"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.
Continuous or recurrent positive SARS-CoV-2 PCR (Polymerase Chain Reaction) tests have been reported in patients weeks or months after recovery from an initial infection (1–14). Although bona fide re-infection of SARS-CoV-2 after recovery has been reported lately (15), cohort-based studies with strict quarantine on subjects recovered from COVID-19 suggested “re-positive” cases were not caused by re-infection (16,17).
We further confirmed that purified SARS-CoV-2 RNA from infected cells can be reverse-transcribed in vitro by lysates of cells expressing either LINE-1 or HIV-1 RT (Fig. S2c-d).
In the same cell population, a significantly higher fraction (~35%) of infected cells overexpressing LINE-1, as indicated by LINE-1 ORF1p immunostaining, showed nuclear N signals than cells not overexpressing LINE-1 (~12%) (Fig. 2e).
Expression analysis using LINE-1 specific primers (33,34) showed a ~3-4-fold up-regulation of LINE-1 in Calu3 cells when infected by SARS-CoV-2 (Fig. 3c). Moreover, PCR analysis on Calu3 cellular DNA showed retro-integration of SARS-CoV-2 N sequences after infection (Fig. 3d-e), possibly by the activated LINE-1 reverse transcriptase.
We treated cells with cytokine-containing conditioned media from Myeloid, Microglia, or CAR-T cell cultures and found a ~2-3-fold upregulation of endogenous LINE-1 expression by PCR analysis (Fig. 3f, S5b). Expressed LINE-1 protein (ORF1p) was also confirmed by immunofluorescence staining (Fig. 3g-h, S5a). In summary, our results show induced LINE-1 expression in cells stressed by viral infection or exposed to cytokines, suggesting a molecular mechanism for SARS-CoV-2 retro-integration in human cells.
Moreover, our results suggest that the integrated SARS-CoV-2 sequences can be transcribed, as shown by RNA-Seq and smRNA-FISH data, providing a possible explanation for the presence of viral sequences at later times after initial virus exposure and in the absence of detectable infectious virus (1–14). The retro-inserted SARS-CoV-2 sequences are most likely sub-genomic fragments, as the integration junctions are mostly enriched at the N sequence (Fig. 1d-e), excluding the production of infectious virus. Our data may also explain that patients, after recovery from disease symptoms, may become again positive for viral sequences as detected by PCR (1,8–14).
LINE-1 proteins have been shown as nucleic acid chaperones with high RNA binding affinity (39), therefore it is perhaps not surprising that they can retro-integrate exogenous viral RNAs. From an evolutionarily perspective, retro-integration of viral RNA by LINE-1 could be an adaptive response by the host to provide sustaining antigen expression possibly enhancing protective immunity. Conversely, retro-integration of viral RNAs could be detrimental and cause a more severe immune response in patients such as a “cytokine storm” or auto-immune reactions."
SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome. 2020-12-12
https://www.biorxiv.org/content/10.1101/2020.12.12.422516v1.full
https://archive.ph/XWC52
"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.
Continuous or recurrent positive SARS-CoV-2 PCR (Polymerase Chain Reaction) tests have been reported in patients weeks or months after recovery from an initial infection (1–14). Although bona fide re-infection of SARS-CoV-2 after recovery has been reported lately (15), cohort-based studies with strict quarantine on subjects recovered from COVID-19 suggested “re-positive” cases were not caused by re-infection (16,17).
We further confirmed that purified SARS-CoV-2 RNA from infected cells can be reverse-transcribed in vitro by lysates of cells expressing either LINE-1 or HIV-1 RT (Fig. S2c-d).
In the same cell population, a significantly higher fraction (~35%) of infected cells overexpressing LINE-1, as indicated by LINE-1 ORF1p immunostaining, showed nuclear N signals than cells not overexpressing LINE-1 (~12%) (Fig. 2e).
Expression analysis using LINE-1 specific primers (33,34) showed a ~3-4-fold up-regulation of LINE-1 in Calu3 cells when infected by SARS-CoV-2 (Fig. 3c). Moreover, PCR analysis on Calu3 cellular DNA showed retro-integration of SARS-CoV-2 N sequences after infection (Fig. 3d-e), possibly by the activated LINE-1 reverse transcriptase.
We treated cells with cytokine-containing conditioned media from Myeloid, Microglia, or CAR-T cell cultures and found a ~2-3-fold upregulation of endogenous LINE-1 expression by PCR analysis (Fig. 3f, S5b). Expressed LINE-1 protein (ORF1p) was also confirmed by immunofluorescence staining (Fig. 3g-h, S5a). In summary, our results show induced LINE-1 expression in cells stressed by viral infection or exposed to cytokines, suggesting a molecular mechanism for SARS-CoV-2 retro-integration in human cells.
Moreover, our results suggest that the integrated SARS-CoV-2 sequences can be transcribed, as shown by RNA-Seq and smRNA-FISH data, providing a possible explanation for the presence of viral sequences at later times after initial virus exposure and in the absence of detectable infectious virus (1–14). The retro-inserted SARS-CoV-2 sequences are most likely sub-genomic fragments, as the integration junctions are mostly enriched at the N sequence (Fig. 1d-e), excluding the production of infectious virus. Our data may also explain that patients, after recovery from disease symptoms, may become again positive for viral sequences as detected by PCR (1,8–14).
LINE-1 proteins have been shown as nucleic acid chaperones with high RNA binding affinity (39), therefore it is perhaps not surprising that they can retro-integrate exogenous viral RNAs. From an evolutionarily perspective, retro-integration of viral RNA by LINE-1 could be an adaptive response by the host to provide sustaining antigen expression possibly enhancing protective immunity. Conversely, retro-integration of viral RNAs could be detrimental and cause a more severe immune response in patients such as a “cytokine storm” or auto-immune reactions."