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Click [Medical side] Follow what we are sharing with you today is a relatively new and interesting article, the title is "SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome" (SARS-CoV-2 RNA is reverse-transcribed And integrated into the human genome), the research comes from the Whitehead Institute of Biomedicine, Massachusetts Institute of Technology.
In order to let the public know first, the researcher published a preprint on December 13, 2020.
It has not yet been peer-reviewed, but the research ideas of the article are worth learning.
Abstract The long-term shedding of SARS-CoV-2 RNAs and PCR re-yang have been widely reported in patients, but these patients are most often asymptomatic infections.
The research group studied the possibility of reverse transcription and integration of SARS-CoV-2 RNAs into the human genome, and the transcription of the integrated sequence may be the cause of positive PCR detection.
In order to support this hypothesis, the research team found chimeric transcripts composed of virus fusion to cell sequence in the published data set of SARS-CoV-2 infection of cultured cells and patient primary cells, and the integration of the genome into the genome.
The transcription of the viral sequence is consistent.
In order to experimentally confirm the possibility of viral reverse transcription integration, the research group described that SARS-CoV-2 RNAs can be reverse transcribed in human cells by reverse transcriptase (RT) from the LINE-1 element or HIV-1 RT.
Evidence that these DNA sequences can be integrated into the cell’s genome and subsequently be transcribed.
SARS-CoV-2 infection or cytokine exposure can induce the expression of human endogenous LINE-1, suggesting the molecular mechanism of SARS-CoV-2 reversing integration.
This new feature of SARS-CoV-2 infection may explain why patients can still produce viral RNA after recovery, and provide a new hypothesis for RNA virus replication.
Background introduction Method 1.
Cell culture and plasmid transfection 2.
SARS-CoV-2 infection 3.
Immunofluorescence staining and single molecule RNA-FISH4, RNA sequence data analysis 5.
Conditioned medium production and processing flowchart Research results 1.
Virus The expression of cell chimeric transcripts in infected cultured cells and patient-derived cells is consistent with the genome integration of the virus sequence.
The RNA-Seq data of SARS-CoV-2 infected cells showed a large number of host virus chimeric reading codes (Figure 1a- c).
These occurred in a variety of sample types, including cells and organoids from lung/heart/brain/gastric tissues, as well as BALF cells isolated directly from COVID-19 patients (Figure 1c).
Chimeric reading abundance and viral RNA levels were positively correlated in all sample types (Figure 1c).
Most chimeric ligations map to the SARS-CoV-2 nucleocapsid (N) sequence (Figure 1d-e).
These analyses support the hypothesis that the SARS-CoV-2rna may be reversely integrated into the genome of the infected cell to produce chimeric viral cell transcripts.
2.
In cells that overexpress reverse transcriptase, SARS-CoV-2rna can be reverse transcribed and integrated into the human genome.
Overexpress human LINE-1 or HIV-1 reverse transcriptase (RT) in HEK293T cells → use SARS-CoV-2 infection of transfected cells → 2 days later → PCR or fluorescence in situ hybridization (FISH) to detect the virus sequence of the cells (Figure 2a).
In the cells overexpressing human LINE-1 or HIV-1 RT (Figure 2b), there were no positive gel bands in untransfected or uninfected cells.
These cells all expressed three types of RT (Figure 2c).
N-RNA-FISH signal was detected in the nucleus of cells overexpressing LINE-1 (Figure 2d).
The proportion of infected cells overexpressing LINE-1 (approximately 35%) was significantly higher than that of cells not overexpressing LINE-1 (approximately 12%) (Figure 2e).
Infected but untransfected cells also showed nuclear N signals, albeit at a lower frequency (~10%; Figure 2e).
SARS-CoV-2 RNA integrates through the cell's endogenous RT activity.
3.
SARS-CoV-2 infection induces human endogenous LINE-1 expression and the relationship between cytokines and post-integration.
In the published RNA sequence data of cells infected with SARS-CoV-2, the expression of LINE-1 is significantly up-regulated, and Related to chimeric reading abundance (Figure 3a-b).
Although the upregulation in Calu3 was not higher than that in NHBE, several LINE-1 elements were upregulated compared to one in NHBE (Figure 3a).
When SARS-CoV-2 was infected, LINE-1 in Calu3 cells was up-regulated 3-4 times (Figure 3c).
Reverse integration of SARS-CoV-2n sequence after infection (Figure 3d-e).
The cells were treated with conditioned medium containing cytokines from myeloid, microglia or CAR-T cell culture, and PCR analysis revealed that the expression of endogenous LINE-1 was up-regulated by 2-3 times (Figure 3f).
The expressed LINE-1 protein (ORF1p) was also confirmed by immunofluorescence staining (Figure 3g-h).
The expression of LINE-1 was induced in cells infected with the virus or exposed to cytokines, which indicates the molecular mechanism of the reverse integration of SARS-CoV-2 in human cells.
Editor's summary 1.
