ABS 107: Virus-derived circRNAs from SARS-CoV-2 NSP13

Gabriella Meshesha ¹ ² ³ , Elysse N. Grossi-Soyster ³ , Peter Sarnow ³

¹ Stanford Summer Research Program
² Department of Biology, Johns Hopkins University
³ Department of Microbiology and Immunology, Stanford University

Van Wickle (2025) Volume 1, ABS 1017

Introduction: Circular RNA molecules (circRNAs) are a looped form of selected exons and introns from linear RNA produced in humans and viruses. SARS-CoV-2, the (+)ssRNA coronavirus which causes COVID, is predicted to generate a multitude of viral circRNAs (VcircRNAs) located within several genes responsible for encoding different proteins important to the viral lifecycle. The junction site of each circle is the unique sequence joining the circRNA’s 5’ and 3’ ends, which is not found in linear RNA, thus its detection is an important step in confirming its circular form. VcircRNAs may serve a role in the SARS-CoV-2 pathogenesis, therefore the existence and function of specific circRNAs must be confirmed. The NSP13 gene is involved in viral replication, encoding for the helicase protein necessary in unwinding dsRNA. We hypothesize that the NSP13 gene, found within ORF1b of SARS-CoV-2, generates two VcircRNAs.

Methods: To investigate these two distinct VcircRNAs from NSP13, RNA was extracted from human-derived A549-ACE2 epithelial lung cells infected with the SARS-CoV-2 WA.1 strain. Rolling circle amplification, PCR, gel electrophoresis, cloning, and DNA sequencing technologies were utilized to confirm the computationally predicted NSP13 VcircRNAs.

Results: Detection of the VcircRNA1's junction site suggests the presence of VcircRNAs in SARS-CoV-2 infected cells, but further investigation is necessary to confirm if VcircRNA1 and 2 have the potential to regulate NSP13 expression and viral replication.

Discussion: Continued interrogation of the expression of NSP13 VcircRNAs is required to reveal the functions of SARS-CoV-2 VcircRNAs and the possible introduction of more effective, efficient diagnostics, treatments, and preventative interventions in clinical settings for COVID-19 patients.