The parapoxvirus Orf virus inhibits dsDNA-mediated type I IFN expression via STING-dependent and STING-independent signalling pathways.

Type I interferons (IFNs) are critical in the host defence against viruses. They induce hundreds of interferon-stimulated genes (ISGs) many of which have an antiviral role. Poxviruses induce IFNs via their pathogen-associated molecular patterns, in particular, their genomic DNA. In a majority of cell types, dsDNA is detected by a range of cytoplasmic DNA sensors that mediate type I IFN expression via stimulator of interferon genes (STING). Orf virus (ORFV) induces cutaneous pustular skin lesions and is the type species of the Parapoxvirus genus within the Poxviridae family. The aim of this study was to investigate whether ORFV modulates dsDNA-induced type I IFN expression via STING-dependent signalling pathways in human dermal fibroblasts (hNDF) and THP-1 cells. We showed that ORFV infection of these cell types treated with poly(dA:dT) resulted in strong inhibition of expression of IFN-ß. In hNDFs, we showed using siRNA knock-down that STING was essential for type I IFN induction. IFN-ß expression was further reduced when both STING and RIG-I were knocked down. In addition, HEK293 cells that do not express STING or Toll-like receptors also produce IFN-ß following stimulation with poly(dA:dT). The 5' triphosphate dsRNA produced by RNA polymerase III specifically results in the induction of type I IFNs through the RIG-I receptor. We showed that ORFV infection resulted in strong inhibition of IFN-ß expression in HEK293 cells stimulated with poly(dA:dT). Overall, this study shows that ORFV potently counteracts the STING-dependent and STING-independent IFN response by antagonizing dsDNA-activated IFN signalling pathways.

The parapoxvirus Orf virus inhibits IFN-ß expression induced by dsRNA.

Orf virus (ORFV) is the type species of the Parapoxvirus genus that belongs to the Poxviridae family. Type I interferons (IFN) are critical in the host defence against viruses. They induce hundreds of interferon stimulated genes (ISGs) many of which have an antiviral role. The ability of ORFV to modulate type I IFN production was undertaken to investigate whether ORFV could inhibit IFN-ß expression via dsRNA dependant signalling pathways. HEK293 cells are known to lack DNA pattern-recognition receptors and Toll-like receptors however, they do express the cytosolic dsRNA receptors RIG-I and MDA5. HEK293 cells were shown to produce high levels of IFN-ß when cells were stimulated with poly(I:C) and this was shown to be predominantly via RIG-I-dependant signalling as confirmed by siRNA knock-down of RIG-I. Further we showed that HEK293 cells are permissive for ORFV and caused potent inhibition of IFN-ß transcription when cells were stimulated with poly(I:C) post-viral infection. Studies using heat inactivated ORFV suggested that de novo synthesis of early genes was required. In addition our findings showed that the ORFV encoded factor ORF020, that is known to bind dsRNA, is involved in antagonising IFN expression. Overall, this study has shown for first time the ability of ORFV to counteract type I IFN expression by antagonising dsRNA-activated RIG-I signalling.

The parapoxvirus Orf virus ORF116 gene encodes an antagonist of the interferon response.

Orf virus (ORFV) is the type species of the Parapoxvirus genus of the Poxviridae family. Genetic and functional studies have revealed ORFV has multiple immunomodulatory genes that manipulate innate immune responses, during the early stage of infection. ORF116 is a novel gene of ORFV with hitherto unknown function. Characterization of an ORF116 deletion mutant showed that it replicated in primary lamb testis cells with reduced levels compared to the wild-type and produced a smaller plaque phenotype. ORF116 was shown to be expressed prior to DNA replication. The potential function of ORF116 was investigated by gene-expression microarray analysis in HeLa cells infected with wild-type ORFV or the ORF116 deletion mutant. The analysis of differential cellular gene expression revealed a number of interferon-stimulated genes (ISGs) differentially expressed at either 4 or 6 h post infection. IFI44 showed the greatest differential expression (4.17-fold) between wild-type and knockout virus. Other ISGs that were upregulated in the knockout included RIG-I, IFIT2, MDA5, OAS1, OASL, DDX60, ISG20 and IFIT1 and in addition the inflammatory cytokine IL-8. These findings were validated by infecting HeLa cells with an ORF116 revertant recombinant virus and analysis of transcript expression by quantitative real time-PCR (qRT-PCR). These observations suggested a role for the ORFV gene ORF116 in modulating the IFN response and inflammatory cytokines. This study represents the first functional analysis of ORF116.

Rescue of a Vaccinia Virus Mutant Lacking IFN Resistance Genes K1L and C7L by the Parapoxvirus Orf Virus.

Type 1 interferons induce the upregulation of hundreds of interferon-stimulated genes (ISGs) that combat viral replication. The parapoxvirus orf virus (ORFV) induces acute pustular skin lesions in sheep and goats and can reinfect its host, however, little is known of its ability to resist IFN. Vaccinia virus (VACV) encodes a number of factors that modulate the IFN response including the host-range genes C7L and K1L. A recombinant VACV-Western Reserve (WR) strain in which the K1L and C7L genes have been deleted does not replicate in cells treated with IFN-ß nor in HeLa cells in which the IFN response is constitutive and is inhibited at the level of intermediate gene expression. Furthermore C7L is conserved in almost all poxviruses. We provide evidence that shows that although ORFV is more sensitive to IFN-ß compared with VACV, and lacks homologues of KIL and C7L, it nevertheless has the ability to rescue a VACV KIL- C7L- gfp+ mutant in which gfp is expressed from a late promoter. Co-infection of HeLa cells with the mutant and ORFV demonstrated that ORFV was able to overcome the block in translation of intermediate transcripts in the mutant virus, allowing it to progress to late gene expression and new viral particles. Our findings strongly suggest that ORFV encodes a factor(s) that, although different in structure to C7L or KIL, targets an anti-viral cellular mechanism that is a highly potent at killing poxviruses.