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Exogenous coronavirus interacts with endogenous retrotransposon in human cells

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Background: There is an increased global outbreak of diseases caused by coronaviruses affecting respiratory tracts of birds and mammals. Recent particularly dangerous coronaviruses are MERS-CoV, SARS-CoV and SARS-CoV-2, causing respiratory illness and even failure of several organs. However, profound impact of coronavirus on host cells remains elusive. Results: Here, we go deep into transcriptome of MERS-CoV, SARS-CoV and SARS-CoV-2 infected human lung-derived cells, and observed that infection of these coronaviruses all induced increase of retrotransposon expression through upregulation of TET genes. Similar upregulation of retrotransposon was also observed in SARS-CoV-2 infected human intestinal organoids. Retrotransposon upregulation will lead to increased genome instability and more frequent readthrough from retrotransposon to dysregulate gene expression. People with higher basal level of retrotransposon like cancer patients and aged people will have increased risk of symptomatic infection. Additionally, we show evidence supporting long-term epigenetic inheritance of retrotransposon upregulation. We also observed significant amount of chimeric transcripts of retrotransposon and SARS-CoV-2 RNA for potential human genome invasion of viral fragments, with the front and the rear part of SARS-CoV-2 genome being easier to form chimeric RNA, and this may apply for other coronaviruses. Here we suggest that primers and probes for nucleic acid detection should be designed in the middle of virus genome to identify live virus with higher probability. Conclusions: In summary, we propose that infection of coronaviruses especially SARS-CoV-2 induce retrotransposon activation, formation of chimeric coronavirus-retrotransposon RNA, and elicits more severe symptoms in patients with underlying diseases. More attention may need to be paid to potential harm contributed by retrotransposon dysregulation in treatment of coronavirus-infected patients.
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Exogenous coronavirus interacts with endogenous
retrotransposon in human cells
Ying Yin
Huazhong University of Science and Technology
Xiao-zhao Liu
Huazhong University of Science and Technology
Ximiao He ( XimiaoHe@hust.edu.cn )
Huazhong University of Science and Technology
Li-quan Zhou ( zhouliquan@hust.edu.cn )
Huazhong University of Science and Technology
Research Article
Keywords: Coronavirus, Retrotransposon, SARS-CoV-2, TET, LINE
DOI: https://doi.org/10.21203/rs.3.rs-40063/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
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Abstract
Background: There is an increased global outbreak of diseases caused by coronaviruses affecting
respiratory tracts of birds and mammals. Recent particularly dangerous coronaviruses are MERS-CoV,
SARS-CoV and SARS-CoV-2, causing respiratory illness and even failure of several organs. However,
profound impact of coronavirus on host cells remains elusive.
Results: Here, we go deep into transcriptome of MERS-CoV, SARS-CoV and SARS-CoV-2 infected human
lung-derived cells, and observed that infection of these coronaviruses all induced increase of
retrotransposon expression through upregulation of TET genes. Similar upregulation of retrotransposon
was also observed in SARS-CoV-2 infected human intestinal organoids. Retrotransposon upregulation
will lead to increased genome instability and more frequent readthrough from retrotransposon to
dysregulate gene expression. People with higher basal level of retrotransposon like cancer patients and
aged people will have increased risk of symptomatic infection. Additionally, we show evidence supporting
long-term epigenetic inheritance of retrotransposon upregulation. We also observed signicant amount of
chimeric transcripts of retrotransposon and SARS-CoV-2 RNA for potential human genome invasion of
viral fragments, with the front and the rear part of SARS-CoV-2 genome being easier to form chimeric
RNA, and this may apply for other coronaviruses. Here we suggest that primers and probes for nucleic
acid detection should be designed in the middle of virus genome to identify live virus with higher
probability.
Conclusions: In summary, we propose that infection of coronaviruses especially SARS-CoV-2 induce
retrotransposon activation, formation of chimeric coronavirus-retrotransposon RNA, and elicits more
severe symptoms in patients with underlying diseases. More attention may need to be paid to potential
harm contributed by retrotransposon dysregulation in treatment of coronavirus-infected patients.
