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Research Article
REVERSE-TRANSCRIBED SARS-COV-2 RNA CAN INTEGRATE INTO THE GENOME OF CULTURED
HUMAN CELLS AND CAN BE EXPRESSED IN PATIENT-DERIVED TISSUES
Liguo Zhang, Alexsia Richards, View ORCID ProfileM. Inmaculada Barrasa, View
ORCID ProfileStephen H. Hughes, View ORCID ProfileRichard A. Young, and Rudolf
Jaenisch
1. aWhitehead Institute for Biomedical Research, Cambridge, MA 02142;
2. bHIV Dynamics and Replication Program, Center for Cancer Research, National
Cancer Institute, Frederick, MD 21702;
3. cDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA
02142
See allHide authors and affiliations
PNAS May 25, 2021 118 (21) e2105968118; https://doi.org/10.1073/pnas.2105968118
Liguo Zhang
aWhitehead Institute for Biomedical Research, Cambridge, MA 02142;
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Alexsia Richards
aWhitehead Institute for Biomedical Research, Cambridge, MA 02142;
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M. Inmaculada Barrasa
aWhitehead Institute for Biomedical Research, Cambridge, MA 02142;
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* ORCID record for M. Inmaculada Barrasa
Stephen H. Hughes
bHIV Dynamics and Replication Program, Center for Cancer Research, National
Cancer Institute, Frederick, MD 21702;
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* ORCID record for Stephen H. Hughes
Richard A. Young
aWhitehead Institute for Biomedical Research, Cambridge, MA 02142;
cDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA
02142
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* ORCID record for Richard A. Young
Rudolf Jaenisch
aWhitehead Institute for Biomedical Research, Cambridge, MA 02142;
cDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA
02142
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* For correspondence: jaenisch@wi.mit.edu
1. Contributed by Rudolf Jaenisch, April 19, 2021 (sent for review March 29,
2021; reviewed by Anton Berns and Anna Marie Skalka)
This article has been updated
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SIGNIFICANCE
An unresolved issue of SARS-CoV-2 disease is that patients often remain positive
for viral RNA as detected by PCR many weeks after the initial infection in the
absence of evidence for viral replication. We show here that SARS-CoV-2 RNA can
be reverse-transcribed and integrated into the genome of the infected cell and
be expressed as chimeric transcripts fusing viral with cellular sequences.
Importantly, such chimeric transcripts are detected in patient-derived tissues.
Our data suggest that, in some patient tissues, the majority of all viral
transcripts are derived from integrated sequences. Our data provide an insight
into the consequence of SARS-CoV-2 infections that may help to explain why
patients can continue to produce viral RNA after recovery.
ABSTRACT
Prolonged detection of severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported
in patients after recovery from COVID-19, but some of these patients do not
appear to shed infectious virus. We investigated the possibility that SARS-CoV-2
RNAs can be reverse-transcribed and integrated into the DNA of human cells in
culture and that transcription of the integrated sequences might account for
some of the positive PCR tests seen in patients. In support of this hypothesis,
we found that DNA copies of SARS-CoV-2 sequences can be integrated into the
genome of infected human cells. We found target site duplications flanking the
viral sequences and consensus LINE1 endonuclease recognition sequences at the
integration sites, consistent with a LINE1 retrotransposon-mediated,
target-primed reverse transcription and retroposition mechanism. We also found,
in some patient-derived tissues, evidence suggesting that a large fraction of
the viral sequences is transcribed from integrated DNA copies of viral
sequences, generating viral–host chimeric transcripts. The integration and
transcription of viral sequences may thus contribute to the detection of viral
RNA by PCR in patients after infection and clinical recovery. Because we have
detected only subgenomic sequences derived mainly from the 3′ end of the viral
genome integrated into the DNA of the host cell, infectious virus cannot be
produced from the integrated subgenomic SARS-CoV-2 sequences.
