VCOM Louisiana Research Day Program

Biomedical Research

Ariana E. Faraji, OMS-III; Kasia Michalak, MS; Lin Kang, PhD; Stephen DiGiuseppe, PhD; Pawel Michalak, PhD Edward Via College of Osteopathic Medicine, Monroe, Louisiana 03 INVESTIGATING THE ROLE OF INTERNALLY TRANSCRIBED SPACERS OF RIBOSOMAL RNA IN VIRAL INFECTIONS

Context: Ribosome biogenesis is the process of making ribosomes, which involves generating and processing of ribosomal RNAs (rRNAs). The 18S, 5.8S, and 28S rRNAs are transcribed into a single precursor, where each rRNA is separated by non-coding spacer regions. Between 18S and the 5.8S rRNA is Internal Transcribed Spacer (ITS) 1 and between 5.8S and 28S rRNA is ITS2. The spacers are released by endonuclease activity with average fragments sizes ranging from 150 – 250 for ITS2 detected in eukaryotic cells. Despite this, investigating biological roles of ITS fragments have been mostly ignored because they are presumed to be degraded. Interestingly, we recently discovered that eukaryotic 45S rRNA gene clusters have several striking Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-like features, such as palindromic organization of highly conserved sequences across species (18S, 5.8S, and 28S rDNA). In addition, we uncovered significant sequence homology within ITSs to a variety of viral genomes including the Alphaherpesvirus, herpes simplex virus 1 (HSV-1). It was recently reported that ribosome biogenesis restricts innate immune response to infection of a Betaherpesvirus, human cytomegalovirus (HCMV). Based on these recent findings, we hypothesize that ITS fragments

serve a hitherto unappreciated role in regulation of the innate immune response in the context of viral infections. Objective: To evaluate biological functions of ITSs in eukaryotic cells and their immune responses to viral infections. Methods: The ITS1 and ITS2 regions were sequenced using Sanger and Nanopore sequencing. We generated siRNA sequences targeting regions found across ITS1 and ITS2 sequences and a scramble control. We transfected siRNAs into normal dermal human fibroblasts (NHDFs). Next, NHDFs were infected at a low multiplicity of infection (MOI) with viruses from different viral families including KOS strain HSV-1, WR strain of vaccinia virus (VacV), and Indiana strain of vesicular stomatitis virus (VSV). Viruses were titered on BSC40 cells and viral proteins were detected via Western blot analysis from total protein lysates post infection. Results: Knockdown of ITS2 exhibited differential increases of titers for each virus tested when compared to scramble control siRNA (n = 3). We also detected differential increases in viral protein levels that agree with our viral titers.

Conclusion: Our preliminary data suggests that ITS2 fragments may serve a previously unappreciated role in restricting viral replication. Interestingly, not all ITS2 fragments demonstrated the same levels of restriction implying that there may be viral specificity. Whether that restriction is due to innate immune impairment is still unknown, but we plan to investigate the role of innate immune pathways after depleting ITS fragments in our future studies. Eukaryotic ITSs contain fragments of virus-like sequences that may play a role in restricting viral replication, which may provide insight into a new paradigm for antiviral innate immunity.

15 2023 Via Research Recognition Day