Background
Emerging coronaviruses often spread rapidly from person to person and there seems to be an increased
global outbreak of related diseases. MERS-CoV and SARS-CoV are two identied rare coronavirus strains
which cause not only severe lung infection but also serious complications (1-4). More recently,
coronavirus disease named COVID-19 caused by a novel coronavirus SARS-CoV-2 is expanding globally
and rapidly, resulting in emerging health issues (5-8). Although cell receptors and the routes of infection
of these coronaviruses have been identied (8-11) complicated impact on human cells is far from clear.
Transposable Elements (TEs) are mobile DNA elements in virtually all eukaryotes and comprise more
than 40% of human genome (12). They can self-replicate and insert into various locations inside genome.
Dysregulation of TE may lead to various illnesses like inammatory diseases (13). The only active
member in TE is retrotransposon which can “copy and paste” themselves through RNA intermediate.
Expression of most of retrotransposon members is suppressed in somatic cells and they are only active
in brains, germ cells, early embryos and pathological conditions (14). About 5% of newborn babies show
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a new retrotransposon integration event (15). Abnormally upregulation of retrotransposons cause
insertions, deletions and inversions in genome (16, 17), resulting in compromised genetic stability and
even cell death (18, 19). Accumulated evidence in recent years also proved their importance in
orchestration of gene expression (20), regulation of chromatin structure (21) and modulation of
developmental program (22, 23).
Long interspersed nuclear elements (LINEs) are common autonomous retrotransposons and comprise
about 17% of human genome (24). Some LINE-1 elements can be transcribed and translated in cells.
After reverse transcription of LINE-1 RNA, they can be integrated back into genome (25). Naturally, LINEs
expression is repressed in most cell types. Its RNA is mainly heritable during early embryogenesis
because of its enrichment and high retrotransposition activity in early embryos (26). Transgenic mouse
model carrying mouse/human LINE-1 retrotransposition reporter demonstrated that this activity creates
somatic mosaicism during development (27). Besides LINEs, short interspersed nuclear elements (SINEs)
and long terminal repeats (LTRs) are also enriched retrotransposons in human genome, and mobilization
of SINEs relies on LINE-1-encoded proteins (12).
In our study, we analyzed publicly available transcriptome data of human cells infected with coronavirus
MERS-CoV, SARS-CoV and SARS-CoV-2, and observed enhanced expression of TEs including several
retrotransposons, as well as inammation, immunity and apoptosis related genes. We further noticed
potential fusion of SARS-CoV-2 RNA with retrotransposon transcripts especially LINEs and SINEs.
Therefore, further examinations on genome and transcriptome of cells from patients and studying
models will be valuable to evaluate potential crosstalk between coronavirus and retrotransposons.
Results And Discussion
Coronavirus infection disturbs diverse biological processes in human cells and can stimulate ACE2
expression through IRF1 and STAT1
Coronaviral infection led to not only respiratory failure but also multiple organ dysfunction syndromes,
indicating that there are common pathways for coronavirus to impact human cells (28). Transcriptome
analysis may provide valuable information on how human cells react with coronavirus entry.
To examine whether coronavirus infection disturbs expression of specic gene sets in human cells, we
analyzed public available RNA-seq data of human lung-derived cells with infection of MERS-CoV, SARS-
CoV, and SARS-CoV-2. Through comparison of transcriptomes before and after infection, we identied
thousands of dysregulated genes (adjusted p-value < 0.05) for each group (Fig. 1A). Among those
dysregulated genes, we found that 26 genes were commonly upregulated after infection of the three
coronaviruses (Fig. 1B), but very few genes were identied to be commonly downregulated (Fig. 1C). GO
analysis of the 26 commonly upregulated genes demonstrated enrichment on inammation, immunity
and apoptosis related pathways (Fig. 1B). Through relative viral sequence content in transcriptome, we
found that the three coronaviruses can infect various human lung-derived cells (Fig. 1D), however, low
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dose of coronavirus or using NHBE cells for infection were not successful to support coronavirus
replication (Fig. S1).
