LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells
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...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-02-01
|
| Series: | Viruses |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1999-4915/15/3/629 |
| _version_ | 1797608614219743232 |
|---|---|
| author | Liguo Zhang Punam Bisht Anthony Flamier M. Inmaculada Barrasa Max Friesen Alexsia Richards Stephen H. Hughes Rudolf Jaenisch |
| author_facet | Liguo Zhang Punam Bisht Anthony Flamier M. Inmaculada Barrasa Max Friesen Alexsia Richards Stephen H. Hughes Rudolf Jaenisch |
| author_sort | Liguo Zhang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-11T05:47:01Z |
| format | Article |
| id | doaj.art-52c8cc1cbfb84c2dafca1dbb29c0f082 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-11T05:47:01Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-52c8cc1cbfb84c2dafca1dbb29c0f0822023-11-17T14:22:07ZengMDPI AGViruses1999-49152023-02-0115362910.3390/v15030629LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected CellsLiguo Zhang0Punam Bisht1Anthony Flamier2M. Inmaculada Barrasa3Max Friesen4Alexsia Richards5Stephen H. Hughes6Rudolf Jaenisch7Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USAWhitehead Institute for Biomedical Research, Cambridge, MA 02142, USAWhitehead Institute for Biomedical Research, Cambridge, MA 02142, USAWhitehead Institute for Biomedical Research, Cambridge, MA 02142, USAWhitehead Institute for Biomedical Research, Cambridge, MA 02142, USAWhitehead Institute for Biomedical Research, Cambridge, MA 02142, USAHIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USAWhitehead Institute for Biomedical Research, Cambridge, MA 02142, USASARS-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.https://www.mdpi.com/1999-4915/15/3/629SARS-CoV-2LINE1retrotranspositionWGSenrichment sequencingRNA transfection |
| spellingShingle | Liguo Zhang Punam Bisht Anthony Flamier M. Inmaculada Barrasa Max Friesen Alexsia Richards Stephen H. Hughes Rudolf Jaenisch LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells Viruses SARS-CoV-2 LINE1 retrotransposition WGS enrichment sequencing RNA transfection |
| title | LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells |
| title_full | LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells |
| title_fullStr | LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells |
| title_full_unstemmed | LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells |
| title_short | LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells |
| title_sort | line1 mediated reverse transcription and genomic integration of sars cov 2 mrna detected in virus infected but not in viral mrna transfected cells |
| topic | SARS-CoV-2 LINE1 retrotransposition WGS enrichment sequencing RNA transfection |
| url | https://www.mdpi.com/1999-4915/15/3/629 |
| work_keys_str_mv | AT liguozhang line1mediatedreversetranscriptionandgenomicintegrationofsarscov2mrnadetectedinvirusinfectedbutnotinviralmrnatransfectedcells AT punambisht line1mediatedreversetranscriptionandgenomicintegrationofsarscov2mrnadetectedinvirusinfectedbutnotinviralmrnatransfectedcells AT anthonyflamier line1mediatedreversetranscriptionandgenomicintegrationofsarscov2mrnadetectedinvirusinfectedbutnotinviralmrnatransfectedcells AT minmaculadabarrasa line1mediatedreversetranscriptionandgenomicintegrationofsarscov2mrnadetectedinvirusinfectedbutnotinviralmrnatransfectedcells AT maxfriesen line1mediatedreversetranscriptionandgenomicintegrationofsarscov2mrnadetectedinvirusinfectedbutnotinviralmrnatransfectedcells AT alexsiarichards line1mediatedreversetranscriptionandgenomicintegrationofsarscov2mrnadetectedinvirusinfectedbutnotinviralmrnatransfectedcells AT stephenhhughes line1mediatedreversetranscriptionandgenomicintegrationofsarscov2mrnadetectedinvirusinfectedbutnotinviralmrnatransfectedcells AT rudolfjaenisch line1mediatedreversetranscriptionandgenomicintegrationofsarscov2mrnadetectedinvirusinfectedbutnotinviralmrnatransfectedcells |