* SARS-CoV-2
* reverse transcription
* LINE1
* genomic integration
* chimeric RNAs
Continuous or recurrent positive severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) PCR tests have been reported in samples taken from patients weeks
or months after recovery from an initial infection (1⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓–17).
Although bona fide reinfection with SARS-CoV-2 after recovery has recently been
reported (18), cohort-based studies with subjects held in strict quarantine
after they recovered from COVID-19 suggested that at least some “re-positive”
cases were not caused by reinfection (19, 20). Furthermore, no
replication-competent virus was isolated or spread from these PCR-positive
patients (1⇓–3, 5, 6, 12, 16), and the cause for the prolonged and recurrent
production of viral RNA remains unknown. SARS-CoV-2 is a positive-stranded RNA
virus. Like other beta-coronaviruses (SARS-CoV-1 and Middle East respiratory
syndrome-related coronavirus), SARS-CoV-2 employs an RNA-dependent RNA
polymerase to replicate its genomic RNA and transcribe subgenomic RNAs
(21⇓⇓–24). One possible explanation for the continued detection of SARS-CoV-2
viral RNA in the absence of virus reproduction is that, in some cases, DNA
copies of viral subgenomic RNAs may integrate into the DNA of the host cell by a
reverse transcription mechanism. Transcription of the integrated DNA copies
could be responsible for positive PCR tests long after the initial infection was
cleared. Indeed, nonretroviral RNA virus sequences have been detected in the
genomes of many vertebrate species (25, 26), with several integrations
exhibiting signals consistent with the integration of DNA copies of viral mRNAs
into the germline via ancient long interspersed nuclear element (LINE)
retrotransposons (reviewed in ref. 27). Furthermore, nonretroviral RNA viruses
such as vesicular stomatitis virus or lymphocytic choriomeningitis virus (LCMV)
can be reverse transcribed into DNA copies by an endogenous reverse
transcriptase (RT), and DNA copies of the viral sequences have been shown to
integrate into the DNA of host cells (28⇓–30). In addition, cellular RNAs, for
example the human APP transcripts, have been shown to be reverse-transcribed by
endogenous RT in neurons with the resultant APP fragments integrated into the
genome and expressed (31). Human LINE1 elements (∼17% of the human genome), a
type of autonomous retrotransposons, which are able to retro-transpose
themselves and other nonautonomous elements such as Alu, are a source of
cellular endogenous RT (32⇓–34). Endogenous LINE1 elements have been shown to be
expressed in aged human tissues (35) and LINE1-mediated somatic
retrotransposition is common in cancer patients (36, 37). Moreover, expression
of endogenous LINE1 and other retrotransposons in host cells is commonly
up-regulated upon viral infection, including SARS-CoV-2 infection (38⇓–40).
In this study, we show that SARS-CoV-2 sequences can integrate into the host
cell genome by a LINE1-mediated retroposition mechanism. We provide evidence
that the integrated viral sequences can be transcribed and that, in some patient
samples, the majority of viral transcripts appear to be derived from integrated
viral sequences.
RESULTS
INTEGRATION OF SARS-COV-2 SEQUENCES INTO THE DNA OF HOST CELLS IN CULTURE.
We used three different approaches to detect genomic SARS-CoV-2 sequences
integrated into the genome of infected cells. These approaches were Nanopore
long-read sequencing, Illumina paired-end whole genomic sequencing, and Tn5
tagmentation-based DNA integration site enrichment sequencing. All three methods
provided evidence that SARS-CoV-2 sequences can be integrated into the genome of
the host cell.