Innovations (1) SARS-CoV-2 RNAs can be reverse transcribed and integrated into reverse transcriptases from multiple sources, such as activated human LINE-1 or co-infected retrovirus (HIV) In the human genome; (2), the expression of LINE-1 can be induced when SARS-CoV-2 is infected or exposed to cytokines; (3) the integrated SARS-CoV-2 sequence can be transcribed, and SRAS- The CoV-2 sequence is likely to be a subgenomic fragment, excluding the production of infectious virus; (4) After the patient recovers from the symptoms of the disease, the virus sequence detected by PCR may again be positive.
2.
Clinical significance From an evolutionary point of view, the reverse integration of viral RNA through LINE-1 may be an adaptive response of the host, providing continuous antigen expression and possibly enhancing protective immunity.
On the contrary, reverse integration of viral RNA may be harmful and cause more severe immune responses in patients, such as "cytokine storm" or autoimmune reactions.
3.
Limitations and potential research directions (1) The results of this research group may also be related to the current clinical trials of antiviral therapy.
Relying on PCR testing to assess the effect of treatment on virus replication and viral load may not reflect the effect of treatment on inhibiting virus replication, because PCR testing can detect viral transcripts from viral sequences that are stably integrated into the genome rather than infectious viruses.
(2) Whether these integrated SARS-CoV-2 sequences can express viral antigens.
If this is the case, assess whether the viral antigens expressed from the integrated viral fragments can trigger an immune response in the patient, thereby affecting the course and treatment of the disease; (3) The immune response may vary depending on the individual's underlying conditions.
(4) The research of this research group may also be applicable to other common pathogenic RNA viruses, such as dengue fever, Zika virus or influenza virus.
These viruses may undergo reverse integration and may affect the progression of the disease. The END country’s natural experience sharing solicitation fund application season is now open, and the "Medical Formula" is now officially open to fans! The content must be original and first published, relevant to the country and nature.
Once adopted, there will be a generous remuneration (300-2000 yuan).
For details, please see the next post.
"Medical side" has always been committed to serving "medical people", pushing the most cutting-edge and most valuable original clinical and scientific research articles to clinicians and scientific researchers.
The medical department has launched "Laboratory Basics", "SCI Writing Skills", "Document Intensive Reading and Analysis", "Easy Learning of Medical English", "National Natural Science Foundation of China", "Clinical Data Mining", "Gene Data Mining", "R Language Tutorial", "Medical Statistics", "Minimally Invasive Animal Experiment Training" and other special courses, if you need to know the detailed tweets of the courses, you can follow the "Medical Party" public account and click on "Exquisite Topics" to enter the Tencent classroom: https://medfun .
ke.
qq.
com NetEase Cloud Classroom: http://study.
163.
com/u/ykt1467466791112 Customer Service Tel: 15821255568 Customer Service WeChat: yixuefang1234
In order to let the public know first, the researcher published a preprint on December 13, 2020.
It has not yet been peer-reviewed, but the research ideas of the article are worth learning.
Abstract The long-term shedding of SARS-CoV-2 RNAs and PCR re-yang have been widely reported in patients, but these patients are most often asymptomatic infections.
The research group studied the possibility of reverse transcription and integration of SARS-CoV-2 RNAs into the human genome, and the transcription of the integrated sequence may be the cause of positive PCR detection.
In order to support this hypothesis, the research team found chimeric transcripts composed of virus fusion to cell sequence in the published data set of SARS-CoV-2 infection of cultured cells and patient primary cells, and the integration of the genome into the genome.
The transcription of the viral sequence is consistent.
In order to experimentally confirm the possibility of viral reverse transcription integration, the research group described that SARS-CoV-2 RNAs can be reverse transcribed in human cells by reverse transcriptase (RT) from the LINE-1 element or HIV-1 RT.
Evidence that these DNA sequences can be integrated into the cell’s genome and subsequently be transcribed.
SARS-CoV-2 infection or cytokine exposure can induce the expression of human endogenous LINE-1, suggesting the molecular mechanism of SARS-CoV-2 reversing integration.
This new feature of SARS-CoV-2 infection may explain why patients can still produce viral RNA after recovery, and provide a new hypothesis for RNA virus replication.
Background introduction Method 1.
Cell culture and plasmid transfection 2.
SARS-CoV-2 infection 3.
Immunofluorescence staining and single molecule RNA-FISH4, RNA sequence data analysis 5.
Conditioned medium production and processing flowchart Research results 1.
Virus The expression of cell chimeric transcripts in infected cultured cells and patient-derived cells is consistent with the genome integration of the virus sequence.
The RNA-Seq data of SARS-CoV-2 infected cells showed a large number of host virus chimeric reading codes (Figure 1a- c).
These occurred in a variety of sample types, including cells and organoids from lung/heart/brain/gastric tissues, as well as BALF cells isolated directly from COVID-19 patients (Figure 1c).
Chimeric reading abundance and viral RNA levels were positively correlated in all sample types (Figure 1c).
Most chimeric ligations map to the SARS-CoV-2 nucleocapsid (N) sequence (Figure 1d-e).
These analyses support the hypothesis that the SARS-CoV-2rna may be reversely integrated into the genome of the infected cell to produce chimeric viral cell transcripts.