ACE2 is the cell receptor of SARS-CoV-2 (8, 9). Differently from robust expression of ACE2 in Calu-3 cells,
ACE2 expression was undetectable in A549 cells, but after SARS-CoV-2 infection, low level of ACE2 was
observed (Fig. 1E). This indicates that transcription factors responding to coronavirus infection induced
ACE2 expression. Recent report showed that ACE2 can be stimulated by interferon, and proposed IRF1
and STAT1-binding sites near ACE2 transcription start site (Fig. S2) (29). Here, we noticed that expression
of both IRF1 and STAT1 were increased after SARS-CoV-2 infection, and ACE2 expression was
signicantly reduced when IRF1 was depleted in virus-infected human cells or STAT1 was depleted in
interferon-treated human cells (Fig. 1E). These results conrmed that IRF1 and STAT1 are essential
upstream activators of ACE2 upon virus infection. So we propose that SARS-CoV-2 might enter human
cells with low eciency by bulk-phase endocytosis in A549 cells, inducing IRF1 and STAT1 expression
which further enhances ACE2 expression to facilitate receptor-mediated viral entry. Therefore, IRF1 and
STAT1 seem to be two promising drug targets to limit coronavirus entry through ACE2.
Coronavirus infection enhanced retrotransposon expression in human lung-derived cells
Next, we ask whether TE expression is impacted by coronavirus infection. We rst examined
transcriptome of human lung adenocarcinoma cell line Calu-3 after 24-hr infection of MERS-CoV (30). We
observed that TE expression was generally activated after coronavirus infection (Fig. 2A). Further
examination documented that subfamilies of LINEs, SINEs, LTRs were differentially upregulated by
coronavirus (Fig. 2B). LINE-1 is the mostly well-studied autonomous retrotransposon. Most LINE-1
elements are inactivated in somatic cells, but some escape variously evolved silencing mechanisms.
Hence, we ask whether evolutionarily old and young retrotransposons were impacted by coronavirus
infection differently. We compared the ratio of fold change of specic LINE-1 element expression ordered
by predicted evolutionary ages, and found that older and younger LINE-1 elements were similarly
inuenced (Fig. 2C) (31). One of the major mechanisms for LINE-1 silencing is DNA methylation, and we
examined expression of genes encoding DNA methyltransferases (DNMTs) and Ten-eleven translocation
(TET) enzymes mediating active DNA demethylation. We observed that Tet genes were generally
upregulated after coronavirus infection (Fig. 2D), and upregulated DNA demethylation activity may lead
to demethylation of retrotransposon promoters. This result supports that increased retrotransposon
expression was caused by genome-wide DNA demethylation. We obtained similar results in MERS-
CoV/SARS-CoV infected MRC5 cells which are noncancerous human lung broblast cells (Fig. 2A-D).
Recent COVID-19 outbreak is caused by the novel coronavirus SARS-CoV-2. Here, we explored
transcriptomes of SARS-CoV-2 infected A549 and Calu-3 cells. Similar to MERS-CoV and SARS-CoV
infection, we found general increase of multiple transposable elements (Fig. 2A-B), no biased impact of
older and younger LINE-1 elements by SARS-CoV-2 infection (Fig. 2C). SARS-CoV-2 infection also causes
upregulation of TET gene expression (Fig. 2D). Similarly, SARS-CoV-2 was identied to have the
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capability of infecting human intestinal organoids (Fig. 2E) and increased retrotransposon expression
can also be observed post infection in a time-dependent manner (Fig. 2F).
Therefore, upregulation of retrotransposon seems to be a common event induced by coronavirus
infection, possibly through enhancing global DNA demethylation activity. Despite of similar upregulation
of retrotransposon families triggered by the three coronaviruses, individual retrotransposons are
differently dysregulated, and this may cause various phenotypes in human cells. Note that above results
were from 24-hr infection of coronaviruses, and impact of long-term infection should be more severe.
Moreover, retrotransposon is able to encode proteins and can form retrovirus-like particles (26), so
electron microscopy examination of coronavirus-infected samples may need to discriminate coronavirus
from retrovirus-like particles because of upregulation of retrotransposons.