To increase the likelihood of detecting rare integration events, we transfected
HEK293T cells with LINE1 expression plasmids prior to infection with SARS-CoV-2
and isolated DNA from the cells 2 d after infection (SI Appendix, Fig. S1A). We
detected DNA copies of SARS-CoV-2 nucleocapsid (NC) sequences in the infected
cells by PCR (SI Appendix, Fig. S1B) and cloned the complete NC gene (SI
Appendix, Fig. S1D) from large-fragment cell genomic DNA that had been
gel-purified (SI Appendix, Fig. S1C). The viral DNA sequence (NC) was confirmed
by Sanger sequencing (Dataset S1). These results suggest that SARS-CoV-2 RNA can
be reverse-transcribed, and the resulting DNA could be integrated into the
genome of the host cell.
To demonstrate directly that the SARS-CoV-2 sequences were integrated into the
host cell genome, DNA isolated from infected LINE1-overexpressing HEK293T cells
was used for Nanopore long-read sequencing (Fig. 1A). Fig. 1 B–D shows an
example of a full-length viral NC subgenomic RNA sequence (1,662 bp) integrated
into the cell chromosome X and flanked on both sides by host DNA sequences.
Importantly, the flanking sequences included a 20-bp direct repeat. This target
site duplication is a signature of LINE1-mediated retro-integration (41, 42).
Another viral integrant comprising a partial NC subgenomic RNA sequence that was
flanked by a duplicated host cell DNA target sequence is shown in SI Appendix,
Fig. S2 A–C. In both cases, the flanking sequences contained a consensus
recognition sequence of the LINE1 endonuclease (43). These results indicate that
SARS-CoV-2 sequences can be integrated into the genomes of cultured human cells
by a LINE1-mediated retroposition mechanism. Table 1 summarizes all of the
linked SARS-CoV-2–host sequences that were recovered. DNA copies of portions of
the viral genome were found in almost all human chromosomes. In addition to the
two examples given in Fig. 1 and SI Appendix, Fig. S2, we also recovered
cellular sequences for 61 integrants for which only one of the two host–viral
junctions was retrieved (SI Appendix, Fig. S2 D–F and Table 1; Nanopore reads
containing the chimeric sequences summarized in Dataset S2). Importantly, about
67% of the flanking human sequences included either a consensus or a variant
LINE1 endonuclease recognition sequence (such as TTTT/A) (SI Appendix, Fig. S2
D–F and Table 1). These LINE1 recognition sequences were either at the chimeric
junctions that were directly linked to the 3′ end (poly-A tail) of viral
sequences, or within a distance of 8–27 bp from the junctions that were linked
to the 5′ end of viral sequences, which is within the potential target site
duplication. Both results are consistent with a model in which LINE1-mediated
retroposition provides a mechanism to integrate DNA copies of SARS-CoV-2
subgenomic fragments into host genomic DNA. About 71% of the viral sequences
were flanked by intron or intergenic cellular sequences and 29% by exons (Fig.
1F and Table 1). Thus, the association of the viral sequences with exons is much
higher than would be expected for random integration into the genome [human
genome: 1.1% exons, 24% introns, and 75% intergenic DNA (44)], suggestive of
preferential integration into exon-associated target sites. While previous
studies showed no preference for LINE1 retroposition into exons (45, 46), our
finding suggests that LINE1-mediated retroposition of some other RNAs may be
different. We noted that viral–cellular boundaries were frequently close to the
5′ or 3′ untranslated regions (UTRs) of the cellular genes, suggesting that
there is a preference for integration close to promoters or poly(A) sites in our
experimental system.
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Fig. 1.
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.
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Table 1.
Summary of the human-CoV2 chimeric sequences obtained by Nanopore DNA sequencing
of infected LINE1-overexpressing HEK293T cells
To confirm the integration of SARS-CoV-2 sequences into genomic DNA by another
method, we subjected DNA isolated from LINE1-transfected and SARS-CoV-2–infected
HEK293T cells to Illumina paired-end whole-genome sequencing, using a Tn5-based
library construction method (Illumina Nextera) to avoid ligation artifacts.
