And putting it into the real-life context: e.g., viral reactivation, persistent immune activation, permanent DNA changes?
A few days ago, a preprint titled “SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome” was released in bioRxiv. Judging from the title, we can tell it would spark some controversy and debate.
“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,” the authors summarized. “This novel feature of SARS-CoV-2 infection may explain why patients can continue to produce viral RNA after recovery and suggest a new aspect of RNA virus replication.”
In simpler language, the study shows that human cells can turn coronavirus’s RNAs into DNAs — in a process called reverse transcription — which are integrated into the human genome. The human genome can then read and translate this new piece of DNA into new proteins. This article will evaluate the pre-print study findings, and what they mean in real-life contexts.
What the study did and found
The study is led by Rudolf Jaenisch, a biology professor at Massachusetts Institute of Technology, Whitehead Institute for Biomedical Research’s founding member, and pioneer in gene modification research. Other authors are from the Whitehead Institute for Biomedical Research and Harvard University. So, at least we can be assured that researchers with credible expertise in world-leading research institutions did the study.
1. Chimeric transcripts
By analyzing published datasets of gene profiles of cells infected with SARS-CoV-2, the study found evidence of chimeric transcripts — a mix of human and SARS-CoV-2 genes — in cells and organoids of the lungs, heart, brain, and stomach. Such chimeric transcripts were also detected in cells in lung fluid samples isolated from Covid-19 patients. These chimeric transcripts suggest that SARS-CoV-2 genes can latch into human genes — becoming a chimeric gene. In biology, a chimera means a single organism that contains genes from two or more organisms.
This supports the hypothesis that the human genome can make SARS-CoV-2-human chimeric transcripts, which may be transcribed into new proteins.
Source: Rodríguez-Martín et al. (2017). BMC Genomics. An illustration of chimeric transcripts.
2. LINE-1 and HIV-1 RT
HIV-1 reverse transcriptase (RT) belongs to the human immunodeficiency virus type 1 (HIV-1). In contrast, long interspersed element 1 (LINE-1) is derived from evolutionary ancient virus remnants that make up about 17% of the human genome. Both HIV-1 RT and LINE1 can integrate parts of certain virus’s genes into the human genome.
The researchers overexpress LINE-1 or HIV-1 RT activities in human cells, and then infected these cells with SARS-CoV-2. Using various genetic tools, they confirmed the presence of SARS-CoV-2 genes (coding for nucleocapsid protein) in the genome inside the human cell nucleus. This did not happen with uninfected human cells. In human cells with low LINE-1 or HIV-1 RT expression, SARS-CoV-2 genes were also detected in the genome but at lower levels.
These analyses show that LINE-1 (that human cells normally express) and HIV-1 RT can integrate SARS-CoV-2 genes into the human genome inside the cell nucleus.
Source: Kazazian & Moran (2017). The New England journal of medicine. Panel A shows protein-coding exons and classes of human transposable elements as a percentage of the genome. For instance, LINEs make up 21% of the human genome, of which 17% belong to LINE-1.
3. Emphasis on LINE-1 and gene-protein transcription
The study then decodes what kind of genes are transcribed (into proteins) by the human cells. And they found that the gene coding for the SARS-CoV-2 nucleocapsid protein was part of the genetic sequence undergoing transcription (into proteins) activities in the human cells. Such transcription activities further correlated with the expression of LINE-1.
Moving on, the study went back to analyzing the published datasets of gene profiles of human cells infected with SARS-CoV-2. This time they looked for any increase in LINE-1 expression. Indeed, LINE-1 expression correlated with the expression of SARS-CoV-2-human chimeric transcripts. Next, by analyzing the genomes of these published datasets, the study also found traces of SARS-CoV-2 genes.
These results show that human cells infected with SARS-CoV-2 exhibit an increase in LINE-1 expression, which integrates SARS-CoV-2 genes into the cell’s genome. These integrated genes can then produce SARS-CoV-2-human chimeric transcripts, which can be transcribed into new proteins.
4. The cytokine involvement
Finally, since Covid-19 is characterized by cytokine storm, they wanted to see if inflammation would induce LINE-1 expression. So, they exposed human cells to cytokines (made by other immune cells) and then confirmed that LINE-1 expression did heighten. This result shows that inflammatory cytokines also enhance LINE-1 expression, in addition to SARS-CoV-2 infection, which goes back to point 3.
Putting into context
1. Study limitations
As with any studies, results must be interpreted in light of their limitations. The current study is only done in cultured human cells, as well as with public datasets. So, results still need to be supported in animal models.
Moreover, the study experimentally induces a high expression of LINE-1 and HIV-1 RT in the human cells. In human cells with normal levels of LINE-1 and HIV-1 RT expression, the genomic integration of SARS-CoV-2 genes was not that efficient, the study found.
Indeed, one critique an HIV-1 expert has raised is that conditions for genomic integration in experimental settings can be optimized to be highly efficient. So, the same efficiency may not happen with living organisms. With this in mind, let’s proceed to the broader implications of this study.