Upregulation of retrotransposon may be long-term memorized epigenetically
We then ask whether retrotransposon upregulation can be long-term inherited through several generations
of cell divisions. We found the mouse model of transgenerational epigenetic inheritance of acquired traits
may provide molecular insights into this question.
tRNA-derived small RNAs (tsRNAs) in sperm were reported to transmit abnormal epigenetic information
into preimplantation embryo, and epigenetic abnormality was further inherited to adult tissue, causing
metabolic disorders (32). Two kinds of tsRNAs were previously identied to regulate retrotransposon LTR
(33), so we ask whether abnormal retrotransposon activity is inheritable during this process. We analyzed
the transcriptome of cleavage mouse embryo and adult islet originated from zygote with injection of
tsRNA of sperm from normal or high-fat diet (HFD) male mice. We found that LINE, SINE and LTR
retrotransposons were all upregulated in 8-cell embryo when HFD tsRNA was injected (Fig. 3A). Notably,
LTR retrotransposon also showed upregulation in adult islet (Fig. 3B). Further analysis on LTR families
supported that upregulation of ERV1 expression was inherited from early embryo (Fig. 3C) to adult islet
(Fig. 3D), probably through DNA methylation inheritance at ERV1 locus. Therefore, above result indicates
that enhancement of retrotransposon expression, ERV1 in this case, can be long-term inherited through
several generations of cell cycles, even from cleavage-stage early embryos to adult tissues, with change
of DNA methylation as the potential molecular mechanism (Fig. 3E).
SARS-CoV-2 RNA forms chimeric transcripts with retrotransposon RNA especially LINE for potential
insertion into host genome
Coronaviruses are RNA viruses and are not supposed to integrate into host genome by themselves.
However, it was reported that several RNA viruses have capacity to recombine with retrotransposons to
invade host genome (34, 35). Regarding contribution of SARS-CoV-2 RNA to total transcriptome in
infected Calu-3 cells to be as high as 15.32% (Fig. 1D), we explored in the transcriptome the potential
chimeric transcripts of SARS-CoV-2 and cellular RNA, and obtained subtranscriptome with chimeric reads.
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We found that 0.23% of SARS-CoV-2 RNA formed chimeric transcripts with non-TE genes and 0.14% with
TE (Fig. 4A). Surprisingly, TE-virus chimeric reads contribute 37.36% to total mapped chimeric reads,
while TE reads are only 2.83% in total mapped reads (Fig. 4B), indicating that TE is much more ecient to
form chimeric transcripts with SARS-CoV-2 RNA than non-TE genes. We randomly extracted reads from
subtranscriptome of chimeric transcripts of SARS-CoV-2 and cellular RNA, and conrmed identity of the
chimeric reads (Fig. 4C).
We further analyzed distribution of TE subfamilies in total transcriptome and subtranscriptome with
chimeric reads, and found that reads of retrotransposon LINE, SINE and LTR were all enriched in the
subtranscriptome of chimeric reads (Fig. 4D). Unexpectedly, only LINE RNA was overrepresented in
subtranscriptome with chimeric reads than in total transcriptome, and further analysis showed that virus-
LINE-1 was overrepresented in virus-LINE reads (Fig. 4E). This demonstrates high eciency of LINE
family especially LINE-1 in forming chimeric transcript with SARS-CoV-2 RNA. LINE-1 is autonomous
retrotransposon with retrotransposition activity, and RNA-RNA ligation mediated by endogenous RNA
ligase RtcB was previously reported for LINE-1 to carry other types of RNA for host genomic invasion (36),
so similar mechanisms may apply for SARS-CoV-2 transcripts. Further analysis of human genome from
SARS-CoV-2 infected human cells or biopsies will be particularly important to identity existence of
integration of coronavirus RNA into human genome.
Moreover, to identify which region of SARS-CoV-2 RNA prone to form chimeric transcripts with cellular
RNA, we aligned total transcriptome and subtranscriptome to SARS-CoV-2 genome, and viewed on IGV to
nd that the front and the rear parts, especially the rear part of coronavirus RNA were biased in forming
chimeric transcripts (Fig. 4F). That means the front and the rear parts of SARS-CoV-2 fragments are
easier to be inserted into human genome for prolonged expression, indicating that people even positive
for Nucleic Acid Test may just have infection history, and not really carry live coronavirus but only silent
viral fragments. Taken together, we suggest that primers and probes for SARS-CoV-2 testing are designed
in middle of the SARS-CoV-2 genome.
The model of coronavirus-retrotransposon interaction
Based on above analysis, we propose that coronavirus infection may increase retrotransposon
expression through modulating TET activity to reduce global DNA methylation. Increased retrotransposon
RNA may further form chimeric transcripts with coronavirus RNA, and integrate viral genomic fragments
into human genome. Moreover, enforced retrotransposon expression may be harmful and probably long-
term inherited (Fig. 5A).
