The role of the viral host response modifiers SPI-2 and N1L in mousepox pathogenesis

Abstract

The Orthopoxvirus genus includes highly-pathogenic viruses such as variola virus and ectromelia virus (ECTV). iECDTV is a natural pathogen of mice, causing mousepox and approximately 25% of its gene products are thought to be mediators of host immune evasion that target diverse processes.such as cellular signalling, intrinsic and extrinsic cell death pathways and components of the innate immune response. I studied the role of SPI-2 and NiL genes in mousepox and for that purpose recombinant ECTV were generated using GFP expression and blasticidin resistance as selection markers for transient dominant selection. The deletion of either or both SPI2 and NiL genes did not affect virus growth in vitro.

The serine proteinase inhibitor-Z (SPI-2) is a host response modifier known to inhibit caspase-8, caspase-I and granzyme B in vitro. I found that the ECTV SPI-2 protein is a potent inhibitor of infected target cell lysis induced by virus-immune cytotoxic T cells mediated by the death receptor pathway (caspase-dependent), but this viral protein has no effect on the granule exocytosis pathway. In the absence of SPI-2 protein, ECTV is attenuated in the infection of mousepox-susceptible mice; resulting in lower viral loads in the liver, decreased spleen pathology and substantially improved host survival. The main in vivo effect found of SPI-2 expression by ECTV-infected cells is the prevention of protective NK cell responses in a susceptible mouse infection. Reduced proportions and total numbers of NK cells expressing granzyme B were found in the spleen and blood of mice infected with wild-type (wt) ECTV compared to mice infected with SPI-2 mutant. Moreover, NK ex-vivo cytotoxicity was decreased in mice infected with wt ECTV compared to mice infected with mutant virus. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defences.

The vaccinia virus NIL protein is a virulence factor known as a NF-KB signalling inhibitor and an antiapoptotic protein of the bc1-2 family, and was shown to inhibit NK cell responses in vivo. In the absence of NIL, ECTV is highly attenuated and susceptible mice survived mutant virus infection with doses at least 1000 higher than the lethal dose of wt virus. The viral loads in the spleen, liver and popliteal draining lymph nodes were greatly reduced in mice infected with mutant viruses compared to mice infected with wt virus, with reductions in the order of 105-fold in the spleen. Interestingly, serum IFN-y and IL-6 and the proportion ofNK and CD8 T cells expressing granzyme B in the spleen of mice infected with mutant viruses were reduced compared to mice infected with wt virus, suggesting that the attenuation observed in the ECTV NILL\ mutants may not simply be associated with improved cytotoxic immune responses. Show more