2.
In cells that overexpress reverse transcriptase, SARS-CoV-2rna can be reverse transcribed and integrated into the human genome.
Overexpress human LINE-1 or HIV-1 reverse transcriptase (RT) in HEK293T cells → use SARS-CoV-2 infection of transfected cells → 2 days later → PCR or fluorescence in situ hybridization (FISH) to detect the virus sequence of the cells (Figure 2a).
In the cells overexpressing human LINE-1 or HIV-1 RT (Figure 2b), there were no positive gel bands in untransfected or uninfected cells.
These cells all expressed three types of RT (Figure 2c).
N-RNA-FISH signal was detected in the nucleus of cells overexpressing LINE-1 (Figure 2d).
The proportion of infected cells overexpressing LINE-1 (approximately 35%) was significantly higher than that of cells not overexpressing LINE-1 (approximately 12%) (Figure 2e).
Infected but untransfected cells also showed nuclear N signals, albeit at a lower frequency (~10%; Figure 2e).
SARS-CoV-2 RNA integrates through the cell's endogenous RT activity.
3.
SARS-CoV-2 infection induces human endogenous LINE-1 expression and the relationship between cytokines and post-integration.
In the published RNA sequence data of cells infected with SARS-CoV-2, the expression of LINE-1 is significantly up-regulated, and Related to chimeric reading abundance (Figure 3a-b).
Although the upregulation in Calu3 was not higher than that in NHBE, several LINE-1 elements were upregulated compared to one in NHBE (Figure 3a).
When SARS-CoV-2 was infected, LINE-1 in Calu3 cells was up-regulated 3-4 times (Figure 3c).
Reverse integration of SARS-CoV-2n sequence after infection (Figure 3d-e).
The cells were treated with conditioned medium containing cytokines from myeloid, microglia or CAR-T cell culture, and PCR analysis revealed that the expression of endogenous LINE-1 was up-regulated by 2-3 times (Figure 3f).
The expressed LINE-1 protein (ORF1p) was also confirmed by immunofluorescence staining (Figure 3g-h).
The expression of LINE-1 was induced in cells infected with the virus or exposed to cytokines, which indicates the molecular mechanism of the reverse integration of SARS-CoV-2 in human cells.
Editor's summary 1.
Innovations (1) SARS-CoV-2 RNAs can be reverse transcribed and integrated into reverse transcriptases from multiple sources, such as activated human LINE-1 or co-infected retrovirus (HIV) In the human genome; (2), the expression of LINE-1 can be induced when SARS-CoV-2 is infected or exposed to cytokines; (3) the integrated SARS-CoV-2 sequence can be transcribed, and SRAS- The CoV-2 sequence is likely to be a subgenomic fragment, excluding the production of infectious virus; (4) After the patient recovers from the symptoms of the disease, the virus sequence detected by PCR may again be positive.
2.
Clinical significance From an evolutionary point of view, the reverse integration of viral RNA through LINE-1 may be an adaptive response of the host, providing continuous antigen expression and possibly enhancing protective immunity.
On the contrary, reverse integration of viral RNA may be harmful and cause more severe immune responses in patients, such as "cytokine storm" or autoimmune reactions.
3.
Limitations and potential research directions (1) The results of this research group may also be related to the current clinical trials of antiviral therapy.
Relying on PCR testing to assess the effect of treatment on virus replication and viral load may not reflect the effect of treatment on inhibiting virus replication, because PCR testing can detect viral transcripts from viral sequences that are stably integrated into the genome rather than infectious viruses.
(2) Whether these integrated SARS-CoV-2 sequences can express viral antigens.
If this is the case, assess whether the viral antigens expressed from the integrated viral fragments can trigger an immune response in the patient, thereby affecting the course and treatment of the disease; (3) The immune response may vary depending on the individual's underlying conditions.
(4) The research of this research group may also be applicable to other common pathogenic RNA viruses, such as dengue fever, Zika virus or influenza virus.
These viruses may undergo reverse integration and may affect the progression of the disease. The END country’s natural experience sharing solicitation fund application season is now open, and the "Medical Formula" is now officially open to fans! The content must be original and first published, relevant to the country and nature.
Once adopted, there will be a generous remuneration (300-2000 yuan).
For details, please see the next post.
"Medical side" has always been committed to serving "medical people", pushing the most cutting-edge and most valuable original clinical and scientific research articles to clinicians and scientific researchers.
The medical department has launched "Laboratory Basics", "SCI Writing Skills", "Document Intensive Reading and Analysis", "Easy Learning of Medical English", "National Natural Science Foundation of China", "Clinical Data Mining", "Gene Data Mining", "R Language Tutorial", "Medical Statistics", "Minimally Invasive Animal Experiment Training" and other special courses, if you need to know the detailed tweets of the courses, you can follow the "Medical Party" public account and click on "Exquisite Topics" to enter the Tencent classroom: https://medfun .
ke.
qq.
com NetEase Cloud Classroom: http://study.
163.
com/u/ykt1467466791112 Customer Service Tel: 15821255568 Customer Service WeChat: yixuefang1234