TE is widely expressed in human tissues (Fig. 5B), with highest enrichment in early human embryos (Fig.
5C). The cells used in this study are mainly derived from human lung and also robustly express TE (Fig.
5D). Moreover, TE subfamilies are variable in different cell types (Fig. 5E-G), suggesting extensive but
specic phenotype upon global retrotransposon upregulation.
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The rst concern regarding global retrotransposon upregulation is genome instability. Retrotransposition
activity is high in early embryo (26) and brain (37) during normal development, so potential integration of
coronavirus sequence into human genome is suggested to be scrutinized for these cells. It was also
reported that retrotransposon upregulation is positively correlated with tumor progression (38), causing
genomic deletion, translocation and duplication (39). What’s more, increased expression of
retrotransposon LINE-1 contributes to age-associated inammation in several tissues (40). Additionally,
vapers and smokers demonstrated higher retrotransposon expression and hypomethylation at associated
loci (41). Also, people with neurological disorders may have higher retrotransposon expression and
retrotransposition activity (42). These reports not only show that upregulation of retrotransposon
expression may cause several diseases, but also indicate that persons with higher basal level of
retrotransposons are supposed to be more susceptible to coronavirus infection and have increased risk of
symptomatic infection. In support of this, recent analysis of SARS-CoV-2 patients showed that cancer
patients (43) and aged people (44) get more severe symptoms after infection. Therefore, inhibition of
reverse transcriptase activity in human cells may be necessary during pharmaceutical treatment of
coronavirus-infected patients, especially those with higher basal level of retrotransposons.
The second concern regarding global retrotransposon upregulation is disturbance of retrotransposon
adjacent gene expression. Accumulated evidence shows that retrotransposons are not just genomic
fossils, but have molecular functions. For example, physically adjacent retrotransposon activates gene
promoter of TMEM156 by readthrough mechanism (Fig. 5H, Fig. S3). Also, transcripts of LINEs, SINEs
and low-complexity repeats physically interacted with specic genomic areas to play distinct roles (45).
The third concern regarding global retrotransposon upregulation is whether coronavirus RNA can enter
nucleus and associate with specic genomic regions through sequence homology, similar like the
behavior of retrotransposon RNA (21, 45). Blast analysis in NCBI using SARS-CoV-2 genome showed no
similar sequence in human genome. We further used CENSOR program (46) to analyze the SARS-CoV-2
genome and all predicted candidate repetitive elements are less than 200bp. Therefore, no evidence
supports that SARS-CoV-2 RNA has the ability to recognize human genome by homologous sequence
even these transcripts enter nucleus by chance.
Conclusions
Taken together, we demonstrate that coronavirus infection increases retrotransposon expression in
human cells, possibly through global DNA hypomethylation, and increased retrotransposon RNA may
further form chimeric transcripts with coronavirus RNA for integration of viral genomic fragments into
human genome. These enhanced retrotransposon transcripts may be long-term inherited to harm host
organs. Therefore, we propose that retrotransposon upregulation induced by coronavirus infection has
signicant contributions to coronavirus caused symptoms, and suggest careful transcriptome
examination and genetic tests in future investigations on coronavirus-infected patients.
Methods
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Cell types used for transcriptome study of coronavirus infection
Cell types below are used in this study. Calu-3, human lung cancer cell; MRC5, human fetal lung strain;
A549, human adenocarcinomic alveolar basal epithelial cell; NHBE, primary human bronchial epithelial
cell. Each group above has three replicates. For human intestinal organoids, each group has two
replicates.
RNA-seq data processing
Raw reads were processed with cutadapt v1.16 to perform quality trimming with default parameters
except for: quality-cutoff =20, pair-lter=both. To include as many non-uniquely mapped reads as
possible, trimmed reads were rstly aligned to human/mouse genome (hg19/mm10) by STAR (v2.5.1b)
with default settings including parameters ‘--winAnchorMultimapmax 2000 --outFilterMultimapNmax
1000 ’. RSEM was used to calculate FPKM value of genes. The annotation and fasta sequences for
consensus transposable element sequences were downloaded from Repbase (version 20.01) (47). TE
transcript with default parameters was used to get counts for transposable elements. For RNA-seq
alignment of coronavirus genomes, MERS-CoV (NC_019843), SARS-CoV (NC_004718) and SARS-CoV-2
(NC_045512) genomes were downloaded from NCBI, and trimmed reads were aligned to coronavirus
genome by STAR (v2.5.1b) using default parameters. To identify potential chimeric transcripts of
coronavirus and cellular transcripts from single-end RNA-seq data, 30nt fastq reads from each end were
extracted from raw fastq reads and both were aligned to human and SARS-CoV-2 genomes respectively.
