Project description:Transcription profiling of mouse CD4+ and CD8+ T cells extracted from GFP-Egr2 knockin (Egr2 Kin) and hCD2-Cre / Egr2loxP/loxP / Egr3-/- Egr2/3 DKO) mice 7 days after infection with vaccinia virus.
Project description:Viral infection both activates stress signaling pathways and redistributes ribosomes away from host mRNAs to translate viral mRNAs. The intricacies of this ribosome shuffle from host to viral mRNAs are poorly understood Here, we uncover a role for the ribosome associated quality control (RQC) factor, ZNF598, during vaccinia virus mRNA translation. ZNF598 acts on collided ribosomes to ubiquitylate 40S subunit proteins uS10 and eS10 initiating RQC-dependent nascent chain degradation and ribosome recycling We show that vaccinia infection in human cells enhances uS10 ubiquitylation indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells which either lack ZNF598 or express a ubiquitylation deficient version of uS10 Using SILAC-based proteomics and concurrent RNAseq analysis, we determine that translation and not transcription of vaccinia virus mRNAs is compromised in cells with deficient RQC activity. Additionally, vaccinia virus infection reduces cellular RQC activity, suggesting that co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.
Project description:Neutrophils are innate immune cells involved in the elimination of pathogens. Recent studies showed that neutrophils can also act as antigen presenting cells (APC) to induce adaptive immune responses. Specific neutrophil populations (Na and Nb) with different levels of APC markers and in vitro distinct capacity to generate vaccinia virus (VACV)-specific CD8 T cell responses have been shown. However, how these neutrophil subtypes exert their role in vivo and how manipulation of Nb/Na ratio can influence vaccine-mediated immune responses are not known. In the context of VACV infection, we characterized HIV-specific CD8 T cell responses, delineated neutrophil transcriptomic profiles and described neutrophil dynamic behaviors in the spleen. By splenic multiphoton intravital microscopy (MV-IVM), we could distinguish both neutrophil subtypes and found that they showed distinct migratory and motility patterns and different ability to interact with CD8 T cells and move towards them. After analysis of adhesion, inflammatory and migration receptors from peritoneal cavity, blood and spleen, we defined that Nb neutrophils overexpressed the trans-endothelial migration marker a4b1integrin compared to Na subtype. Finally, by inhibiting a4b1integrin, we increased the Nb/Na ratio in peritoneal cavity and spleen, and in turn enhanced CD8 T cell responses to HIV-antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system. These results are relevant in vaccine vector design that could induce distinct neutrophil subtypes and, in consequence modulate antigen-specific T cell responses.
Project description:Comparsion of the transcriptomes of naive, resting memory and activated memory CD4+ T lymphocytes. Peripheral blood lymphocytes were collected at baseline and day 13 post-inoculation of a healthy subject administered with vaccinia virus. Cells were isolated by FACs using antibodies against CD4, CD45RO and CD38.
Project description:Ribosomes are highly abundant cellular machines that perform the essential task of translating the genetic code into proteins. Cellular translation activity is finely tuned and proteostasis insults, such as those incurred upon viral infection, activate stress signaling pathways that result in translation reprogramming. Viral infection selectively shuts down host mRNA while redistributing ribosomes for selective translation of viral mRNAs. The intricacies of this selective ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome associated quality control (RQC) factor ZNF598, a sensor for collided ribosomes, as a critical factor for vaccinia virus mRNA translation. Collided ribosomes are sensed by ZNF598, which ubiquitylates 40S subunit proteins uS10 and eS10 and thereby initiates RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection in human cells enhances uS10 ubiquitylation indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells which either lack ZNF598 or contain a ubiquitylation deficient version of uS10. Using SILAC-based proteomics and concurrent RNAseq analysis, we determine that host translation of vaccinia virus mRNAs is compromised in cells that lack RQC activity as compared to control cells whereas there was little evidence of differences in host or viral transcription. Additionally, vaccinia virus infection resulted in a loss of cellular RQC activity, suggesting that ribosomes engaged in viral protein production recruit ZNF598 away from its function in host translation. Thus, co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.
Project description:RNA-seq analysis identified differentially regulated genes in purified NK cells isolated from naïve mice or those vaccinated with vaccinia virus. DEGs were compared with