ORCID
https://orcid.org/0000-0002-3998-476X
Date of Award
Spring 2026
Language
English
Embargo Period
5-7-2028
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
College/School/Department
Department of Biological Sciences
Program
Biology
First Advisor
Gabriele Fuchs
Committee Members
Cara T. Pager, Thomas Begley, Alan Chen
Keywords
Translation, Circular RNA, Internal Ribosome Entry Site (IRES), Shunting, SARS-CoV-2 5' UTR
Subject Categories
Molecular Biology
Abstract
The ongoing pandemic caused by the SARS-CoV-2 coronavirus has sparked researchers' interest in understanding viral replication and translation. Structural analysis showed that the SARS-CoV-2 5′ UTR is highly structured, similar to other well-known Internal Ribosomal Entry Sites (IRESs). Based on analysis of the SARS-CoV-2 5′ UTR, Miao et al. (2021) proposed that translation of the positive-sense single stranded RNA Coronavirus open reading frame (ORF) 1a may be mediated by an unconventional translation initiation mechanism rather than exclusive canonical translation initiation. Following translation of SARS-CoV-2 ORF1a, canonical cap-dependent translation of cellular mRNAs is shut down by the viral NSP1 protein through binding to the ribosome channel in the 40S ribosomal subunit. However, translation of certain IRES-containing mRNAs and the SARS-CoV-2 genomic RNA continues as the SL1 in the SARS-CoV-2 5ʹ UTR alleviates the ribosome blockage by NSP1. In addition, the genomic RNAs of coronaviruses have a conserved upstream open-reading frame (uORF) with unknown function .
We hypothesized that SARS-CoV-2 5′ UTR is able to use a non-canonical mode of translation initiation. To test for the presence of an IRES, we inserted the SARS-CoV-2 5′ UTR sequence into a dual luciferase reporter containing a Renilla luciferase (Rluc) and Firefly luciferase (Fluc) reporters. After transfection on HeLa cells, luciferase activity was measured. We have data showing translation mediated by the SARS-CoV-2 5′ UTR in a dual-luciferase construct, suggesting that the SARS-CoV-2 5′ UTR contains weak IRES activity. The IRES activity of the SARS-CoV-2 5′ UTR was further validated using control experiments, including a promoterless plasmid and a circular RNA reporter. Moreover, mutational disruption of the SARS-CoV-2 5′ UTR RNA structure disrupted luciferase translation. Through mutagenesis, we identified that RNA stem-loops (SLs) 2, 3, and 5 within the 5′ UTR are critical for non-canonical translation. Additionally, deletion of SL4 to SL5abc, containing an annotated uORF in SLs 4 and 4.5, resulted in an engineered IRES sequence with enhanced cap-independent translation but strongly reduced canonical translation.
Previous studies identified an increased number of ribosome footprints that map to a CUG start codon located at positions 59–61 rather than the downstream AUG start codons of the annotated uORF and the main ORF. Curiously, mutation of this CUG start codon to either AUG or CCG reduced cap-dependent and -independent translation, suggesting that the CUG unlikely functions as a traditional uORF but that it serves a critical function in translation initiation. To test the efficiency of translation initiation from the CUG uORF, we inserted a HiBiT protein tag downstream and in-frame with CUG 59–61. Surprisingly, we only measured very low nanoluciferase units, suggesting that the CUG uORF is inefficiently translated. Deletion of SL4, the start codon of the annotated uORF, and SL4.5, which contains four stop codons in three different reading frames, slightly increased translation. Together, this data suggests that cap-dependently translating ribosomes use ribosome shunting and skip the CUG uORF to reach the main ORF AUG start codon. Overall, this study implies that the SARS-CoV-2 5′ UTR can utilize multiple modes of translation initiation, namely, noncanonical cap-independent translation via an IRES and cap-dependent ribosome recruitment followed by ribosome shunting.
License
This work is licensed under the University at Albany Standard Author Agreement.
Recommended Citation
Lin, Shu Jun, "Noncanonical Translation Initiation of SARS-CoV-2 5ʹ UTR" (2026). Electronic Theses & Dissertations (2024 - present). 475.
https://scholarsarchive.library.albany.edu/etd/475