SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome

  1. Liguo Zhang
  2. Alexsia Richards
  3. Andrew Khalil
  4. Emile Wogram
  5. Haiting Ma
  6. Richard A. Young
  7. Rudolf Jaenisch

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Abstract

Prolonged SARS-CoV-2 RNA shedding and recurrence of PCR-positive tests have been widely reported in patients after recovery, yet these patients most commonly are non-infectious 1–14 . Here we investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the human genome and that transcription of the integrated sequences might account for PCR-positive tests. In support of this hypothesis, we found chimeric transcripts consisting of viral fused to cellular sequences in published data sets of SARS-CoV-2 infected cultured cells and primary cells of patients, consistent with the transcription of viral sequences integrated into the genome. To experimentally corroborate the possibility of viral retro-integration, we describe evidence that SARS-CoV-2 RNAs can be reverse transcribed in human cells by reverse transcriptase (RT) from LINE-1 elements or by HIV-1 RT, and that these DNA sequences can be integrated into the cell genome and subsequently be transcribed. Human endogenous LINE-1 expression was induced upon SARS-CoV-2 infection or by cytokine exposure in cultured cells, suggesting a molecular mechanism for SARS-CoV-2 retro-integration in patients. This novel feature of SARS-CoV-2 infection may explain why patients can continue to produce viral RNA after recovery and suggests a new aspect of RNA virus replication.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.12.422516: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were permeabilized with 0.5% (v/v) Triton X-100/PBS, blocked with 4% (w/v) BSA/CMF-PBS at RT for 1 hr, incubated with 1:200 diluted anti-LINE-1 ORF1p mouse monoclonal antibody (clone 4H1, Sigma MABC1152, Lot 3493991), and then with 1:400 diluted Donkey-anti-Mouse-Alexa Fluor 594 second antibody (Invitrogen 21203)
    anti-LINE-1
    suggested: (LSBio (LifeSpan Cat# LS-C130455-100, RRID:AB_10847571)
    Experimental Models: Cell Lines
    SentencesResources
    Calu3 cells were obtained from ATCC (HTB-55) and cultured in EMEM (ATCC 30-2003) supplemented with 10% heat-inactivated FBS (Hyclone, SH30396.03) following ATCC’s method.
    Calu3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    SARS-CoV-2 infection: SARS-CoV-2 USA-WA1/2020 (Gen Bank: MN985325.1) was obtained from BEI Resources and expanded and tittered on Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    For microglia stimulation, microglia differentiation media was exchanged with HEK293T media (DMEM + 10% heat-inactivated FBS + final 2mM L-Glutamine) and supplemented with 100 hg/ml lipopolysaccharide (LPS, Sigma Aldrich L4391-1MG) or PBS.
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmid for HIV-1 reverse transcriptase expression: pCMV-dR8.2 dvpr was a gift from Bob Weinberg (Addgene plasmid # 8455; http://n2t.net/addgene:8455; RRID:Addgene_8455)41
    detected: RRID:Addgene_8455)
    Addgene plasmid # 51288; http://n2t.net/addgene:51288; RRID:Addgene_51288)42; EF06R (5’UTR-LINE-1) was a gift from Eline Luning Prak (Addgene plasmid # 42940; http://n2t.net/addgene:42940; RRID:Addgene_42940)43.
    detected: RRID:Addgene_51288)
    detected: RRID:Addgene_42940)
    Software and Algorithms
    SentencesResources
    qPCR plots were generated with Prism 8 (Prism)
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    All figure panel images were prepared using FIJI software (ImageJ, NIH) and Adobe Illustrator 2020 (Adobe), showing deconvolved single z-slices.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)
    To measure the LINE-1 ORF1p immuno-staining signal intensity, we projected cell optical sections (sum, 42 slices) with the “z projection” function in FIJI.
    FIJI
    suggested: (Fiji, RRID:SCR_002285)
    To identify human – SARS-CoV-2 chimeric reads, raw sequencing reads were aligned to concatenated human and SARS-CoV-2 genomes plus transcriptomes by STAR (version 2.7.1a)47.
    STAR
    suggested: (STAR, RRID:SCR_015899)
    Differential expression was analyzed using EdgeR package (version 3.30.3)49,50 in R (version 4.0.3)46.
    EdgeR
    suggested: (edgeR, RRID:SCR_012802)
    PMPs were harvested from suspension during medium exchange and plated in microglia differentiation media over 7-14 days to produce microglia like cell monocultures (Neurobasal (Life Technologies 21103049) supplemented with Gem21 NeuroPlex without Vitamin A (GeminiBio, 400-161)
    NeuroPlex
    suggested: (NeuroPlex, RRID:SCR_016193)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. ScreenIT

