxA and U937-MxA cells leads to inhibition of SFV multiplication. (A) Western blot analysis of cytoplasmic extracts from various HEp-2-MxA and U937-MxA clonal lines. Samples (50 μg of protein per lane) were separated in SDS–10% polyacrylamide gels and subsequently blotted onto nitrocellulose membranes. The blots were immunostained with a monoclonal mouse anti-MxA antibody. The bands were visualized with enhanced chemiluminescence reagents (Amersham). Quantitation of the optical density was carried out with the video documentation system E.A.S.Y (Herolab, Wiesloch, Germany). The relative MxA levels are shown as arbitrary expression units. (B) Reduction of SFV yields by MxA. The different clonal lines were infected with SFV at an MOI of 0.1. Viral yields in culture supernatants 24 h postinfection were determined by the TCID₅₀ method.

FIG. 3

Inhibition of SFV protein synthesis in HEp-2 cells expressing MxA. HEp-2-neo control cells (A and B) and cells of the clonal line HEp-2-MxA 71.2 (C through F) were infected with SFV at an MOI of 2. Cells were fixed 24 h postinfection and subjected to double immunofluorescence analysis with a confocal laser scanning light microscope. Cells were simultaneously immunostained with a mouse monoclonal anti-recombinant MxA antibody (A, C, and E) and a polyclonal rabbit anti-SFV C protein serum (B, D, and F).

FIG. 4

Accumulation of SFV C protein is inhibited in infected cells stably expressing MxA protein. Monolayer cultures of HEp-2-MxA (A) and U937-MxA (B) clonal lines were infected with SFV at an MOI of 3. HEp-2-neo and U937 cells were used as controls. Infected cells were harvested 7 h postinfection. Western blots of whole-cell extracts (200 μg per lane) were immunostained with a polyclonal rabbit anti-SFV C protein antiserum.

FIG. 5

Reduced accumulation of SFV genomic 49S and subgenomic 26S RNAs in infected cells expressing MxA protein. Monolayer cultures of different clonal lines of HEp-2-MxA (A) and U937-MxA cells (B) were infected with SFV at an MOI of 3, and total cell RNA was prepared 5 h postinfection. HEp-2-neo and U937 cells served as controls. HEp-2 cells were either left untreated or pretreated with IFN-α2 for 18 h prior to infection (A). (C) To examine RNA accumulation throughout the course of infection, the clonal lines HEp-2-MxA 71.2 and HEp-neo were infected with SFV at an MOI of 1 and total cell RNA was prepared 5, 24, 48, and 72 h postinfection (C). The RNA preparations (20 μg per lane) were subjected to Northern blot analysis with radiolabeled genomic cDNA of SFV. Hybridization to a radiolabeled 0.8-kb fragment of human GAPDH cDNA was used as a control (C, lower section). The blots were exposed to X-ray film.

FIG. 6

Structural proteins are not required for the inhibition of SFV replication by MxA protein. Eighty-percent confluent monolayer cultures of the clonal lines HEp-2-MxA 71.2, 44.12, and 34.36 were cotransfected with 3 μg of in vitro-transcribed RNA from the plasmid pSFV3-lacZ and 3 μg of pSV2-CAT expression plasmid. Clones 3T3-neo, 3T3-MxA, 3T3-Mx1, and HEp-2-neo were used as controls. The recombinant RNA coding for the SFV replicase and the bacterial gene LacZ drives its own replication and capping, resulting in the production of heterologous β-Gal protein. For the quantitative determination of β-Gal and CAT colorimetric enzyme, immunoassays were used.

FIG. 7

Accumulation of pSFV3-lacZ replicon RNA is strongly reduced in HEp-2 cells lines expressing MxA protein. Eighty-percent confluent monolayer cultures of the clonal lines HEp-2-MxA 71.2, 44.12, and 34.36 were transfected with 5 μg of in vitro-transcribed RNA from the plasmid pSFV3-lacZ. Clone HEp-2-neo was used as a control. RNA (20 μg per lane) was prepared 24 h after transfection and subjected to Northern blot analysis with a radiolabeled cDNA coding for nsP1 to nsP4. The blots were exposed to X-ray film.