UPDATE 14. May 2021: MIT Study: SARS-CoV-2 Integrates into the Human Genome

UPDATE 10. May 2021: Researchers show SARS-CoV-2 genes can be integrated into the human genome

Do coronavirus genes slip into human chromosomes?

Further evidence supports controversial claim that SARS-CoV-2 genes can integrate with human DNA

Lab studies of genetically engineered human cells suggest the RNA (blue) of SARS-CoV-2 could convert to DNA in infected people and slip into their chromosomes. CLAUS LUNAU / SCIENCE SOURCE

By Jon Cohen - 06. May

A team of prominent scientists has doubled down on its controversial hypothesis that genetic bits of the pandemic coronavirus can integrate into our chromosomes and stick around long after the infection is over. If they are right—skeptics have argued that their results are likely lab artifacts—the insertions could explain the rare finding that people can recover from COVID-19 but then test positive for SARS-CoV-2 again months later.

Stem cell biologist Rudolf Jaenisch and gene regulation specialist Richard Young of the Massachusetts Institute of Technology, who led the work, triggered a Twitter storm in December 2020, when their team first presented the idea in a preprint on bioRxiv. The researchers emphasized that viral integration did not mean people who recovered from COVID-19 remain infectious. But critics charged them with stoking unfounded fears that COVID-19 vaccines based on messenger RNA (mRNA) might somehow alter human DNA. (Janesich and Young stress that their results, both original and new, in no way imply that those vaccines integrate their sequences into our DNA.)

Researchers also presented a brace of scientific criticisms, some of which the team addresses in apaper released online today by the Proceedings of the National Academy of Sciences (PNAS). “We now have unambiguous evidence that coronavirus sequences can integrate into the genome,” Jaenisch says.

SARS-CoV-2, the virus that causes COVID-19, has genes composed of RNA, and Jaenisch, Young, and co-authors contend that on rare occasions an enzyme in human cells may copy the viral sequences into DNA and slip them into our chromosomes. The enzyme, reverse transcriptase, is encoded by LINE-1 elements, sequences that litter 17% of the human genome and represent artifacts of ancient infections by retroviruses. In their original preprint, the researchers presented test tube evidence that when human cells spiked with extra LINE-1 elements were infected with the coronavirus, DNA versions of SARS-CoV-2’s sequences nestled into the cells’ chromosomes.

Many researchers who specialize in LINE-1 elements and other “retrotransposons” thought the data were too thin to support the claim. “If I would have had this data, I would have not submitted to any publication at that point,” says Cornell University’s Cedric Feschotte, who studies endogenous retrovirus chunks in the human genome. He and others also said they expected higher quality work coming from scientists of the caliber of Jaenisch and Young. In two subsequent studies, both posted on bioRxiv, critics presented evidence that the supposed chimeras of human and viral DNA traces are routinely created by the very technique the group used to scan for them in chromosomes. As one report concluded, the human-virus sequences “are more likely to be a methodological product, [sic] than the result of genuine reverse transcription, integration and expression.”

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.

In the new PNAS paper, the team provides evidence that artifacts alone can’t explain the detected levels of virus-human chimeric DNA. The scientists also show that portions of LINE-1 elements flank the integrated viral genetic sequence, further supporting their hypothesis. And they have collaborated with one of the original skeptics, Stephen Hughes of the National Cancer Institute, who suggested an experiment to clarify whether the integration was real or noise, based on the orientation of the integrated viral sequences relative to the human ones. The results support the original hypothesis, says Hughes, a co-author of the new paper. “That analysis has turned out to be important,” he says.

“The integration data in cell culture is much more convincing than what was presented in the preprint, but it’s still not totally clean,” says Feschotte, who now calls Jaenisch’s and Young’s hypothesis “plausible.” (SARS-CoV-2, he notes, can also persist in a person for months without integrating its genes.)

The real question is whether the cell culture data have any relevance to human health or diagnostics. “In the absence of evidence of integration in patients, the most I can take away from these data is that it is possible to detect SARS-CoV-2 RNA retroposition events in infected cell lines where L1 is overexpressed,” Feschotte says. “The clinical or biological significance of these observations, if any, is a matter of pure speculation at this point.”