Viral DNA reads were concentrated at the 3′ end of the SARS-CoV-2 genome (SI
Appendix, Fig. S3). We recovered 17 viral integrants (sum of two replicates), by
mapping human–viral chimeric DNA sequences (Fig. 1E and Table 2, chimeric
sequences summarized in Dataset S3); 7 (41%) of the junctions contained either a
consensus or a variant LINE1 recognition sequence in the cellular sequences near
the junction (Fig. 1E and Table 2), consistent with a LINE1-mediated
retroposition mechanism. Similar to the results obtained from Nanopore
sequencing, about 76% of the viral sequences were flanked by intron or
intergenic cellular sequences and 24% by exons (Fig. 1F and Table 2).
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Table 2.
Summary of the human-CoV2 chimeric sequences obtained by Illumina paired-end
whole-genome DNA sequencing of infected LINE1-overexpressing HEK293T cells
About 32% of SARS-CoV-2 sequences (6/21 integration events in Nanopore, 4/10 in
Illumina data) were integrated at LINEs, short interspersed nuclear elements, or
long terminal repeat elements without evidence for a LINE1 recognition site,
suggesting that there may be an alternative reverse transcription/integration
mechanism, possibly similar to that reported for cells acutely infected with
LCMV, which resulted in integrated LCMV sequences fused to intracisternal A-type
particle (IAP) sequences (29).
To assess whether genomic integration of SARS-CoV-2 sequences could also occur
in infected cells that did not overexpress RT, we isolated DNA from
virus-infected HEK293T and Calu3 cells that were not transfected with an RT
expression plasmid (Fig. 2A). Tn5 tagmentation-mediated DNA integration site
enrichment sequencing (47, 48) (Fig. 2B and SI Appendix, Fig. S4A) detected a
total of seven SARS-CoV-2 sequences fused to cellular sequences in these cells
(sum of three independent infections of two cell lines), all of which showed
LINE1 recognition sequences close to the human–SARS-CoV-2 sequence junctions
(Fig. 2 C–F and SI Appendix, Fig. S4 B–D, chimeric sequences summarized in
Dataset S4).
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Fig. 2.
Evidence for integration of SARS-CoV-2 cDNA in cultured cells that do not
overexpress a reverse transcriptase. (A) Experimental workflow. (B) Experimental
design for the Tn5 tagmentation-mediated enrichment sequencing method used to
map integration sites in the host cell genome. (C) A human–viral chimeric read
pair supporting viral integration. The reads are aligned with the human (blue)
and SARS-CoV-2 (magenta) genomic sequences. The arrows indicate the read
orientations relative to the human and SARS-CoV-2 genomes as shown in D and E.
Sequence of the viral primer used for enrichment is shown with green highlight
in the read (corresponding to the green arrow illustrated in B). Sequences that
could be mapped to both genomes are shown in purple. (D) Alignment of the read
pair in C with the human genome (chromosome 15, blue arrow). The highlighted
(light blue) region of the human sequence is enlarged to show the LINE1
recognition sequence (underlined) with a 19-base poly-dT sequence (purple
highlight) that could be annealed by the viral poly-A tail for “target-primed
reverse transcription.” Additional 5-bp human sequence (GAATG, blue) was
captured in read 2 (C), supporting a bona fide integration site. (E) Alignment
of the read pair in C with the SARS-CoV-2 genome (magenta). The viral primer
sequence is shown with green highlight. (F) Summary of seven human–viral
chimeric sequences identified by the enrichment sequencing method in the two
cell lines showing the integrated human chromosomes, LINE1 recognition sequences
close to the chimeric junction, and human genomic features at the read junction.
EXPRESSION OF VIRAL–CELLULAR CHIMERIC TRANSCRIPTS IN INFECTED CULTURED CELLS AND
PATIENT-DERIVED TISSUES.
