Project description:By doing ChIP-seq with antibodies against two P23-45 RNA polymerases gp64 and gp96 we showed that both RNA polymerases interact with the phage genome at the early stage of infection. Gp96 interacts with pre-early P23-45 genes, while gp64 interacts with pre-early, early and some middle-stage genes.
Project description:Rabbit haemorrhagic disease virus (RHDV) belongs to the family Caliciviridae, genus Lagovirus and is used in Australia as a biocontrol tool to keep the population of european rabbits low. This virus has a positive-sense single-stranded RNA genome that encodes structural (capsid) and non-structural proteins. Due to the lack of an established cell culture system for this virus, some of the non-structural proteins are yet awaiting characterisation and their function is unknown. This work was aimed at the identification of cellular interactors of RHDV proteins p23 and RNA-dependent RNA polymerase (RdRp). p23 has an unknown function and RdRp is the main enzyme that replicates viral genome. SILAC-labeled rabbit kidney (RK13) cells were transfected with the FLAG-tagged viral proteins and GFP-transfected cells served as an internal control. For example, two replicates of heavy Arg and Lys labeled cells were transfected with protein p23 and two replicates of light Arg and Lys labeled cells were transfected with GFP. For the third replicate this was swapped: 'heavy' cells were transfected with GFP and 'light' cells were transfected with p23. After that, cells were harvested, lysed in mild conditions and applied onto anti-FLAG antibody resin for immunoprecipitation. Eluates from the resin were processed for mass spectrometry processing.
