Abstract
In common with other poxviruses, orf virus (ORFV) is believed to induce drastic changes in the host intracellular environment through expression of an arsenal of virulence factors. The ankyrin repeat (ANK) proteins are examples of such factors. ANK proteins are ubiquitous in eukaryotes and participate in protein-protein interactions. ANK proteins in viruses are rare with the exception of poxvirus. Many poxviral ANK proteins also carry an F-box motif which mediates interaction with the cellular SCF1 ubiquitin ligase complexes. It has been proposed that ORFV ANK proteins recruit substrate proteins to SCF1 complexes for subsequent ubiquitination. This study aimed to identify the binding partners of ORFV ANK proteins and changes in the cell transcriptome and proteome early in ORFV infection so as to assist in elucidating the functions of the ORFV ANK proteins. This study harnessed the power of high throughput next generation sequencing (RNA-seq) and both qualitative and quantitative mass spectrometry.
This study presents a characterisation of the ORFV ANK proteins during ORFV infection, including their timing of expression, localisation, and interaction with SCF1 components. Additionally, the use of proteasome inhibitor confirmed the importance of a function ubiquitin proteasome system to ORFV replication.
Transcriptomic studies revealed dramatic changes in host gene expression early in ORFV infection of HeLa cells, including genes involved in cell cycle related processes. These processes were highlighted in studies using an ORFV deleted of its proposed cell cycle regulator gene, PACR. Additionally, infection with a recombinant ORFV deleted of the gene encoding one of its ANK proteins (ORFV ANK008KO) revealed involvement of ANK008 in functions like cellular replication, apoptosis initiation, transcription regulation, and angiogenesis.
Mass spectrometry was used to identify proteins co-precipitating with 3 of the 5 ORFV ANK proteins when these viral proteins were expressed from their natural promoters, during infection with recombinant ORFVs. Co-precipitates of each ORV ANK protein included components of the SCF1 complex, as predicted. Further analyses revealed evidence of wider links between both ORFV ANK008 and ANK129 and the ubiquitin proteasome pathway. ANK129 also showed evidence of possible activities in cellular proliferation.
SWATH quantitative mass spectrometry was used in pairwise quantitative comparisons of mock-infected, ORFV-infected, and ORFV ANK008KO-infected HeLa cells. This approach built on the model that ORFV ANKs may promote the proteasomal degradation of specific cellular proteins. The data identified a group of cellular proteins whose abundance appears to be directly influenced by ANK008. For example, macrophage migration inhibition factor (MIF), an important regulator of innate immunity, was reduced during ORFV infection and ANK008 at very least contributed to that reduction. Network and pathway analyses of these proteins revealed links to cell cycle, cellular metabolism and apoptosis, reflecting results of the transcriptome analyses. The SWATH data from ORFV infection support PACR’s involvement in cell cycle in a manner that provides ORFV with an environment rich in nucleotides. Additionally, links to nucleotide metabolism were also made for ANK008 in both the qualitative and quantitative mass spectrometry, suggesting a novel function for ANK008.
Finally, the transcriptomic and proteomic data were combined to provide a comprehensive understanding of the temporal expression of all ORFV genes. This study presents a robust examination of ORFV-host interactions and the functions of ORFV ANK proteins. The results provide numerous exciting areas to pursue in future research.