To investigate the possibility that SARS-CoV-2 sequences integrated into the
genome can be expressed, we analyzed published RNA-seq data from
SARS-CoV-2–infected cells for evidence of chimeric transcripts (49). Examination
of these datasets (50⇓⇓⇓⇓–55) (SI Appendix, Fig. S5) revealed a number of
human–viral chimeric reads (SI Appendix, Fig. S6 A and B). These occurred in
multiple sample types, including cultured cells and organoids from
lung/heart/brain/stomach tissues (SI Appendix, Fig. S6B). The abundance of the
chimeric reads positively correlated with viral RNA level across the sample
types (SI Appendix, Fig. S6B). Chimeric reads generally accounted for
0.004–0.14% of the total SARS-CoV-2 reads in the samples. A majority of the
chimeric junctions mapped to the sequence of the SARS-CoV-2 NC gene (SI
Appendix, Fig. S6 C and D). This is consistent with the finding that NC RNA is
the most abundant SARS-CoV-2 subgenomic RNA (56), making it the most likely
target for reverse transcription and integration. However, recent data showed
that up to 1% of RNA-seq reads from SARS-CoV-2–infected cells can be
artifactually chimeric as a result of RT switching between RNA templates, which
can occur during the cDNA synthesis step in the preparation of a RNA-seq library
(57). Thus, because there is a mixture of host mRNAs and positive-strand viral
mRNAs in infected cells, the identification of genuine chimeric viral–cellular
RNA transcripts is compromised by the generation of artifactual chimeras in the
assays.
We reasoned that the orientation of an integrated DNA copy of SARS-CoV-2 RNA
should be random with respect to the orientation of the targeted host gene,
predicting that about half the viral DNAs that were integrated into an expressed
host gene should be in an orientation opposite to the direction of the host cell
gene’s transcription (Fig. 3A). As predicted, ∼50% of viral integrants in human
genes were in the opposite orientation relative to the host gene in our Nanopore
dataset (integration at human genes with LINE1 recognition sequences, Fig. 3B).
Thus, for chimeric transcripts derived from integrated viral sequences, we would
expect that ∼50% of the chimeric transcripts should contain negative-strand
viral sequences linked to positive-strand host RNA sequences. We therefore
determined the fraction of the viral and human–viral chimeric transcripts in
infected cultured cells/organoids and in patient-derived tissues containing
negative-strand viral RNA sequences.
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Fig. 3.
Negative-strand viral RNA-seq reads suggest that integrated SARS-CoV-2 sequences
are expressed. (A) Schema predicting fractions of positive- or negative-strand
SARS-CoV-2 RNA-seq reads that are derived from viral (sub)genomic RNAs or from
transcripts of integrated viral sequences. The arrows (Right) showing the
orientation of an integrated SARS-CoV-2 (magenta) positive strand relative to
the orientation of the host cellular gene (blue). (B) Fractions of SARS-CoV-2
sequences integrated into human genes with same (n = 15) or opposite (n = 13)
orientation of the viral positive strand relative to the positive strand of the
human gene. A total of 28 integration events at human genes with LINE1
endonuclease recognition sequences were identified from our Nanopore DNA
sequencing of infected LINE1-overexpressing HEK293T cells (Fig. 1A). (C)
Fraction of total viral reads that are derived from negative-strand viral RNA in
acutely infected cells or organoids (see SI Appendix, Table S1 for details). (D)
Fraction of human–viral chimeric reads that contain viral sequences derived from
negative-strand viral RNA in acutely infected cells or organoids (see SI
Appendix, Table S1 for details). (E) Fraction of total viral reads that are
derived from negative-strand viral RNA in published patient RNA-seq data
(autopsy FFPE samples, GSE150316, samples with no viral reads or of low library
strandedness quality not included; see SI Appendix, Table S2 for details;
reanalysis results consistent with the original publication). (F) Fraction of
human–viral chimeric reads that contain viral sequences derived from
negative-strand viral RNA in published patient RNA-seq data (autopsy FFPE
samples, GSE150316; see SI Appendix, Table S2 for details). (G) Fraction of
total viral reads that are derived from negative-strand viral RNA in published
patient RNA-seq data (BALF samples, GSE145926; see SI Appendix, Table S3 for
details). The red dashed lines in E–G indicate the level at which 50% of all
viral reads (E and G) or viral sequences in human–viral chimeric reads (F) were
from negative-strand viral RNAs, a level expected if all the viral sequences
were derived from integrated sequences.