Non-viral end of the chimeric reads were mapped to consensus transposable element sequences using
STAR with parameters ‘--winAnchorMultimapmax 2000 --outFilterMultimapNmax 1000’ to get counts of
transposons. Integrative Genomics Viewer (IGV) was used for snapshot of transcriptome. R package
Deseq2 was used to get differential expressed genes. Metascape was used to visualize functional
proles of genes and gene clusters (48). Graphs were created by R or Excel. Images were organized by
Adobe Illustrator.
Accession number
RNA sequencing data of coronavirus-infected human lung-derived cells are from GSE122876
(transcriptome of MERS-CoV-infected Calu-3 cells; single-read; MOI 2, 24 hours) (30), GSE56192
(transcriptome of MERS-CoV and SARS-CoV infected MRC5 cells; paired-end; MOI 2, 24 hours),
GSE147507 (transcriptome of SARS-CoV-2 infected A549 cells, Calu-3 cells, and NHBE cells; MOI 2, 24
hours) (49). RNA sequencing data of SARS-CoV-2-infected human intestinal organoids are from
GSE149312 (MOI 1, 24 and 60 hours, grown in differentiation medium) (50). SARS-CoV-2 infected Calu-3
cells were used to identify chimeric transcripts of coronavirus and cellular RNA. RNA sequencing data of
IRF1 knockout and control human hepatocytes infected with hepatitis A virus are from GSE114916. RNA
sequencing data of STAT1 knockout and control human HepG2 cells treated by IFN are from GSE98372
(51). RNA sequencing data of human tissues and cell types are from GSE83115 (52). RNA sequencing
data of human early embryos and embryonic stem cells are from GSE36552 (53). RNA sequencing data
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of 8-cell mouse embryos and adult mouse islet developed from zygotes with injection of sperm tsRNAs
from high-fat-diet males are from GSE75544 (32).
List Of Abbreviations
TE, transposable element
LINE, long interspersed nuclear element
SINE, short interspersed nuclear element
LTR, long terminal repeat
Declarations
Acknowledgements
We thank Dr. Bing Li from Shanghai Jiao Tong University for assistance of data analysis on
retrotransposon expression.
Authors’ contributions
Li-quan Zhou and Ximiao He conceived and designed the project. Ying Yin analyzed the data and wrote
the manuscript. Xiao-zhao Liu performed analysis on chimeric transcripts. Li-quan Zhou and Ximiao He
revised the manuscript. All authors have read and approved the nal manuscript.
Funding
This work was supported by National Key R&D Program of China [2018YFC1004502, 2018YFC1004001],
National Natural Science Foundation of China [NSFC 31771661].
Availability of data and materials
RNA sequencing data of MERS-CoV-infected Calu-3 cells (30) are obtained from NCBI Sequence Read
Archive with BioProject ID: PRJNA506733 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA506733/).
RNA sequencing data of MERS-CoV-infected and SARS-CoV-infected MRC5 cells are obtained from NCBI
Sequence Read Archive with BioProject ID: PRJNA233943
(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA233943). RNA sequencing data of SARS-CoV-2 infected
A549 cells, Calu-3 cells, and NHBE cells (49) are obtained from NCBI Sequence Read Archive with
BioProject ID: PRJNA615032 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA615032). RNA sequencing
data of SARS-CoV-2-infected human intestinal organoids (50) are obtained from NCBI Sequence Read
Archive with BioProject ID: PRJNA628628 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA628628/).