Jaenisch’s and Young’s team do report hints of SARS-CoV-2 integration in tissue from living and autopsied COVID-19 patients. Specifically, the researchers found high levels of a type of RNA that is only produced by integrated viral DNA as the cell reads its sequence to make proteins. But, Young acknowledges, “We do not have direct evidence for that yet.”

Harmit Malik, a specialist in ancient viruses in the human genome at the Fred Hutchinson Cancer Research Center, says it’s a “legitimate question” to ask why people who should have cleared the virus sometimes have positive polymerase chain Reaction tests for its sequences. But he also remains unconvinced that the explanation is integrated virus. “Under normal circumstances, there is so little reverse transcription machinery available” in human cells, Malik says.

The controversy has grown decidedly more civil since December. Both Young and Jaenisch say they received more intense criticism for their preprint than any studies in their careers, in part because some researchers worried it played into the hands of vaccine skeptics spreading false claims about the newly authorized mRNA vaccines. “If there ever was a preprint that should be deleted, it is this one! It was irresponsible to even put it up as a preprint, considering the complete lack of relevant evidence. This is now being used by some to spread doubts about the new vaccines,” Marie-Louise Hammarskjöld, a microbiologist at the University of Virginia, posted in a comment on bioRxiv at the time.

And what of the original journal submission? “They rejected it,” Jaenisch says.

doi:10.1126/science.abj3287

Science  14 May 2021:

Vol. 372, Issue 6543, pp. 674-675
DOI: 10.1126/science.372.6543.674

Science's COVID-19 reporting is supported by the Heising-Simons Foundation

View Abstract

Author:

Jon Cohen

Jon Cohen is a staff writer for Science. Email Jon

Science’s COVID-19 reporting is supported by the Heising-Simons Foundation.

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MIT Study: SARS-CoV-2 Integrates into the Human Genome

 MIT Study: SARS-CoV-2 Integrates into the Human Genome17. May 2021

In the early months of the COVID-19 pandemic, healthcare workers analyzing test results began noticing something strange: patients who had already recovered from COVID-19 would sometimes inexplicably test positive on a PCR test weeks or even months later. 

Although people can catch COVID-19 for a second time, this did not appear to be the case for these patients; no live viruses were isolated from their samples, and some studies found these false positive results even while holding participants in quarantine. Also, RNAs generally have a short life—most only stick around for a few minutes—so it was unlikely for positive tests to be the result of residual RNAs.

Now, a new paper from the lab of Whitehead Institute Member and MIT professor of biology Rudolf Jaenisch may offer an answer to why some patients continue to test positive after recovery from COVID-19. In the paper, published online in PNAS, Jaenisch and collaborators show that genetic sequences from the RNA virus SARS-CoV-2 can integrate into the genome of the host cell through a process called reverse transcription. These sections of the genome can then be “read” into RNAs, which could potentially be picked up by a PCR test. 

SARS-CoV-2 is not the only virus that integrates into the human genome. Around 8 percent of our DNA consists of the remnants of ancient viruses. Some viruses, called retroviruses, rely on integration into human DNA in order to replicate themselves.

“SARS-CoV-2 is not a retrovirus, which means it doesn't need reverse transcription for its replication,” says Whitehead Institute postdoc and first author Liguo Zhang. “However, non-retroviral RNA virus sequences have been detected in the genomes of many vertebrate species, including humans.”

With this in mind, Zhang and Jaenisch began to design experiments to test whether this viral integration could be happening with the novel coronavirus. With the help of Jaenisch lab postdoc Alexsia Richards, the researchers infected human cells with coronavirus in the lab and then sequenced the  DNA from infected cells two days later to see whether it contained traces of the virus’ genetic material. 

To ensure that their results could be confirmed with different methodology, they used three different DNA sequencing techniques. In all samples, they found fragments of viral genetic material (though the researchers emphasize that none of the inserted fragments was enough to recreate a live virus).

Zhang, Jaenisch and colleagues then examined the DNA flanking the small viral sequences for clues to the mechanism by which they got there. In these surrounding sequences, the researchers found the hallmark of a genetic feature called a retrotransposon. 

