ena-STUDY-CBMSO-04-08-2016-10:34:32:882-109
RNA-SEQ BASED TRANSCRIPTOME ANALYSIS OF THE INTERFERON HOST RESPONSE UPON VACCINIA VIRUS INFECTION.
Evasion of interferon (IFN)-mediated antiviral immunity is critical for a successful virus infection. So poxviruses have evolved diverse molecular strategies to counteract IFN host response activity at different levels. In the case of VACV, one of these is to encode a soluble IFN-a/ß binding protein (IFNBP) which located at cell surface binds type-I IFN molecules with high affinity, preventing their interaction with the host cell receptor. In the present work, we have dissected by RNA-Seq the viral modulation of the IFN-based host response at the transcriptional level. RNA from VACV-WR infected murine L929 cells was extracted at 0, 4 and 9 h post-infection in the absence or presence of IFN-alpha and/or recombinant purified IFNBP. To examine the immunomodulatory activity of the IFNBP, the transcriptome analysis from cells infected with a VACV-WR deletion mutant lacking the IFNBP was included. RNA libraries were prepared and paired-end sequencing performed using the Illumina HiSeq system. After removal of low quality reads, over 100M high quality reads per sample were obtained, which could be mapped either to the VACV or mus musculus strain C57/BL6 reference genomes. Then, analysis of differential gene expression and GO pathway enrichment analysis were performed to reveal the molecular mechanisms of action. We could validate the experiment identifying the expected transcriptional changes after IFN-induced signalling in the transcriptome from IFN-treated cells. The addition of recombinant IFNBP to cells prior to IFN completely reverted these IFN-induced changes to basal levels found in untreated cells. The addition of recombinant IFNBP to cell cultures did not result in significant activation of any cellular pathway, in spite of the IFNBP attaching to the cell surface. Finally, to detect and analyze those changes in host gene expression after viral infection of cultures, treated or not with IFN, analysis of differential gene expression and GO pathway enrichment analysis were performed and will be discussed.
Evasion of interferon (IFN)-mediated antiviral immunity is critical for a successful virus infection. So poxviruses have evolved diverse molecular strategies to counteract IFN host response activity at different levels. In the case of VACV, one of these is to encode a soluble IFN-a/ß binding protein (IFNBP) which located at cell surface binds type-I IFN molecules with high affinity, preventing their interaction with the host cell receptor. In the present work, we have dissected by RNA-Seq the viral modulation of the IFN-based host response at the transcriptional level. RNA from VACV-WR infected murine L929 cells was extracted at 0, 4 and 9 h post-infection in the absence or presence of IFN-alpha and/or recombinant purified IFNBP. To examine the immunomodulatory activity of the IFNBP, the transcriptome analysis from cells infected with a VACV-WR deletion mutant lacking the IFNBP was included. RNA libraries were prepared and paired-end sequencing performed using the Illumina HiSeq system. After removal of low quality reads, over 100M high quality reads per sample were obtained, which could be mapped either to the VACV or mus musculus strain C57/BL6 reference genomes. Then, analysis of differential gene expression and GO pathway enrichment analysis were performed to reveal the molecular mechanisms of action. We could validate the experiment identifying the expected transcriptional changes after IFN-induced signalling in the transcriptome from IFN-treated cells. The addition of recombinant IFNBP to cells prior to IFN completely reverted these IFN-induced changes to basal levels found in untreated cells. The addition of recombinant IFNBP to cell cultures did not result in significant activation of any cellular pathway, in spite of the IFNBP attaching to the cell surface. Finally, to detect and analyze those changes in host gene expression after viral infection of cultures, treated or not with IFN, analysis of differential gene expression and GO pathway enrichment analysis were performed and will be discussed.