Abstract

Copyback viral genomes (cbVGs) are truncated non-protein coding viral genomes produced when an RNA virus polymerase detaches from the replication template and resumes elongation downstream, copying back a sequence complementary to the nascent strand. cbVGs can stimulate antiviral immunity and compete for viral proteins to suppress virus replication, but technical limitations of PCR-based approaches have painted an incomplete picture of the diverse cbVG populations generated during infection. Here, we sought to improve our understanding of cbVG molecular structure by harnessing long-read RNA sequencing tools. To do this, we developed a robust and reliable pipeline to sequence and analyze native full-length cbVGs from minimal RNA input without RT-PCR–introduced errors. We applied this approach to a recombinant Sendai virus stock, uncovering cbVGs with break positions occurring at or near position one in the reference genome. Sequencing cbVG RNA directly validated predictions from short-read next-generation sequencing and supported prior RT-PCR results, without introducing RT- or PCR-associated artifacts. The optimized platform was then applied to Respiratory Syncytial Virus (RSV), a major cause of severe respiratory disease in which cbVGs are associated with distinct clinical outcomes. Long-read sequencing of hundreds of RSV cbVG species from virus stocks and infected cells revealed unexpected heterogeneity at their 3′ termini. Specifically, we identified 3′ terminal extensions consistent with polymerase-mediated back-priming, an activity previously described for standard viral genomes but not for cbVG templates. Notably, many cbVGs exhibited extensions that were longer and more complex than those reported for full-length genomes, and they were generated only in the presence of viral polymerase. Collectively, this work reveals new principles of cbVG molecular biology by elucidating underappreciated structural and functional diversity of cbVGs, with implications for cbVG replication, stability, encapsidation, and interactions with both viral and host factors.

Committee Chair

Carolina López

Committee Members

Kristine Wylie; Megan Baldridge; Sean Whelan; Tamara Doering

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

3-11-2026

Language

English (en)

Author's ORCID

https://orcid.org/0000-0001-7826-3503

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Available for download on Thursday, September 10, 2026

Included in

Virology Commons

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