Sometimes called “jumping genes,” transposons are sections of DNA that can move from one region of the genome to another. They are often activated to “jump” in conditions of high stress or during cancer or aging, and are powerful agents of genetic change. 

One common transposon in the human genome is called the LINE1 retrotransposon, which is made up of a powerhouse combination of DNA-cutting machinery and reverse transcriptase, an enzyme that creates DNA molecules from an RNA template (like the RNA of SARS-CoV-2). 

“There’s a very clear footprint for LINE1 integration,” Jaenisch says. “At the junction of the viral sequence to the cellular DNA, it makes a 20 base pair duplication.” 

Besides the duplication, another feature as evidence for LINE1-mediated integration is a LINE1 endonuclease recognition sequence. The researchers identified these features in nearly 70 percent of the DNAs that contained viral sequences, but not all, suggesting that the viral RNA may be integrating into cellular DNA via multiple mechanisms. 

To screen for viral integration outside of the lab, the researchers analyzed published datasets of RNA transcripts from different types of samples, including COVID-19 patient samples. With these datasets, Zhang and Jaenisch were able to calculate the fraction of genes that were transcribed in these patients’ cells which contained viral sequences that could be derived from integrated viral copies. The percentage varied from sample to sample, but for some, a relatively large fraction of viral transcripts seem to have been transcribed from viral genetic material integrated into the genome. 

A previous draft of the paper with this finding was published online on the preprint server bioRxiv. However, recent research revealed that at least some of the viral-cellular reads could be the product of misleading artefacts of the RNA sequencing method. In the present paper, the researchers were able to eliminate these artefacts that could have been obscuring the results. 

Instead of simply tallying transcripts that contained viral material, the researchers looked at which direction the transcripts had been read. If the viral reads were the result of live viruses or existing viral RNAs in the cell, the researchers would expect that most of the viral transcripts would have been read in the correct orientation for the sequences in question; in acutely infected cells in culture, more than 99 percent are in the correct orientation. If the transcripts were the product of random viral integration into the genome, however, there would be a near 50-50 split—half the transcripts would have been read forwards, the other half backwards, relative to the host genes.

“This is what we saw in some patient samples,” says Zhang. “It suggests that much of the viral RNA in some samples could be transcribed from integrated sequences.” 

Because the dataset they used was quite small, Jaenisch emphasizes that more information is needed to establish exactly how common this phenomenon is in real life and what it might mean for human health. 

It is possible that only a very few human cells experience any kind of viral integration at all. In the case of another RNA virus that integrates into the host cell genome, only a fraction of a percent of infected cells (between .001 and .01) contained integrated viral DNA. For SARS-CoV-2, the frequency of integration in humans is still unknown.

“The fraction of cells which have the integrating with could be very small,” says Jaenisch. “But even if it's rare, there are more than 140 million people who have been infected already, right?”

In the future, Jaenisch and Zhang plan to investigate whether the fragments of SARS-CoV-2 genetic material could be made into proteins by the cell.

“If they do, and trigger immune responses, it may provide continuous protection against the virus,” Zhang says.

They also hope to investigate whether these integrated sections of DNA could be partly to blame for some of the long-term autoimmune consequences that some COVID-19 patients experience.

“At this point, we can only speculate,” says Jaenisch. “But one thing we do think we can explain is why some patients are long-term PCR positive.”

Email

Republished courtesy of MIT.  Photo: An image of lung cancer cells infected with the SARS-CoV-2 virus. Blue represents DNA, green shows the SARS-CoV-2 nucleocapsid protein, and red represents double-stranded RNA, which occurs when the virus replicates its genome. A new study from the Jaenisch lab suggests that some virus RNA can be reverse transcribed and inserted into the human genome, which may explain why some patients continue to test positive for COVID-19 even after recovery. Credit: Alexsia Richards/Whitehead Institute

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Researchers show SARS-CoV-2 genes can be integrated into the human genome

By  - 10. May 2021

Researchers in the United States have shown that genes from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – the causative agent of coronavirus disease 2019 (COVID-19) – can be integrated into the genome of infected human cells.

The team says the viral RNA can be expressed as chimeric transcripts with fused cellular and viral sequences.

“Importantly, such chimeric transcripts are detected in patient-derived tissues,” writes the team from the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts and the National Cancer Institute in Frederick, Maryland.

