CG dinucleotide-mediated immune responses during West Nile virus infection

ABSTRACT Viruses contain genetic material in DNA or RNA composed of nucleotides. For RNA, these nucleotides are A (adenine), C (cytosine), G (guanine), and U (uracil). Two nucleotides linked by a phosphate molecule, such as UA or CG, are called dinucleotides. Many RNA viruses, including flaviviruses, have evolved to mimic the low cytosine-phosphate-guanine (CG) dinucleotide content of vertebrate genomes to evade recognition by host zinc finger antiviral protein 1 (ZAP), which binds CG-rich or CG-enriched non-self RNA and targets it for degradation. Many viruses have also evolved reduced uracil-phosphate-adenine (UA) dinucleotide content to evade RNA- degrading enzymes and possibly ZAP. Enrichment of CG and UA dinucleotides in viral RNA is an emerging promising vaccine approach. In addition to vaccines, dinucleotide enrichment is an emerging approach for oncolytic viruses. A critical knowledge gap for the further rational development of safe and protective vaccines and effective oncolytic viruses is the lack of understanding of innate cellular immune responses to viruses carrying CG/UA-enriched RNA. Thus, in this project, we will study how peripheral and lymphoid dendritic cells (DCs) interact with CG-, UA-, and CG/UA-enriched viruses. Understanding interactions with DCs is significant because they are essential for initiating the early activation of inflammatory monocytes and adaptive T cell responses. Moreover, many viruses, such as flaviviruses like West Nile virus (WNV), have evolved to impair DC activation and DC-mediated stimulation of T cell antiviral responses. We will leverage characterized CG-, UA-, and CG/UA-enriched WNV variants, as well as the WNV-human peripheral DC-T cell interaction model. We will also use the conventional and ZAP knockout C57BL/6J mouse models to study interactions with lymphoid DCs. Unlike many human viruses, WNV is virulent in conventional C57BL/6J mice, which facilitates immune response studies. Classical virology and flow cytometry assays, bulk RNA-seq, and state-of-the-art single-cell RNA-seq will be used in the project. We will provide the first insights into how CG-, UA-, and CG/UA dinucleotide-enriched viruses interact with essential DC populations, and whether dinucleotide enrichment can overcome DC impairment and T cell dysfunction induced by wild-type virus infection. This project will advance the rational development of enriched vaccines and oncolytic viruses that selectively activate dinucleotide-specific beneficial immune pathways.