You might recall that preprint claiming SARS-CoV2 would integrate into host’s genome, from the lab of the MIT professor Rudolph Jaenisch at Whitehead Institute. It implied serious consequences for the pandemic, suggesting an infected person would never get rid of the virus, somewhat similar to HIV. The preprint was harshly criticised for its weak science, some demanded its removal for emboldening the antivax movement. Surely now the critics were debunked themselves, because the study passed peer review in the elite journal Proceedings of the National Academy of Sciences of the United States of America (PNAS)!
Oh wait, it did not pass any independent peer review. Being an Academy member, the MIT professor Jaenisch “contributed” the paper, meaning he asked two friendly colleagues, one of them his former postdoc, to write some nice recommendations which were supplied for the antiquated “contributed” track at PNAS.
This was the original preprint, from from December 2020, done in collaboration with Jaenisch’s Whitehead Institute colleague, Richard Young:
Liguo Zhang, Alexsia Richards, Andrew Khalil, Emile Wogram, Haiting Ma, Richard A. Young, Rudolf Jaenisch SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome bioRxiv (2020) doi: 10.1101/2020.12.12.422516
The claim was that the ancient retroviral LINE1 elements residing inside the human genome would somehow get activated and cause a retrotranscription of the SARS-CoV2 RNA genome, thus allowing its genomic integration. People once infected with the coronavirus would still produce its genes like the spike protein, which would result in positive PCR tests results labelling these former COVID-19 patients as active virus careers. Who knows, maybe they would even produce a functional virus, all their lives? A very worrisome thought.
Jaenisch’s preprint was criticised not only on Twitter or in the bioRxiv comment section, like here by Cedric Feschotte. Another preprint Yan et a bioRxiv 2021 declared in February 2021: “the observed HVC [host-virus chimeric] events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes.“. Yet another Kazachenka and Kassiotis, bioRxiv 2021 declared:
“human-SARS-CoV-2 chimeric reads found in RNA-seq data may arise during library preparation and do not necessarily signify SARS-CoV-2 reverse transcription, integration in to host DNA and further transcription.“
The Whitehead Institute preprint’s working model of a mechanism was that the coronavirus infection is associated with a dangerous mega-inflammation often called the “cytokine storm” (a popular target of therapies, read here), and it would be this cytokine storm which can somehow re-activate dormant LINE1 elements and cause the genome integration of the virus.
This is where the preprint’s successor, the newly published and “peer reviewed” PNAS paper, gets interesting.
Liguo Zhang, Alexsia Richards, M. Inmaculada Barrasa, Stephen H. Hughes, Richard A. Young, and Rudolf Jaenisch Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues PNAS (2021) doi: 10.1073/pnas.2105968118
The “peer reviewed” study concludes:
“Our data suggest that, in some patient tissues, the majority of all viral transcripts are derived from integrated sequences. Our data provide an insight into the consequence of SARS-CoV-2 infections that may help to explain why patients can continue to produce viral RNA after recovery.”
The mechanism behind SARS-CoV2 genome integration, as provided in the PNAS paper, was:
“we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral–host chimeric transcripts.”
No mention of the cytokine storm anymore. What happened? Here a clue: during the “peer review” process between preprint and published PNAS paper, the manuscript mysteriously lost not just the cytokine storm model, but also 3 authors, all postdocs at the Jaenisch lab at Whitehead Institute: Andrew Khalil, Emile Wogram and Haiting Ma.
One of them explained to me:
“The work I contributed to the original draft of the manuscript is no longer included.“
The postdoc added:
“I was/am involved in the myeloid cell-conditioned medium experiments.“
That would mean Figure 3 and Figure S5 of the preprint, which dealt with the cytokine activation of lINE1 elements, and which were dropped from the final paper:
The accompaning text of the preprint explained the concept of the “cytokine storm”:
“Patients infected with SARS-CoV-2 and other corona viruses show evidence of cytokine induction associated with the immune response, and in severe cases experience a cytokine storm35–37, prompting us to investigate whether cytokines alone can induce LINE-1 activation.“
To prove the mechanism, the Whitehead Institute researchers performed the experiments that cancelled postdoc was involved in, and concluded:
“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.“
That Figure 3 used to be quite central to the preprint’s discovery of molecular mechanism of LINE1 activation due to viral infection, as per preprint’s abstract:
“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.”