    SciScore for 10.1101/2020.12.12.422516: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were permeabilized with 0.5% (v/v) Triton X-100/PBS, blocked with 4% (w/v) BSA/CMF- PBS at RT for 1 hr, incubated with 1:200 diluted anti-LINE-1 ORF1p mouse monoclonal antibody (clone 4H1, Sigma MABC1152, Lot 3493991), and then with 1:400 diluted Donkey- anti-Mouse-Alexa Fluor 594 second antibody (Invitrogen 21203)
    anti-LINE-1
    suggested: (LifeSpan Cat# LS-C130455-100, RRID:AB_10847571)
    anti-Mouse-Alexa
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 infection SARS-CoV-2 USA-WA1/2020 (Gen Bank: MN985325.1) was obtained from BEI Resources and expanded and tittered on Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Calu3 cells were cultured in the myeloid conditioned media or HIM media (basal) for two days with daily media change before harvest or fixation.
    Calu3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    After 24 hrs, the microglia conditioned media was collected, centrifugated (1000 rpm 10min) and the supernatant was directly applied to HEK293T cells.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Recombinant DNA
    SentencesResources
    Plasmid for HIV-1 reverse transcriptase expression: pCMV-dR8.2 dvpr was a gift from Bob Weinberg (Addgene plasmid # 8455 ; http://n2t.net/addgene:8455 ; RRID:Addgene_8455)41
    detected: RRID:Addgene_8455)
    Addgene plasmid # 51288 ; http://n2t.net/addgene:51288 ; RRID:Addgene_51288)42; EF06R (5’UTR-LINE-1) was a gift from Eline Luning Prak (Addgene plasmid # 42940 ; http://n2t.net/addgene:42940 ; RRID:Addgene_42940)43.
    detected: RRID:Addgene_51288)
    detected: RRID:Addgene_42940)
    Software and Algorithms
    SentencesResources
    qPCR plots were generated with Prism 8 (Prism)
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    All figure panel images were prepared using FIJI software (ImageJ, NIH) and Adobe Illustrator 2020 (Adobe), showing deconvolved single z-slices.
    FIJI
    suggested: (Fiji, RRID:SCR_002285)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)
    The following STAR parameters31 were used to call chimeric reads unless otherwise specified (Supplementary Figure 1a): --chimOutType Junctions SeparateSAMold WithinBAM HardClip \ --chimScoreJunctionNonGTAG 0 \ --alignSJstitchMismatchNmax -1 -1 -1 -1 \ -- chimSegmentMin 50 \ --chimJunctionOverhangMin 50.
    STAR
    suggested: (STAR, RRID:SCR_015899)
    Differential expression was analyzed using EdgeR package (version 3.30.3)49,50 in R (version 4.0.3)46.
    EdgeR
    suggested: (edgeR, RRID:SCR_012802)
    PMPs were harvested from suspension during medium exchange and plated in microglia differentiation media over 7-14 days to produce microglia like cell monocultures (Neurobasal (Life Technologies 21103049) supplemented with Gem21 NeuroPlex without Vitamin A (GeminiBio, 400-161)
    NeuroPlex
    suggested: (NeuroPlex, RRID:SCR_016193)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2020.12.12.422516v1 on bioRxiv