Investigating the physical interaction between the influenza A virus endoribonuclease PA-X and its target RNAs
Influenza A virus (IAV) results in about 500,000 annual deaths globally. The host immune response to IAV can cause fatal lung damage. The IAV-encoded endoribonuclease PA-X contributes to controlling the host immune response. In model organisms, PA-X-deficient strains elicit a stronger immune response than wild-type strains and are typically more lethal. Recent work from the Gaglia lab has shown that PA-X specifically degrades spliced host mRNA through an unknown mechanism. Evidence also suggests PA-X interacts with host mRNA processing proteins, and is physically brought to spliced mRNAs for degradation. Based on this model, I hypothesize that the physical interaction between PA-X and a mRNA is sufficient for cleavage. I artificially tethered PA-X to mRNA by fusing PA-X to a bacteriophage λN protein and the mRNA of interest (spliced or intronless IFN-λ2 targeted reporter) to the λN target sequence BoxB. I expressed these components in HEK293T cells and quantified PA-X-mediated down-regulation of mRNA by RT-qPCR. I found that λN had no impact on normal PA-X function and therefore did not distort the data. However, the insertion of BoxB in exon 2 of the spliced IFN-λ2 mRNA reporter blocked reporter down-regulation by PA-X. A new location for BoxB insertion must be investigated before any further testing takes place, along with replication of the experiments for statistical analysis. Ultimately, understanding how PA-X degrades mRNA will allow us to better understand how influenza proteins can modulate the host immune response and what this means for the outcome of a viral infection.
Richards, Rachel, "Investigating the physical interaction between the influenza A virus endoribonuclease PA-X and its target RNAs" (2019). Undergraduate Research and Creative Inquiry Symposia. 58.