Since the spread of the new crown virus , there have been frequent cases of re-infection and rejuvenation of recovered patients. Previously, medical experts generally believed that the cause of rejuvenation was the inability of the human immune system to resist the continuous attack of the coronavirus. Recently, a study by the team of MIT molecular biologist Rudolf Jaenisch revealed another possibility.
This study pointed out that SARS-CoV-2 RNA can be reverse transcribed and integrated into the human genome. The simple understanding is that the virus does not disappear in the human body, but is hidden by the reverse transcription combined with human DNA, which may be the cause of the patient's recovery.
Figure | Paper "SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome" (Source: bioRxiv)
Currently, this research paper is published on the preprint platform bioRxiv, once it is online It triggered intense discussions in the industry. It is worth noting that the comments on this research show two diametrically opposite trends. Some experts believe that this is a “inspiring study”, but there are also opinions that question “whether this research has biological significance”.
University of Hong Kong virology expert Professor Jin Dongyan believes that this experiment is "untenable". According to experimental inferences, if RNA viruses can integrate into human DNA through reverse transcription, then the common flu may change the human body's genes. But in fact, modern medicine has never found traces of influenza virus gene integration into human genome .
SARS-CoV-2 RNA can be reverse transcribed and integrated into the human genome
At first, Jaenisch's team was curious about the results of nucleic acid detection of Fuyang after the initial recovery of patients infected with the new crown.Although there are reports that the cause of Fuyang was re-infected with the virus, after strict isolation measures have been taken on the recovered, virus information can still be obtained from them, and these viruses do not have replication and infectious properties.
, as a positive-strand RNA virus, SARS-CoV-2, like SARS-CoV-1, MERS and other type B coronaviruses, uses RNA polymerase to replicate genomic RNA and transcribe subgenomic RNA. However, the replication properties of the virus were not found in the test samples of the survivors. From this, the researchers inferred a possibility that SARS-CoV-2 RNA was reverse transcribed and integrated into the human genome. The transcription of the integrated DNA copy may lead to PCR positive result.
To confirm the above inference, the team used the new coronavirus and human LINE-1 reverse transcriptase or HIV-1 reverse transcriptase through in vitro cell experiments to obtain three findings:
infected cultured cells and (Fuyang) patients The chimeric transcript expression in the cell is consistent with the genome integration of the virus sequence;
In order to study the possibility of virus transcription and integration into the genome of human nucleus , the researchers analyzed the RNA of SARS-CoV-2 infected cells- Seq (transcription sequencing) data. Through the analysis of transcriptional sequencing data of cell samples from lung, heart, brain, stomach and BALF (bronchial cell lavage fluid) of patients diagnosed with COVID-19, it was found that a large number of sample cells may have chimeric sequences of human genome and viral genes .
These combined sequences generally account for 0.004%-0.14% of the total sample, of which the combined sequence from BALF (bronchial cell lavage fluid) cells from severely ill patients with new coronary disease is as high as 69.24%.
Figure | cov2 chimeric reads can be mapped to the ratio of reads in the published RNA-Seq dataset
and most of the chimeric RNA comes from the nucleocapsid (N) of SARS-CoV-2 sequence. The nucleocapsid (N) is the RNA with the highest content in the SARSCoV-2 subgenomic RNA.Therefore, it is most likely to become a target for reverse transcription and integration. The researchers believe that these analyses support that SARS-CoV-2 RNA may be retro-integrated into the genome of infected cells and produce chimeric virus-cell transcripts.
SARS-CoV-2 RNA can be reverse transcribed in cells overexpressing reverse transcriptase and integrated into the human genome;
In order to further confirm the reverse transcription and integration of SARS-CoV-2 RNA, the researchers conducted research in HEK293T cells Express human LINE-1 or HIV-1 reverse transcriptase and infect transduced cells with SARS-CoV-2.
Figure | SARS-CoV-2 RNA can be reverse transcribed and integrated into the host. The expression of reverse transcriptase in cells
Considering that nucleocapsid (N) RNA is the most likely target for reverse transcription integration At this point, the researchers selected 4 sets of nucleocapsid (N) targeted PCR primers to amplify the purified cell DNA, and performed gel electrophoresis analysis on the cell genomic DNA. The researchers not only confirmed the nucleocapsid (N) sequence, but also cloned the full-length nucleocapsid (N) DNA from the genomic DNA (gDNA) of CMV-LINE-1 overexpressing cells, and confirmed it by Sanger sequencing sequence.
2 days after infection, the virus sequence of the cells is detected by PCR or fluorescence in situ hybridization. The results confirmed that in the same group of infected cells overexpressing LINE-1, the signal ratio of nucleocapsid (N) (about 35%) was significantly higher than that of infected cells that did not overexpress LINE-1 (about 12%). The percentage of positive nucleocapsid (N) signals in infected cells transfected with LINE-1 (about 80% transfection efficiency) (about 30%) was significantly higher than that of untransfected cells (13%). Infected but untransfected cells also show nucleocapsid (N) signals, albeit at a lower frequency (approximately 10%).
The above experiment further confirms that SARS-CoV-2 RNA can be reverse transcribed by overexpressed endogenous reverse transcriptase in vitro.
SARS-CoV-2 RNA and cells can induce human LINE-1 expression,Related to reverse transcription integration;
LINE-1 in human body is not only an autonomous retrotransposon, but also a retrotransposon that contributes to non-autonomous elements. Based on this particularity of LINE-1, researchers found that when SARS-CoV-2 was infected, the expression of LINE-1 in Calu3 cells was up-regulated by about 3-4 times. In addition, PCR analysis of Calu3 cell DNA revealed that the reverse transcription integration of the SARS-CoV-2 N sequence after infection may come from the LINE-1 reverse transcriptase activated by the cytokine .
