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Background
Herpesviruses can infect a wide range of animal species. Herpes simplex virus 1 (HSV-1) is one of the eight herpesviruses that can infect humans and is prevalent worldwide. Herpesviruses have evolved multiple ways to adapt the infected cells to their needs, but knowledge about these transcriptional and post-transcriptional modifications is sparse.
Results
Here, we show that HSV-1 induces the expression of about 1000 antisense transcripts from the human host cell genome. A subset of these is also activated by the closely related varicella zoster virus. Antisense transcripts originate either at gene promoters or within the gene body, and they show different susceptibility to the inhibition of early and immediate early viral gene expression. Overexpression of the major viral transcription factor ICP4 is sufficient to turn on a subset of antisense transcripts. Histone marks around transcription start sites of HSV-1-induced and constitutively transcribed antisense transcripts are highly similar, indicating that the genetic loci are already poised to transcribe these novel RNAs. Furthermore, an antisense transcript overlapping with the BBC3 gene (also known as PUMA) transcriptionally silences this potent inducer of apoptosis in cis.
Conclusions
We show for the first time that a virus induces widespread antisense transcription of the host cell genome. We provide evidence that HSV-1 uses this to downregulate a strong inducer of apoptosis. Our findings open new perspectives on global and specific alterations of host cell transcription by viruses.
Visualization of type I immunity using bicistronic IFN-gamma reporter mice in vitro and in vivo
(2006)
IFN-γ is the signature cytokine of Th1 and CD8+ effector cells generated in type I immune responses against pathogens, such as Influenza virus, Sendai virus and the intracellular protozoan parasite Toxoplasma gondii. Understanding the regulation of IFN-γ is critical for the manipulation of immune responses, prevention of immunopathology and for vaccine design. In the present thesis, IFN-γ expression by CD4+ and CD8+ T cells was characterized in detail and the requirement of IFN-γ receptor mediated functions for IFN-γ expression was assessed. Bicistronic IFN-γ-eYFP reporter mice, which allow direct identification and isolation of live IFN-γ expressing cells, were used to visualize IFN-γ expression in vitro and in vivo after infection with the afore mentioned pathogens. Expression of the IFN-γ-eYFP reporter by CD4+ and CD8+ T cells was broadly heterogeneous in vitro and in vivo after infection. Increased expression of the reporter correlated positively with the abundance of IFN-γ transcripts and IFN-γ protein production upon stimulation. eYFP reporter brightness reflected the potential for IFN-γ production, but actual secretion was largely dependent on antigenic stimulation. Increased expression of the reporter also correlated with enhanced secretion of additional proinflammatory cytokines and chemokines and cell surface expression of markers that indicate recent activation. Highly eYFP fluorescent cells were generally more differentiated and their anatomical distribution was restricted to certain tissues. The anatomical restriction depended on the pathogen. IFN-γ expressing CD4+ and CD8+ T cells were generated in IFN-γ receptor deficient reporter mice after infection with Sendai virus or Toxoplasma gondii. However, in the absence of IFN-γ receptor mediated functions, the frequency and brightness of the eYFP reporter expression was altered. Dual BM chimeric mice, reconstituted with wild-type and IFN-γ receptor deficient reporter BM, revealed a T cell-intrinsic requirement for the IFN-γ receptor for optimal IFN-γ expression. Reporter fluorescence intensities were regulated independently of IFN-γ receptor mediated functions. Finally, we propose a model for IFN-γ expression by CD4+ and CD8+ T cells. 2. SUMMARY 10 In summary, the expression of IFN-γ is differentially regulated in CD4+ and CD8+ T cells and after viral or protozoan infections. Additionally, the role of IFN-γ receptor mediated functions for the expression of IFN-γ was determined.