LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells

Liguo Zhang, Punam Bisht, Anthony Flamier, M. Inmaculada Barrasa, Max Friesen, Alexsia Richards, Stephen H. Hughes, Rudolf Jaenisch

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

SARS-CoV-2 sequences can be reverse-transcribed and integrated into the genomes of virus-infected cells by a LINE1-mediated retrotransposition mechanism. Whole-genome sequencing (WGS) methods detected retrotransposed SARS-CoV-2 subgenomic sequences in virus-infected cells overexpressing LINE1, while an enrichment method (TagMap) identified retrotranspositions in cells that did not overexpress LINE1. LINE1 overexpression increased retrotranspositions about 1000-fold as compared to non-overexpressing cells. Nanopore WGS can directly recover retrotransposed viral and flanking host sequences, but its sensitivity depends on the depth of sequencing (a typical 20-fold sequencing depth would only examine 10 diploid cell equivalents). In contrast, TagMap enriches the host–virus junctions and can interrogate up to 20,000 cells and is able to detect rare viral retrotranspositions in LINE1 non-overexpressing cells. Although Nanopore WGS is 10–20-fold more sensitive per tested cell, TagMap can interrogate 1000–2000-fold more cells and, therefore, can identify infrequent retrotranspositions. When comparing SARS-CoV-2 infection and viral nucleocapsid mRNA transfection by TagMap, retrotransposed SARS-CoV-2 sequences were only detected in infected but not in transfected cells. Retrotransposition in virus-infected cells, in contrast to transfected cells, may be facilitated because virus infection, in contrast to viral RNA transfection, results in significantly higher viral RNA levels and stimulates LINE1 expression by causing cellular stress.

Original languageEnglish
Article number629
JournalViruses
Volume15
Issue number3
DOIs
StatePublished - 25 Feb 2023
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2023 by the authors.

Funding

This work was supported in part by grants from the National Institutes of Health to R.J. [grant numbers 5U19-AI3115135, 5RO1MH104610] and by a generous unrestricted gift from Dewpoint Therapeutics and from Jim Stone. This work was supported in part by funding from the Intramural Research Program, National Institutes of Health, National Cancer Institute, Center for Cancer Research. This work was performed in part in the Ragon Institute BSL3 core, which is supported by the NIH-funded Harvard University Center for AIDS Research (P30 AI060354).

FundersFunder number
National Institutes of Health5U19-AI3115135, 5RO1MH104610
National Cancer Institute
Harvard University Center for AIDS ResearchP30 AI060354

    Keywords

    • LINE1
    • RNA transfection
    • SARS-CoV-2
    • WGS
    • enrichment sequencing
    • retrotransposition

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