Figure | Under SARS-CoV-2 infection and treatment with cytokine-containing conditioned medium, cell line 1 is expressed in human cells as an endogenous reverse transcriptase source
although LINE-1 In the human body, the proportion of the genome reaches 17%, but in fact its activity is low, only 100 copies of 500,000 copies are active. In order to study whether cytokines can induce LINE-1 alone, the researchers cultured bone marrow cells, microglia cells, and CART cells with a medium containing cytokines. PCR analysis found that the expression of endogenous LINE-1 was up-regulated 2- 3 times.
researchers also pointed out that LINE-1 expression was significantly up-regulated in the RNA-Seq data of cells infected with SARS-CoV-2, which was related to the abundance of chimeric sequences. In other words, the new coronavirus sequence integrated into the human genome through reverse transcription may be a subgenomic fragment, because the integrated sequence is mostly concentrated near the nucleocapsid (N), so it is not infectious.
Jaenisch’s team stated that experiments confirmed that SARS-CoV-2 RNA can be reverse transcribed and integrated into the human genome by reverse transcriptase; and the expression of LINE-1 can be affected by SARS-CoV-2 infection or exposure to cytokines. The induction indicates the molecular mechanism by which SARS-CoV-2 reverses integration in patients; the integrated fragment is most likely a SARS-CoV-2 subgenome fragment.SARS-CoV-2 RNA reverse transcription explains why the virus sequence still exists after the virus is exposed, when no infectious virus is detected.
research sparked heated debate, real in-vivo conditions do not support experimental conclusions
researchers concluded that if the LINE-1 sequence naturally produces RT (reverse transcriptase) in human cells , then the body of a patient who is diagnosed with new crown infection There may have been a combination of virus and DNA. In addition, for patients who are simultaneously infected with the new crown and AIDS, this integration change may have already occurred in the body.
This experiment provides a possibility for the nucleic acid to detect the residual virus in patients. At the same time, some concerns have been raised about the effectiveness of current PCR detection and treatment methods for COVID-19.
Figure | New Coronavirus (Source: Science)
"At present, only retroviruses and similar hepatitis B viruses have been found to integrate nucleic acid into the host DNA. Retroviruses must be integrated to continue to replicate And assemble new viruses, but the new coronavirus is not a retrovirus. In this MIT experiment, an excessive amount of reverse transcriptase was artificially added from outside the cell to cause reverse transcription of RNA into DNA. But in fact, the human body There are not so many reverse transcriptases in cells, so this experiment is more inclined to a'hypothesis', which is very different from the real environment and possible actual situations in human cells." Professor Jin Dongyan told DeepTech.
Chinese Academy of Medical Sciences researcher and former Peking Union Medical College professor Wang Chenguang also pointed out that the behavior of new coronaviruses and retroviruses after infecting the host is essentially different. A retrovirus is equivalent to a virus hijacking the host and turning itself into a part of the host's body. After the new coronavirus infects host cells, unlike retroviruses, the virus nucleic acid can only be replicated and protein can be expressed and produced in the host cell, and finally the assembly of virus particles is completed.
The well-known typical retrovirus and HIV,It can be reverse transcribed into DNA through its own RNA-producing enzyme, and then it can enter the human genome through continuous replication and fusion. The new coronavirus is replicated by RNA polymerase, and there will be no reverse transcription to DNA in the middle. In addition, the replication process of the new coronavirus is completed in the cytoplasm, and the DNA is mainly concentrated in the nucleus, so the replication process of the new coronavirus does not touch the DNA.
In fact, in the process of human evolution, a large amount of viral or bacterial RNA has been integrated into human DNA, but most integrated genomes have been proven to be ineffective.
Although there are constant doubts, some experts and scholars have affirmed the experiment. "Science" magazine quoted Robert Gallo, a famous American virologist and HIV discoverer, as commenting that although I doubt the completeness of the experiment, I really like the study and I guess the experiment is correct.
It is worth noting that discussions on this experiment and derived vaccines are constantly escalating. Some scholars pointed out that “viral RNA can be integrated into human DNA through reverse transcription, and mRNA vaccines based on protein synthesis can also be integrated into human DNA through reverse transcription, which will cause human genes to be changed after injection of the vaccine.”
Jin Dongyan explained to DeepTech Considering the characteristics of mRNA, firstly, the life span of mRNA is very short, ranging from tens of minutes to several hours. Moreover, mRNA is easily degraded by RNase in the body and in vitro, even human sweat, tears and saliva have RNase. Therefore, the mRNA vaccine must be stored at minus 70°C to preserve its activity. Secondly, the cells that the mRNA enters also have a lifespan and will naturally die. After the formation of antibodies against the new coronavirus antigen and immune cells, they will also help the body to completely eliminate cells with mRNA vaccines, so these cells cannot stay in the body forever, and will not cause the so-called reverse transcription of the vaccine to DNA and cause genetic changes. .
Wang Chenguang also pointed out that the mRNA nucleic acid vaccine only contains a nucleic acid sequence encoding a protein of the virus. The viral nucleic acid material can neither be integrated into the human genomic DNA, nor can it be replicated and amplified in cells. Not only is the cell not hijacked,Instead, the protein synthesized by mRNA is used to equip a well-armed mercenary to guard against the new coronavirus.
Although the entire experimental process lacks direct evidence that the virus in patients with new coronavirus has integrated into the DNA, this is the main reason why many scholars criticize this research, but this hypothesis does provide certain value for virus research, especially for In the future to prevent and avoid the risk of RNA viruses. Not letting go of every possibility is probably the greatest charm of biology.
Australian National University John Curtin Institute of Medicine, a doctoral student of genetics and epigenetics Zhang Jing also contributed to this article
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