Functional analysis of the Orf virus ORF116 gene: a potential modulator of the interferon response
Orf virus (OV) is the type species of the Parapoxvirus genus. Like other poxviruses, genetic and functional studies have revealed that it has immunomodulatory genes that manipulate the innate immune response, often during the early stage of infection. The main focus of this study was to find the possible function of a novel gene in OV, ORF116, in strain NZ2 using a microarray approach. The ORF116 gene is 696 nts encoding a predicted polypeptide of 231 aa. Northern blotting analysis and reverse transcription-PCR have shown that ORF116 was expressed early. Northern blotting revealed a transcript of 2.1 kb detected in OV-NZ2 infected cells which could be detected as early as 2 h.p.i. by RT-PCR. The deletion of the ORF116 gene, showed that the virus replicated in primary lamb testis (LT) cells and HeLa cells, indicating that this gene is non-essential in cell culture, although viral growth was reduced in both cell types. Moreover, the deletion mutant produced smaller plaques on LT cells and induced a slower cytopathic effect in HeLa cells. Microarray expression profiling in HeLa-infected cells was conducted to identify genes whose expression altered with the presence of ORF116 and also to analyze the global change in gene expression induced by OV-NZ2 or OV-NZ2Δ116 at early times post-infection. Major findings showed that 3000 and 2200 transcripts were altered in their expression in OV-NZ2- and OV-NZ2Δ116-infected HeLa cells at 6 h.p.i. compared with the mock infection control. Furthermore, two genes IL-6 and IFI16 showed a similar expression pattern of up-regulation and down-regulation respectively relative to mock infection. The analysis of differential cellular gene expression has shown 68 genes differentially expressed at either 4 or 6 h.p.i. Of all of the genes that were analyzed by microarray, IFI44 showed the greatest differential expression between wild type and knockout virus. IFI44 expression was 4.17-fold higher in the knockout compared with the wild type at 4 h.p.i. It was also found that other interferon-stimulated genes were up-regulated to higher levels in the knockout which included RIG-I, MDA5, IFIT2, IFIT1, OAS1, OASL and ISG20. The differential expression of these genes was validated by qRT-PCR which suggests that ORF116 has a role in subverting the interferon (IFN) effector response. This study represents the first functional analysis of ORF116 gene encoded by a parapoxvirus.
Advisor: Fleming , Stephen
Degree Name: Master of Science
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: Orf; virus; ORF116; IFN; Immunomodulation
Research Type: Thesis