Now, the preprint may be all about cytokines, but the PNAS paper mentions the word “cytokine” only once:
“It is not known how many antigen-presenting cells are needed to elicit an antigen response, but derepressed LINE1 expression, induced by viral infection or by exposure to cytokines (38⇓–40), may stimulate SARS-CoV-2 integration into the genome of infected cells in patients.“
No, the references 38-40 do not include Jaenisch’s previous preprint. In fact, that preprint is mentioned only once, in passing:
“To investigate the possibility that SARS-CoV-2 sequences integrated into the genome can be expressed, we analyzed published RNA-seq data from SARS-CoV-2–infected cells for evidence of chimeric transcripts (49).“
The postdoc who was once a coauthor said:
“I believe the cytokine work and many of the other data removed from the preprint are still ongoing“
If the work is ongoing, then without this postdoc, apparently? I asked Jaenisch what happened to the 3 preprint authors and the cytokine storm. The MIT professor replied:
“In this paper we addressed the two key questions raised in the preprint in December both of which needed more convincing evidence. The present paper shows clearly that SARS-CoV- sequences can integrate into the genome of cultured cells that were or not transfected with LINE1. The most frequent mechanism of integration is a LINE1-mediated retroposition mechanism because the majority of integrants have all the footprints of this integration process. We felt that the induction of Line 1 by cellular stress, while an interesting and important aspect, needed more data to be totally convincing and we did not address this in the present study (this is why three authors who were involved in these previous experiments but not in the present ones were removed from the author list ). The studies on LIne1 expression are ongoing.“
I wrote back that I understood Jaenisch’s words as dismissal of the cytokine storm mechanism as incorrect. I also argued that even then the removal of the 3 postdocs as co-authors was possibly still not justified. After all, they did contribute to the project experimentally by helping their principal investigator figure out the correct mechanism, or at least dismiss the wrong one. Jaenisch then stated:
“I strongly believe that the LINE1 expression data as presented in the December preprint are correct and significant. But we want to get more data to clearly define what type of elements is induced – our previous results were not conclusive on this issue (and, given the reaction on this ill-fated preprint: we are particularly cautions). The 3 authors who participated in the initial work did not contribute to the PNAS paper so they are not co-authors.“
Wait. Now Jaenisch insists the LINE1 induction via cytokines was “correct and significant“. But he seems not sure if it is specifically LINE1 which causes SARS-CoV2 genome integration, hence the need for “more data to clearly define what type of elements is induced“? But why is the PNAS paper so certain about LINE1 as mechanism then, and concludes in the discussion (without referencing the preprint):
“These and other data are consistent with a target primed reverse transcription and retroposition integration mechanism (41, 42) and suggest that endogenous LINE1 RT can be involved in the reverse transcription and integration of SARS-CoV-2 sequences in the genomes of infected cells.“
It’s all a bit confusing. No wonder the manuscript was previously rejected by another journal. It may be not scientificality convincing enough, while the potential danger of feeding antivaxx paranoia is certainly conceivable. There is this Jaenisch quote in the Science article about the PNAS paper:
“In their new paper, Jaenisch, Young, and colleagues acknowledge that the technique they used accidentally creates human-viral chimeras. “I think it’s a valid point,” Jaenisch says. He adds that when they first submitted the paper to a journal, they knew it needed stronger data, which they hoped to add during the review process. But the journal, like many, requires authors to immediately post all COVID-19 results to a preprint server. “I probably should have said screw you, I won’t put it on bioRxiv. It was a misjudgment,” Jaenisch says.”
Now the crazy thing is that the most heavily criticised aspect of the preprint was NOT about cytokines, but about the reality of SARS-CoV2 genome integration and the alleged role of LINE1 elements therein, which were claimed to be artefacts instead. And yet, the idea of cytokines as mechanism of LINE1 activation, which Jaenisch himself says was correct, was buried, and everyone involved in these experiments thrown off the PNAS paper. Which given PNAS‘ impact factor and status, is no small thing. The Science article ends with:
“And what of the original journal submission? “They rejected it,” Jaenisch says.”
There are actually not many journals which have a preprint mandate at manuscript submission stage, i.e., where posting a preprint is absolutely mandatory to enter peer review. But then again, I was informed that because of the pandemic, many journals mandate that all COVID-19 manuscripts must be first be deposited on a pre-print server and communicated to the WHO before considered for peer review. So unlike I previously speculated, one cannot know which journal Jaenisch first submitted the paper to, but it must have been a big one. Well, PNAS is also very big. In this regard, one of the 3 preprint postdocs who got cancelled told me:
“I was on the original submission, which was based on the preprint that I am listed as an author on.“
So after the manuscript was rejected, Jaenisch could have reconsidered the validity of the whole idea of SARS-CoV2 genome integration. He did reconsider, added another collaboration, and the cytokine data and its 3 contributors were shed. The new manuscript was then contributed to PNAS by Jaenisch, via the journal submission track of same name, meaning he invited his own two peer reviewers and submitted their reports together with the manuscript. A much criticised and often abused track, which turns the whole concept of peer review and editorial gatekeeping on its head.
Surely such a controversial study should have undergone proper peer review before appearing in a high-ranking journal?
Jaenisch however told me:
“I think you fundamentally misunderstand the process of publishing in PNAS: this paper was stringently reviewed, as indicated on the paper, by two eminent scientists, Anton Berns and Ann Skalka, who both have a many decades long track record in retrovirology and in transposon mediated genomic integration issues.“
The point here however is not the eminence of Anton Berns (Jaenisch’s former postdoc) and of Ann Skalka (emeritus director of Fox Chase Cancer Center, where Jaenisch once trained), but that Jaenisch invited their reviews himself. So the MIT professor clarified:
“It is correct that in a contributed paper the NAS member can suggest reviewers. They are carefully and stringently considered by the PNAS board and all correspondence goes between board and reviewer. I suggested the two most competent and eminent scientists in the field I could think of and they agreed to review this paper. It is absolutely wrong to suggest that a PNAS paper is not peer reviewed – the board has very stringent criteria.“
Still doesn’t explain why the contributed track at PNAS, a choice which certainly doesn’t help allay the criticisms. If anything, the moral of the story is about the usefulness of preprints in debunking weak science and in opening the doors to how academic sausages are really made.
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