The replication of SARS-CoV2 RNA requires the synthesis of negative-strand viral
RNA, which serves as template for replication of viral genomic RNA and
transcription of viral subgenomic positive-strand RNA (21). To assess the
prevalence of negative-strand viral RNA in acutely infected cells, we determined
the ratio of total positive to negative-strand RNAs. Between 0 and 0.1% of total
viral reads were derived from negative-strand RNA in acutely infected Calu3
cells or lung organoids [our data and published data (50, 58)] (Fig. 3C and SI
Appendix, Table S1), similar to what has been reported in clinical samples taken
early after infection (59). These results argue that the level of
negative-strand viral RNA is at least 1,000-fold lower than that of
positive-strand viral RNA in acutely infected cells, due at least in part to a
massive production of positive-strand subgenomic RNA during viral replication.
This greatly reduces the likelihood that random template switching during the
reverse transcription step in the RNA-seq library construction would generate a
large fraction of the artifactual chimeric reads that would contain viral
negative-strand RNA fused to cellular positive-strand RNA sequences. We
determined that between 0 and 1% of human–viral chimeric reads contained
negative-strand viral sequences in the acutely infected cells/organoids (Fig. 3D
and SI Appendix, Table S1), consistent with a small fraction of viral reads
being derived from integrated SARS-CoV-2 sequences.
In contrast to the results obtained with acutely infected Calu3 cells or lung
organoids, up to 51% of all viral reads, and up to 42.5% of human–viral chimeric
reads, were derived from the negative-strand SARS-CoV-2 RNA in some
patient-derived tissues [published data (60, 61), patient clinical background
available in the original publications] (Fig. 3 E–G and SI Appendix, Tables S2
and S3). Single-cell analysis of patient lung bronchoalveolar lavage fluid
(BALF) cells from patients with severe COVID [published data (61)] showed that
up to 40% of all viral reads were derived from the negative-strand SARS-CoV-2
RNA (SI Appendix, Fig. S7). Fractions of negative-strand RNA in tissues from
some patients were orders of magnitude higher than those in acutely infected
cells or organoids (Fig. 3 C–G). In fixed (formalin-fixed, paraffin-embedded
[FFPE]) autopsy samples, in 4 out of 14 patients (Fig. 3E and SI Appendix, Table
S2), and in BALF samples, in 4 out of 6 patients (Fig. 3G and SI Appendix, Table
S3), at least ∼20% of the viral reads were derived from negative-strand viral
RNA. In contrast to acutely infected cells (Fig. 3 C and D and SI Appendix,
Table S1), there was little or no evidence for virus reproduction in these
autopsy samples (60). As summarized in SI Appendix, Table S2, there were
negative-strand viral sequences in a large fraction of the human–viral chimeric
reads (up to ∼40%) in samples from one patient. Different samples derived from
the same patient revealed a similarly high fraction of negative viral
strand–human RNA reads. Several other patient samples revealed lower fraction of
negative viral strand RNA–human RNA chimeras, which were, however, still
significantly higher than what was found in acutely infected cells (Fig. 3 D and
F and SI Appendix, Table S1 and S2). Because the ability to identify viral–human
chimeric reads using short-read RNA-seq is limited, our analysis failed to show
significant numbers of chimeric reads in patient BALF samples (SI Appendix,
Table S3). In summary, our data suggest that in some patient-derived tissues,
where the total number of SARS-CoV-2 sequence-positive cells may be small, a
large fraction of the viral transcripts could have been transcribed from
SARS-CoV-2 sequences integrated into the host genome.
