Wednesday, April 14, 2021

It's Not Supposed To Be Possible For RNA To Modify DNA

The central dogma of molecular biology is “DNA makes RNA makes proteins" but a quick read of this paper says different, and, the data are probably good. Moreover, Richard Young and Rudolf Jaenisch have been pioneers in the later phase of modern molecular biology. Bottomline, you'd have to be a total fooking fool to get injected with any of that mRNA therapeutic goop. Strongly recommend downloading the pdf as the technical Karenwaffen is shitting its anti-vax implication panties about now and agitating for censorship of the paper.

biorxiv  |  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-infectious114. 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.

Introduction

Continuous or recurrent positive SARS-CoV-2 PCR tests have been reported in patients weeks or months after recovery from an initial infection114. Although bona fide re-infection of SARS-CoV-2 after recovery has been reported lately15, cohort-based studies with strict quarantine on subjects recovered from COVID-19 suggested “re-positive” cases were not caused by re-infection16,17. Furthermore, no replication-competent virus was isolated or spread from these PCR-positive patients13,5,6,12. The cause for such prolonged and recurrent viral RNA production is unknown. As positive-stranded RNA viruses, SARS-CoV-2 and other beta-coronaviruses such as SARS-CoV-1 and MERS employ an RNA-dependent RNA polymerase to replicate their genomic RNA and transcribe their sub-genomic RNAs1820. One possibility is that SARS-CoV-2 RNAs could be reverse-transcribed and integrated into the human genome, and transcription of the integrated DNA copies could be responsible for positive PCR tests.

Endogenous reverse transcriptase (RT) activity has been observed in human cells, and the products of reverse transcription have been shown to become integrated into the genome21,22. For example, APP transcripts have been shown to be reverse-transcribed by endogenous RT, with resultant APP fragments integrated into the genome of neurons and transcribed22. Human LINE-1 elements (~17% of the human genome), a type of autonomous retrotransposons, are a potential source of endogenous RT, able to retro-transpose themselves and other non-autonomous elements such as Alu21,23.

Results

Expression of viral-cellular chimeric transcripts in infected cultured and in patient-derived cells is consistent with genomic integration of viral sequences

To investigate the possibility of viral integration into virus infected cells we analyzed published RNA-Seq data from SARS-CoV-2-infected cells for evidence of chimeric transcripts, which would be indicative of viral integration into the genome and expression. Examination of these data sets 2430 (Fig. S1a-b) revealed a substantial number of host-viral chimeric reads (Fig. 1a-c, S1c). These occurred in multiple sample types, including cells and organoids from lung/heart/brain/stomach tissues, as well as BALF cells directly isolated from COVID-19 patients (Fig. 1c). Chimeric read abundance was positively correlated with viral RNA level across the sample types (Fig. 1c). Chimeric reads generally accounted for 0.004% - 0.14% of total SARS-CoV-2 reads across the samples, with a 69.24% maximal number of reads in bronchoalveolar lavage fluid cells derived from severe COVID19 patients and near no chimeric reads from patient blood buffy coat cells (corresponding to almost no total SARS-CoV-2 reads). A majority of chimeric junctions mapped to SARS-CoV-2 nucleocapsid (N) sequence (Fig. 1d-e). This is consistent with the finding that nucleocapsid (N) RNA is the most abundant SARS-CoV-2 sub-genomic RNA31, and thus is most likely to be a target for reverse transcription and integration. These analyses support the hypothesis that SARS-CoV-2 RNA may retro-integrate into the genome of infected cells resulting in the production of chimeric viral-cellular transcripts.