Study: Further evidence supports controversial claim that SARS-CoV-2 genes can integrate with human DNA. Image Credit: vchal / Shutterstock

Study: Further evidence supports controversial claim that SARS-CoV-2 genes can integrate with human DNA. Image Credit: vchal / Shutterstock

Rudolf Jaenisch and colleagues say the findings may help to explain why some patients who have recovered from SARS-CoV-2 infection still test positive for the virus months later.

Patients remaining positive for viral RNA is an unresolved issue

Continuous or recurrent SARS-CoV-2-positive tests by polymerase chain reaction (PCR) have been reported in patients weeks or months after they have recovered from COVID-19. However, no infectious virus was isolated or shed from these patients, and the cause of the continued viral RNA production remains unknown.

Like other beta coronaviruses, SARS-CoV-2 uses an RNA-dependent RNA polymerase to replicate its genomic RNA and to transcribe subgenomic RNAs.

One potential explanation for the recurrent detection of viral RNA in the absence of viral replication is that DNA copies of viral subgenomic RNAs may become integrated into the DNA of the host cell via reverse transcription.

Transcription of the integrated DNA copies could be responsible for positive PCR tests long after the initial infection was cleared,” writes Jaenisch and colleagues.

Indeed, nonretroviral RNA virus sequences have been detected in the genomes of many animals. Several integrations in these sequences exhibit signals that are consistent with the integration of DNA copies of viral mRNAs into the germline via long interspersed nuclear element (LINE) retrotransposons.

Furthermore, nonretroviral RNA viruses such as lymphocytic choriomeningitis virus (LCMV) can be reverse transcribed into DNA copies by an endogenous reverse transcriptase. Studies have also shown that DNA copies of the viral sequences can integrate into the DNA of host cells.

“Moreover, expression of endogenous LINE1 and other retrotransposons in host cells is commonly up-regulated upon viral infection, including SARS-CoV-2 infection,” says Jaenisch and the team.

What did the researchers do?

The team used three different approaches to investigate whether SARS-CoV-2 RNA can be reverse transcribed and integrated into the genome of infected human cells in culture. The three approaches used to detect the viral RNA were nanopore long-read sequencing, Illumina paired-end whole genomic sequencing, and Tn5 tagmentation-based DNA integration site enrichment sequencing.

As reported in the Proceedings of the National Academy of Sciences, all three approaches provided evidence that SARS-CoV-2 RNA can be integrated into the genome of the host cell.

DNA copies of SARS-CoV-2 sequences were present in the genome and were shown to be integrated via a LINE1-mediated retroposition mechanism.

SARS-CoV-2 RNA can be reverse transcribed and integrated into the host cell genome. (A) Experimental workflow. (B) Chimeric sequence from a Nanopore sequencing read showing integration of a full-length SARS-CoV-2 NC subgenomic RNA sequence (magenta) and human genomic sequences (blue) flanking both sides of the integrated viral sequence. Features indicative of LINE1-mediated “target-primed reverse transcription” include the target site duplication (yellow highlight) and the LINE1 endonuclease recognition sequence (underlined). Sequences that could be mapped to both genomes are shown in purple with mismatches to the human genomic sequences in italics. The arrows indicate sequence orientation with regard to the human and SARS-CoV-2 genomes as shown in C and D. (C) Alignment of the Nanopore read in B with the human genome (chromosome X) showing the integration site. The human sequences at the junction region show the target site, which was duplicated when the SARS-CoV-2 cDNA was integrated (yellow highlight) and the LINE1 endonuclease recognition sequence (underlined). (D) Alignment of the Nanopore read in B with the SARS-CoV-2 genome showing the integrated viral DNA is a copy of the full-length NC subgenomic RNA. The light blue highlighted regions are enlarged to show TRS-L (I) and TRS-B (II) sequences (underlined, these are the sequences where the viral polymerase jumps to generate the subgenomic RNA) and the end of the viral sequence at the poly(A) tail (III). These viral sequence features (I–III) show that a DNA copy of the full-length NC subgenomic RNA was retro-integrated. (E) A human–viral chimeric read pair from Illumina paired-end whole-genome sequencing. The read pair is shown with alignment to the human (blue) and SARS-CoV-2 (magenta) genomes. The arrows indicate the read orientations relative to the human and SARS-CoV-2 genomes. The highlighted (light blue) region of the human read mapping is enlarged to show the LINE1 recognition sequence (underlined). (F) Distributions of human–CoV2 chimeric junctions from Nanopore (Left) and Illumina (Right) sequencing with regard to features of the human genome.

