Defining early anti-viral pathways that dictate a universal TLR2/6-based anti-viral response to conserved viral architectures.

PROJECT SUMMARY/ABSTRACT Throughout history, there have been emerging viral infections that have caused several epidemics and now we are facing the current global pandemic that poses a serious threat to human health and an immense burden on the economy. The emergence of SARS-CoV-2 has highlighted our need to further understand mechanisms of universal viral recognition and anti-viral response pathways to work toward hindering future outbreaks, such as G4 influenza viruses. The field of innate viral pattern recognition primarily focuses on innate recognition of viral nucleic acids, but some extracellular viral PRRs, such as C-type lectin receptors (CLRs) bind glycoproteins present in the viral envelope. Our recent discovery suggests there may be a more common pattern, namely the repeating protein subunit pattern (RPSP) that serves as a likely conserved mechanism of extracellular pathogen associated molecular pattern (PAMP) recognition for viruses. Recognition of RPSP is dependent on the cell- surface pattern recognition receptor (PRR) Toll-like receptor 2 (TLR2). Increased efforts into understanding how extracellular PRRs are involved in viral recognition have shed light on the role of extracellular viral recognition, but whether there are other common viral patterns recognized prior to viral particle internalization by innate immune cells remains to be fully explored. Our goal for this proposal is to address early roles of signaling components, including co-receptor CD14 and signaling molecules MyD88 and TRAM, in a unique TLR2/6 anti- viral mediated pathway in response to viral RPSP. Our preliminary and published data demonstrate a requirement for TRAM in RPSP internalization and both MyD88 and TRAM in inducing a protective anti-viral state post-TLR2/6-RPSP recognition. We therefore hypothesize that recognition of viral RPSP by TLR2/6 is facilitated by the co-receptor, CD14, resulting in unique anti-viral signaling from the endosome post-RPSP recognition involving crosstalk between MyD88 and TRAM leading to the production of type I IFNs. To address this hypothesis, I propose utilizing established approaches including bacterial clearance assays, cytokine analyses, microscopy, and chemical-crosslinking methods to address a potentially unique TLR signaling pathway involving interaction between MyD88 and TRAM that has not yet been defined for RPSP. Aim 1 will investigate how CD14 mediates the subcellular location of TLR2/6 signaling following RPSP recognition to determine if a CD14-mediated TLR2/6-RPSP response requires endocytosis for anti-viral signaling. Aim 2 will explore if MyD88 and TRAM interaction occurs post-RPSP recognition for anti-viral signaling. Aim 3 will identify how CD14, MyD88 and TRAM signaling mediates the anti-viral response post-RPSP recognition by addressing type I IFN and proinflammatory signaling pathways. Collectively, this data could provide valuable insight into mechanisms of common virus pattern recognition and potentially unique signaling responses involving crosstalk between MyD88 and TRAM to mediate a protective anti-viral state. Thus, determining how viruses can elicit anti-viral responses in immune cells prior to infection is critical for development of novel treatments spanning multiple viruses.