RNA sequencing data of IRF1 knockout and control human hepatocytes infected with hepatitis A virus are
are obtained from NCBI Sequence Read Archive with BioProject ID: PRJNA473130
Page 10/21
(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA473130/). RNA sequencing data of STAT1 knockout
and control human HepG2 cells treated by IFN (51) are obtained from NCBI Sequence Read Archive with
BioProject ID: PRJNA384926 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA384926/). RNA
sequencing data of human tissues and cell types (52) are obtained from NCBI Sequence Read Archive
with BioProject ID: PRJNA324812 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA324812/). RNA
sequencing data of human early embryos and embryonic stem cells (53) are obtained from NCBI
Sequence Read Archive with BioProject ID: PRJNA153427
(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA153427/). RNA sequencing data of 8-cell mouse
embryos and adult mouse islet developed from zygotes with injection of sperm tsRNAs from high-fat-diet
males (32) are obtained from NCBI Sequence Read Archive with BioProject ID: PRJNA304514
(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA304514/).
Competing interests
The authors declare that they have no competing interests.
Ethics approval and consent to participate
Not applicable
Consent for publication
Not applicable
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Figures
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Figure 1
Analysis of transcriptome alteration induced by infection of various coronaviruses. A: MA plot (log ratio
RNA abundance versus log abundance) of RNA-seq data comparing control and coronavirus-infected
cells. Differentially expressed genes with adjusted P<0.05 are highlighted in red. Numbers of up/down-
regulated genes are indicated. Calu-3, MRC5 and A549 are all human cells with lung origin. MERS, MERS-
CoV; SARS, SARS-CoV; CoV2, SARS-CoV-2. B: Venn diagrams document 26 commonly upregulated genes
by different coronavirus infection (left panel). Gene ontology analysis of the 26 genes for enriched
biological processes (right panel). C: Venn diagrams document only 3 commonly downregulated genes
by SARS-CoV-2 infection in two cell types. D: Bar graph indicates percentage of reads mapped to
Page 15/21
coronavirus genome to total mapped reads in human cells infected with coronavirus. E: Bar graphs
demonstrate that SARS-CoV-2 infection caused change of ACE2 expression from below detection to low
level in A549 cells. SARS-CoV-2 infection also caused upregulation of IRF1 and STAT1. IRF1 knockout in
human hepatocytes infected with hepatitis A virus signicantly decreased ACE2 expression. STAT1
knockout in IFN-treated human HepG2 cells signicantly decreased ACE2 expression.
Page 16/21
Page 17/21
Figure 2
Coronavirus infection in human cells enhanced retrotransposon expression. A: Bar graphs show that after
24-hr MERS-CoV/SARS-CoV/SARS-CoV-2 infection in Calu-3/MRC5/A549 cells, expression of TE was
generally increased. B: Heatmap indicates upregulation of several LTR, LINE and SINE elements induced
by MERS-CoV/SARS-CoV/SARS-CoV-2 infection. C: Ratio of LINE1 upregulation was not determined by
evolutionary age of LINE1 elements. Evolutionary age was calculated based on a substitution rate of
0.17%/million years. Mya, million years ago. D: Bar graphs depict expression of genes encoding enzymes
controlling DNA methylation status before and after MERS-CoV/SARS-CoV/SARS-CoV-2 infection in Calu-
3/MRC5/A549 cells. E: Bar graphs show that 24hrs or 60hrs post SARS-CoV-2 infection in human
intestinal organoid, expression of TE was generally increased. F: Bar graph indicates percentage of reads
mapped to SARS-CoV-2 genome to total mapped reads in transcriptome of human intestinal organoids
infected with SARS-CoV-2.
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Figure 3
Potential long-term memory of TE upregulation. A: Expression of TE subfamilies in 8-cell (8C) embryos
developed from normal mouse zygote injected with tsRNA derived from sperm of high-fat-diet (HFD)
male. B: Expression of TE subfamilies in adult islet developed from normal mouse zygote injected with
tsRNA derived from sperm of HFD male. C: Expression of LTR elements in 8C embryos developed from
normal mouse zygote injected with tsRNA derived from sperm of HFD male. D: Expression of LTR
elements in adult islet developed from normal mouse zygote injected with tsRNA derived from sperm of
HFD male. E: Scheme of the process from obtaining tsRNA injected zygote to examining TE expression in
8C embryos and adult islet.
