The type I interferon (IFN) response is a critical early response in host defense against viral infection that leads to the transcriptional induction of the IFN-? promoter. Many viruses encode activities that disrupt this response thus aiding in the establishment of infection and disease progression. Poliovirus is a positive stranded RNA virus that belongs to a large family of related viruses that includes both animal and human pathogens. Poliovirus infection inhibits the ability of cells to respond to exogenously added dsRNA, a strong inducer of the IFN-? promoter, indicating that poliovirus infection suppresses the Type I IFN response. Recent evidence has raised the possibility that components of the nuclear pore complex (NPC) could have roles in this anti-viral response. The NPC is a large structure found embedded in the nuclear envelope that is composed of approximately 30 different proteins called nucleoporins (Nups). One component of the NPC, Nup98, has roles in regulating transcriptional induction of developmental, cell cycle and heat shock genes in Drosophila. Furthermore, Nup98 is induced in response to type I IFN suggesting that it may have a role in host defense mechanisms. Consistent with this possibility multiple viruses interfere with Nup98 function during infection. An example of this is in poliovirus-infected cells where Nup98 is proteolyzed very rapidly following the onset of infection. Currently it is not known if Nup98 has a role in the type I IFN response or if proteolysis of Nup98 during poliovirus infection contributes to evasion of the host immune response and establishment of productive infection. The central hypothesis of this proposal is that Nup98 contributes to efficient activation of the type I IFN response. This hypothesis will be tested by examining the effect of Nup98 depletion on the type I IFN response along with the effect of preventing Nup98 cleavage in poliovirus-infected cells.
Aim 1 will determine if Nup98 is required for efficient induction of the type I interferon response This will be accomplished by knocking down expression of Nup98 using small interfering RNAs and examining the effect this has on transcriptional induction of the type I IFN response. Subsequent experiments using chromatin immunoprecipitation will determine if Nup98 associates with DNA elements of transcriptionally induced genes.
Aim 2 will determine if an RNA virus targets Nup98 to disrupt the type I interferon response. Stable cell lines expressing mutant forms of Nup98 that are resistant to proteolysis by poliovirus will be generated. Cell lines will then be infected with poliovirus and tested for their ability to mount a type I IFN response. These experiments will determine if Nup98 has a role in transcriptional induction of the type I IFN response and if a pathogenic human virus evades this response by targeting Nup98 for degradation. The results of these studies will yield a better understanding of the molecular mechanisms underlying induction of type I IFN and the viral strategies for its subversion.
The synthesis of type I interferons plays a critical role in defending humans against viral pathogens. Experiments are proposed to identify novel mechanisms regulating the production of type I interferon and the processes by which viruses interfere with their synthesis. The results of these studies may identify novel targets for the development of therapeutics that modulate the Type I interferon response and reduce disease associated with virus infection.