Michaël Leroy

Expression of the interferon-alpha/beta-inducible bovine Mx1 dynamin interferes with replication of rabies virus.

Rabies is a fatal anthropozoonotic viral infection of the central nervous system that remains a serious public health problem in many countries. As several animal cases of spontaneous survival to infection were reported and because type 1 interferons were shown to protect against the virus, it was suggested that innate resistance mechanisms exist. Among the antiviral proteins that are synthesized in response to interferon-alpha/beta stimulation, Mx proteins from several species are long known to block the replication of vesicular stomatitis virus (VSV). As both VSV and rabies virus belongs to the Rhabdoviridae family, this study was started with the aim to establish whether the anti-VSV activity of a mammalian Mx protein could be extended to rabies virus. This question was addressed by inoculating the virus onto a bovine Mx1 or human MxA-expressing Vero cell clone. Plaque formation was unambiguously blocked, and viral yields were reduced 100- to 1000-fold by bovine Mx1 expression for both SAG2 and SADB19 viral strains. In opposition, only SAG2 strain could be inhibited by the expression of human MxA protein. The effect of both proteins expression was then evaluated at the viral protein expression level. Again, boMx1 was able to repress protein expression in both strain, whereas only SAG2 proteins were inhibited in human MxA-expressing cells. These results suggest that protection conferred by interferon-alpha/beta against rabies could be, at least partially, attributable to the Mx pathway. Alternatively, bovine Mx1 could be unique in its ability to repress rabies virus which, if confirmed in vivo, would open an avenue for the development of new antirabies therapeutic strategies.

Modulating mouse innate immunity to RNA viruses by expressing the Bos taurus Mx system

Mx proteins are interferon-induced members of the dynamin superfamily of large guanosine triphosphatases. These proteins have attracted much attention because some display antiviral activity against pathogenic RNA viruses, such as members of the orthomyxoviridae, bunyaviridae, and rhabdoviridae families. Among the diverse mammalian Mx proteins examined so far, we have recently demonstrated in vitro that the Bos taurus isoform 1 (boMx1) is endowed with exceptional anti-rabies-virus activity. This finding has prompted us to seek an appropriate in vivo model for confirming and evaluating gene therapy strategies. Using a BAC transgene, we have generated transgenic mouse lines expressing the antiviral boMx1 protein and boMx2 proteins under the control of their natural promoter and short- and long-range regulatory elements. Expressed boMx1 and boMx2 are correctly assembled, as deduced from mRNA sequencing and western blotting. Poly-I/C-subordinated expression of boMx1 was detected in various organs by immunohistochemistry, and transgenic lines were readily classified as high- or low-expression lines on the basis of tissue boMx1 concentrations measured by ELISA. Poly-I/C-induced Madin-Darby bovine kidney cells, bovine turbinate cells, and cultured cells from high-expression line of transgenic mice were found to contain about the same concentration of boMx1, suggesting that this protein is produced at near-physiological levels. Furthermore, insertion of the bovine Mx system rendered transgenic mice resistant to vesicular-stomatitis-virus-associated morbidity and mortality, and embryonic fibroblasts derived from high-expression transgenic mice were far less permissive to the virus. These results demonstrate that the Bos taurus Mx system is a powerful anti-VSV agent in vivo and suggest that the transgenic mouse lines generated here constitute a good model for studying in vivo the various antiviral functions—known and yet to be discovered—exerted by bovine Mx proteins, with priority emphasis on the antirabic function of boMx1.