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Figure 4
Retrotransposon-coronavirus chimeric transcripts were observed in SARS-CoV-2 infected human cells. A:
Bar graph shows relative enrichment of chimeric transcripts of coronavirus and cellular transcripts to
total coronavirus transcripts. B: Examination of ratio of mapped TE reads to non-TE gene reads in total
transcriptome and in subtranscriptome of chimeric reads (between viral and cellular transcripts). C:
Example of chimeric reads with junctions of coronavirus-gene, coronavirus -LINE and coronavirus-SINE.
D: Pie charts demonstrate distribution of TE subfamilies in total transcripts (left panel) and coronavirus-
retrotransposon chimeric transcripts (right panel) in SARS-CoV-2 infected Calu-3 cells. Red arrow
indicates overrepresentation of LINE reads. E: Pie charts demonstrate distribution of LINE members in
total transcripts (left panel) and coronavirus-retrotransposon chimeric transcripts (right panel) in SARS-
CoV-2 infected Calu-3 cells. Red arrow indicates overrepresentation of LINE-1 (L1) reads. F: IGV snapshot
of SARS-CoV-2 transcripts (upper) and chimeric transcripts (between viral and cellular transcripts, lower)
identied in infected Calu-3 cells. SARS-CoV-2 genome was used for alignment. Logarithmic scale is
displayed. The reference panel was obtained from UCSC genome browser.
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Figure 5
Model of how coronavirus may impact retrotransposons to harm human cells. A: Generally, entry of
coronavirus into human cells enhances TET activity for genome-wide DNA hypomethylation to facilitate
retrotransposon enhancement. This may lead to increased fusion between viral and retrotransposon
transcripts, reduced genome stability and increased susceptibility of aged people and cancer patients,
dysregulated TE-adjacent gene expression, and this inuence may be inherited for a long term.
Meanwhile, increased retrovirus-like particles of retrotransposons may be induced. B-G: Endogenous
retrotransposon expression and distribution of subfamilies are variable in human tissues and cells. Bar
graphs indicate percentages of reads mapped to TE to reads mapped to genes in human tissues and
immunocytes (B), human oocytes and early embryos (C), and normal Calu-3, MRC5 and A549 cells (D).
Bar charts (E-G) demonstrate distribution of individual subfamilies of TE in tissues or cell types shown
above. H: An example of retrotransposon-initiated gene expression by readthrough mechanism.
Page 21/21
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
SupplementalFile.docx
... In particular, relatively little is known about the processes following viral infection and why some individuals develop little to no symptoms, while others develop life-threatening or persistent ("long") COVID-19. Recent studies have identified host-virus chimeric (HVC) reads in RNA-sequencing data from SARS-CoV2 infected cells and samples from COVID-19 patients 15,16 . Both studies have suggested that HVC events support potential "human genome invasion" and "integration" by SARS-CoV2. ...
... RNA-sequencing reads that partly align to the host genome and partly align to the viral genome are the signature of HVC events in RNA-sequencing datasets. Recent reports 15,16 suggesting the presence of HVC events in SARS-CoV2-infected cells have been interpreted as supporting viral integration into the human genome as a mechanism of viral persistence. To gain insights into the authenticity of these events, we re-analyzed the 3 available RNA-seq datasets from patients with COVID-19 (n=57 samples) and in vitro SARS-CoV2 infected cells (n=64 samples). ...
... We next quantified the reads that partly mapped to the human genome and partly mapped to the SARS-CoV2 genome (see methods). We found that nearly 0.05-1% of all viral reads are formed of hybrid sequences between host and virus RNAs, a frequency consistent with that recently reported by others 15,16 (Fig. 1C). Infected A549-ACE2 and Calu-3 cells had the highest 105 and is also made available for use under a CC0 license. ...
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Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle, it is biologically unlikely to be in a location where splicing events could result in genome integration. Here, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines, were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ~1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background noise. Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes.
... Genetic and epigenetic mosaicisms of nonpolymorphic Alus as well as polymorphic Alus located in other genes, particularly the apolipoprotein cluster (APOA1, APOC3, APOA4) and HLA loci [91][92][93], could also influence the host response and disease outcomes of COVID-19. Enhanced transcription of Alus during viral infection is a known phenomenon and the expression of Alu and other retrotransposons in human cells have been found to increase during coronavirus infection [94]. Double-stranded RNAs transcribed from Alu elements have been shown to activate antiviral innate immune signaling pathways in mitochondria through MDA5 [95]. ...
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