Investigation of Orf virus resistance to interferon
The interferon (IFN) effector response is induced in cells through the JAK/STAT signaling pathway by type 1, type 2 and type 3 IFNs. This results in the upregulation of hundreds of genes, many of which are involved in combating viral replication. The Poxviridae virus family are large DNA viruses that replicate in the cytoplasm. The most well-known poxvirus is the variola virus the causative agent of smallpox that has devastated the human population over the years. Vaccinia virus (VACV) a closely related member to variola virus was used successfully in a global program to eradicate smallpox. In VACV the genes K1L and C7L have long been known as host range genes. Recently, they have been shown to be critical for blocking the IFN effector response. A recombinant VACV-Western Reserve (WR) in which the KIL and C7L genes have been deleted (vKIL-C7L-) does not replicate in cells treated with IFN-beta nor in HeLa cells. Gene expression in poxviruses is divided into three temporal classes, early, intermediate and late. Early genes are expressed shortly after the virus enters the cell and express polymerases required for intermediate gene transcription, and intermediate genes express polymerases required for late gene transcription. Interestingly, it was discovered that vKIL-C7L- replication was blocked at the step of intermediate viral gene translation. Homologues of these genes are found in almost all poxviruses except Orf virus (ORFV). ORFV is the type species of the Parapoxvirus genus which induces pustular skin lesions in sheep, goats and humans. Only one gene has been found to block the IFN effector response, OV20.0L that targets the IFN-induced double-stranded RNA-dependent protein kinase R (PKR). ORFV does not encode homologues of KIL or C7L. There is a growing body of evidence that suggests that ORFV is able to inhibit the IFN effector response and produces a strong cytopathic effect in cells treated with IFN-α or IFN-γ and replicates in HeLa cells in which the IFN response is constitutive. This suggests that ORFV encodes a gene(s) that may be functionally equivalent to K1L and C7L in their ability to resist the IFN effector response and be able to rescue a mutant virus lacking K1L and C7L (vKIL-C7L-) in cells in which the IFN effector response is present. In my PhD, four approaches were used to test whether any ORFV genes can rescue the vKIL-C7L- mutant. The first approach was to make a VACV recombinant using an ORFV plasmid library. This plasmid library contains the full complement of ORFV genes in a large multi-gene overlapping DNA fragments expressed from their native promoters. Unfortunately, we were unsuccessful in making any recombinants. The second approach was screening the ORFV plasmid library for any plasmids that might provide some rescue for the vKIL-C7L-. The rescue effect was measured by the expression of a lacZ reporter expressed from a VACV early/late promoter from the transfected plasmid. The vKIL-C7L- replication is blocked at the level of intermediate viral gene translation. Therefore, it was thought that if any ORFV plasmids managed to provide some rescue, it will increase the expression of the lacZ reporter from the late promoter. Four ORFV recombinant plasmids showed some evidence of partial rescue to a vKIL-C7L- mutant. This approach was validated using a plasmid expressing C7L from its native promoter which showed a significate increase in beta-galactosidase expression. The third approach to determine if ORFV genes could be identified using a plasmid transfection approach involved a vKIL-C7L- mutant expressing GFP off a late promoter (vK1L-C7L-/GFP+). Having the reporter expressed from a strictly late promoter was thought to provide a more accurate measure of the rescue effect. It was expected that the number of cells expressing GFP from the late promoter will increase if any ORFV plasmids provided rescue for the vK1L-C7L-/GFP+ mutant. Although a plasmid expressing C7L was shown to rescue the vK1L-C7L-/GFP+ mutant, none of the ORFV plasmids were shown to rescue this VACV mutant. The partial rescue seen with the lacZ reporter but not with GFP reporter could be explained by lacZ being expressed from early/late promoter and GFP is expressed from the strictly late promoter. The fourth approach involved a double infection with ORFV and vK1L-C7L-/GFP+. The double infection was done in HeLa cells and our results showed that ORFV can provide some rescue to the vK1L-C7L-/GFP+ allowing it to progress to late gene expression and produce new viral particles. However, ORFV did not rescue the vK1L-C7L-/GFP+ mutant to the same degree as VACV-wild type (wt) did. Finally, we wanted to determine the ability of ORFV and VACV to resist the hIFN-beta effector response. Human dermal fibroblasts were treated with different amounts of hIFN-beta and infected with ORFV or VACV (wt). Compared to untreated cells, ORFV replication was inhibited by 229-fold, while VACV replication was inhibited by 4.7-fold at 63 U/mL of hIFN-beta treatment of the cells. ORFV inhibition was independent of multiplicity of infection (MOI). Our results confirm the ability of ORFV to resist the IFN effector response but not to the same level as VACV. In summary, my PhD study suggests that ORFV encodes factors that are functionally equivalent to K1L and C7L but are not as potent. That could explain the finding that ORFV has less ability to resist the IFN effector response compared with VACV.
Advisor: Fleming, Stephen Bruce; Mercer, Andew
Degree Name: Doctor of Philosophy
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: New Zealand; Orf; virus; interferon; IFN; vaccinia
Research Type: Thesis