2. Viral reactivation
“We found LINE-1 expression can be induced upon SARS-CoV-2 infection or cytokine exposure, suggesting a molecular mechanism responsible for SARS-CoV-2 retro-integration in patients,” the study authors wrote. “Moreover, our results suggest that the integrated SARS-CoV-2 sequences can be transcribed…, 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.”
Sometimes, people get tested positive for SARS-CoV-2 again after two consecutive negative tests, and despite symptom resolution and hospital release. Due to the short time span between the negative and second-positive tests and strict quarantine, scientists have suspected reactivation over reinfection as the cause. While there have been true cases of reinfection, the second infection usually occurs much later as the person has to be exposed to a different SARS-CoV-2 variant found elsewhere in the globe.
The current preprint provides the first evidence that SARS-CoV-2 could reactivate, explaining the odd phenomenon of a second positive test result after two consecutive negative tests. However, as with other viruses capable of reactivation, the second reactivation usually causes a milder disease for reasons such as immune memory or weaker microbe’s virulence, which is consistent with certain cases of suspected cases of Covid-19 reactivation.
Indeed, the preprint study only found evidence of parts of SARS-CoV-2 genes —i.e., coding for the nucleocapsid — being integrated into the human cell’s genome, not the whole SARS-CoV-2 genome. Thus, the transcription process of this integrated SARS-CoV-2 should exclude infectious virions, the authors explained.
3. Infectious vs. non-infectious reactivation
Most RNA viruses, including coronaviruses, replicate in the cell cytoplasm and not in the cell nucleus containing the genome. One exception is HIV-1, an RNA virus that causes AIDS. As HIV-1 can enter and replicate in the cell nucleus, it integrates its genes into the human genome via the HIV-1 RT. This integration allows HIV-1 to stay in the body forever.
But the key point is that HIV-1 genomic integration involves the HIV-1 full genome, which can produce infectious virions. So, HIV-1 reactivation can cause a new infection. In contrast, experiments from the current preprint showed that only certain parts of SARS-CoV-2 got integrated into the human genome. Thus, we can be assured that SARS-CoV-2 would only partially reactivate in a non-infectious manner.
“The work, only reported in a preprint, suggests the pandemic pathogen takes a page from HIV and other retroviruses and integrates its genetic code — but, importantly, just parts of it — into people’s chromosomes,” the journal Science reported. “Because it is [not] all pieces of the coronaviral genome, it can’t lead to infectious RNA or DNA and therefore it is probably biologically a dead end.”
4. Persistent immune activation?
The next issue the authors raised is if such SARS-CoV-2 genomic integration would produce proteins capable of triggering the immune system, which may worsen the clinical outcomes of Covid-19.
But it’s also possible that such immune activation would benefit the human host. As mentioned, LINE-1 constitutes a large chunk of the human genome, which scientists believe may serve as an evolutionary advantage to humans. “From an evolutionary 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,” the authors explained. In other words, by expressing parts of the virus occasionally, immune memory can be better maintained.
“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,” they added. Another possibility is that proteins being made from such integrated genes would just be degraded as cellular waste.
So, at this point, not much is known about the health consequences (if any) of such SARS-CoV-2 genomic integration.
5. Revision of diagnostic tests?
If SARS-CoV-2 genomic integration is harmless to the host — as in no production of infectious virions or no harmful immune responses — then the current diagnostic test for SARS-CoV-2 may need revision or re-interpreted in a different light.
As the authors explained, “The reliance of PCR tests to assess the effect of treatments on viral replication and viral load may not reflect the efficacy of the treatment to suppress viral replication as the PCR assay may detect viral transcripts from viral sequences stably integrated into the genome rather than infectious virus.”
The journal Science agreed, “The phenomenon, if true and frequent, could have profound implications that range from false signals of active infection to misleading results from Covid-19 treatment studies.”
6. Permanent DNA changes?
Assuming the genomic integration of SARS-CoV-2 follows the trajectory of HIV-1, we can assume it’s permanent. But this remains speculation and further investigations are still warranted. But the more important question is whether such genomic integration would cause any health problems in the future.
Another form of permanence, albeit controversial, is whether such genomic integration of SARS-CoV-2 can be passed down to offspring as in children? One example is human herpesvirus type 6 (HHV-6), where it integrates itself into the genome’s telomeres, which can be passed down to offspring genetically. But such genetic inheritance of HHV-6 is harmless that doesn’t trigger any immune responses.
More good news is that most viruses can’t do this; it’s only HHV-6 science knows thus far. Even HIV-1 can only be inherited in a non-genetic manner, such as from the mother’s blood to the child’s. So, the chances are that SARS-CoV-2 can’t as well.
This article discusses a newly released preprint from credible experts that have produced findings needing careful interpretations. The study finds that SARS-CoV-2 infection and cytokines can increase LINE-1 expression in human cells. LINE-1 then integrates parts of SARS-CoV-2 genes into the human genome inside the nucleus, which can then be turned into chimeric transcripts that can be read and translated into new proteins. This novel finding bears practical importance, such as understanding viral reactivation in an infectious or non-infectious manner, the reevaluation of the current diagnostic test, and the possible short-term and life-long health effects of such SARS-CoV-2 genomic integration.
This article was originally published here with modifications.