SARS-CoV-2 RNA can be reverse transcribed and integrated into the host cell genome. (A) Experimental workflow. (B) Chimeric sequence from a Nanopore sequencing read showing integration of a full-length SARS-CoV-2 NC subgenomic RNA sequence (magenta) and human genomic sequences (blue) flanking both sides of the integrated viral sequence. Features indicative of LINE1-mediated “target-primed reverse transcription” include the target site duplication (yellow highlight) and the LINE1 endonuclease recognition sequence (underlined). Sequences that could be mapped to both genomes are shown in purple with mismatches to the human genomic sequences in italics. The arrows indicate sequence orientation with regard to the human and SARS-CoV-2 genomes as shown in C and D. (C) Alignment of the Nanopore read in B with the human genome (chromosome X) showing the integration site. The human sequences at the junction region show the target site, which was duplicated when the SARS-CoV-2 cDNA was integrated (yellow highlight) and the LINE1 endonuclease recognition sequence (underlined). (D) Alignment of the Nanopore read in B with the SARS-CoV-2 genome showing the integrated viral DNA is a copy of the full-length NC subgenomic RNA. The light blue highlighted regions are enlarged to show TRS-L (I) and TRS-B (II) sequences (underlined, these are the sequences where the viral polymerase jumps to generate the subgenomic RNA) and the end of the viral sequence at the poly(A) tail (III). These viral sequence features (I–III) show that a DNA copy of the full-length NC subgenomic RNA was retro-integrated. (E) A human–viral chimeric read pair from Illumina paired-end whole-genome sequencing. The read pair is shown with alignment to the human (blue) and SARS-CoV-2 (magenta) genomes. The arrows indicate the read orientations relative to the human and SARS-CoV-2 genomes. The highlighted (light blue) region of the human read mapping is enlarged to show the LINE1 recognition sequence (underlined). (F) Distributions of human–CoV2 chimeric junctions from Nanopore (Left) and Illumina (Right) sequencing with regard to features of the human genome.

In some tissue samples taken from patients, the team also found evidence suggesting that a large proportion of the viral sequences were transcribed from integrated DNA copies of viral sequences, generating viral–host chimeric transcripts.

These and other data are consistent with a target primed reverse transcription and retroposition integration mechanism and suggest that endogenous LINE1 reverse transcriptase can be involved in the reverse transcription and integration of SARS-CoV-2 sequences in the genomes of infected cells,” writes the team.

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However, approximately 30% of the viral integrants lacked a recognizable nearby LINE1 endonuclease recognition site, thereby indicating that the integration could also occur via another mechanism.

What are the implications of the findings?

Jaenisch and colleagues say the findings raise several questions that require further investigation.

For example, the researchers ask whether integrated SARS-CoV-2 sequences express viral antigens in patients and whether these might influence the clinical course of disease.

If a cell with an integrated and expressed SARS-CoV-2 sequence survives and presents a viral- or neoantigen after the infection is cleared, this might engender continuous stimulation of immunity without producing infectious virus and could trigger a protective response or conditions such as autoimmunity as has been observed in some patients,” they write.

More generally, the integration of viral DNA in somatic cells may represent a consequence of natural infection that could play a role in the effects of other common disease-causing RNA viruses such as dengue and influenza virus, says the team.

The results may also be relevant for clinical trials of antiviral therapies.

If integration and expression of viral RNA are fairly common, reliance on extremely sensitive PCR tests to determine the effect of treatments on viral replication and viral load may not always reflect the ability of the treatment to fully suppress viral replication because the PCR assays may detect viral transcripts that derive from viral DNA sequences that have been stably integrated into the genome rather than infectious virus,” says Jaenisch and colleagues.

Source:

Journal reference:

Author:

Sally Robertson, B.Sc.

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