Analysis of RNA synthesis in MCMV infected Wild type and Tyk2-/- bone marrow-derived macrophages 2 macrophage interferon antiviral cascade

Murine Cytomegalovirus infection of macrophages results in hundreds of alterations in cellular gene expression. Many of these alterations are due to the autocrine and paracrine effects of virus-elicited type I interferons released by infected cells. Key to the function of type I interferons is the JAK-STAT signalling pathway. The extracellular binding of Type I IFN to the type 1 interferon receptor (consisting of IFNAR1 and IFNAR2) results in cytoplasmic activation of Tyrosine Kinase 2 (TYK2) and Janus Kinase 1 (JAK1) and the subsequent phosphorylation of STAT1 and STAT2. Phosphorylated STAT1 and 2 bind IRF9 forming a complex known as ISGF3. ISGF3 is translocated to the nucleus where it directly interacts with Interferon-sensitive regulatory elements in the genomic DNA. This binding results in direct and indirect alterations in the transcription of hundreds of genes. Whilst the functions of Tyk2 and changes in transcript abundance following MCMV infection are well characterised, little is known about changes in RNA synthesis at very early times after infection in intact cells or cells with a defective IFN signalling pathway. Microarrays were used to analyse alterations in transcript synthesis 30 to 60 mins and 360 to 390 minutes after MCMV infection of Wild Type or Tyk2-/- BMDM. Wild-type (WT) or Tyk2-/- Bone Marrow derived macrophages (BMDM) were grown using L929 conditioned medium. On day 7, BMDM were mock-infected (2x 15cm dishes WT BMDM and 2x 15cm dishes Tyk2-/- BMDM) or infected with MCMV (MOI = 1, 2x 15cm dishes WT BMDM and 2x 15cm dishes Tyk2-/- BMDM) for 1h. After 1h, virus and medium were aspirated and fresh medium added to all dishes – designated time=0. After 30 or 360 minutes post infection, 4-thiouridine (4sU) was used to label newly transcribed RNA in 4 dishes of mock or infected WT and Tyk2-/- BMDM (Mock WT, MCMV WT, Mock Tyk2-/-, MCMV Tyk2-/-) for a period of 30 minutes. Newly transcribed RNA was then isolated from each total RNA sample using a protocol described in Dolken et al (PMID: 18658122). For analysis, all 8 newly transcribed RNA samples were hybridized to Affymetrix GeneST v1.0 arrays.