DISCUSSION
We present here evidence that SARS-CoV-2 sequences can be reverse-transcribed
and integrated into the DNA of infected human cells in culture. For two of the
integrants, we recovered “human–viral–human” chimeric reads encompassing a
direct target site repeat (20 or 13 bp), and a consensus recognition site of the
LINE1 endonuclease was present on both ends of the host DNA that flanked the
viral sequences. These and other data are consistent with a target primed
reverse transcription and retroposition integration mechanism (41, 42) and
suggest that endogenous LINE1 RT can be involved in the reverse transcription
and integration of SARS-CoV-2 sequences in the genomes of infected cells.
Approximately 30% of viral integrants analyzed in cultured cells lacked a
recognizable nearby LINE1 endonuclease recognition site. Thus, it is also
possible that integration can occur by another mechanism. Indeed, there is
evidence that chimeric cDNAs can be produced in cells acutely infected with LCMV
by copy choice with endogenous IAP elements during reverse transcription. This
mechanism is expected to create a chimeric cDNA complementary to both LCMV and
IAP. In some cases, the resulting chimeric cDNAs were integrated without the
generation of a target site duplication (29). A recent study has also suggested
that the interaction between coronavirus sequences and endogenous
retrotransposon could be a potential viral integration mechanism (40).
It will be important, in follow-up studies, to demonstrate the presence of
SARS-CoV-2 sequences integrated into the host genome in patient tissues.
However, this will be technically challenging because only a small fraction of
cells in any patient tissues are expected to be positive for viral sequences
(61). Consistent with this notion, it has been estimated that only between 1 in
1,000 and 1 in 100,000 mouse cells infected with LCMV either in culture or in
the animal carried viral DNA copies integrated into the genome (30). In
addition, only a fraction of patients may carry SARS-CoV-2 sequences integrated
in the DNA of some cells. However, with more than 140 million humans infected
with SARS-CoV-2 worldwide (as of April, 2021), even a rare event could be of
significant clinical relevance. It is also challenging to estimate the frequency
of retro-integration events in cell culture assays since infected cells usually
die and are lost before sample collection. For the same reason, no clonal
expansion of integrated cells is expected in acute infection experiments.
Moreover, the chance of integration at the same genomic locus in different
patients/tissues may be low, due to a random integration process.
The presence of chimeric virus–host RNAs in cells cannot alone be taken as
strong evidence for transcription of integrated viral sequences because template
switching can happen during the reverse transcription step of cDNA library
preparation. However, we found that only a very small fraction (0–1%) of
chimeric reads from acutely infected cells contained negative-strand viral RNA
sequences, whereas, in the RNA-seq libraries prepared from some patients, the
fraction of total viral reads, and the fraction of human–viral chimeric reads
that were derived from negative-strand SARS-CoV-2 RNAs was substantially higher.
For retrotransposon-mediated integration events, the orientation of the
reverse-transcribed SARS-CoV-2 RNA should be random with respect to the
orientation of a host gene. Thus, for chimeric RNAs derived from integrated
viral sequences, about half of the chimeric reads will link positive-strand host
RNA sequences to negative-strand viral sequences. In some patient samples,
negative-strand viral reads accounted for 40–50% of the total viral RNA
sequences and a similar fraction of the chimeric reads contained negative-strand
viral RNA sequences, suggesting that the majority if not all of the viral RNAs
in these samples were derived from integrated viral sequences.
It is important to note that, because we have detected only subgenomic sequences
derived mainly from the 3′ end of the viral genome integrated into the DNA of
the host cell, infectious virus cannot be produced from such integrated
subgenomic SARS-CoV-2 sequences. The possibility that SARS-CoV-2 sequences can
be integrated into the human genome and expressed in the form of chimeric RNAs
raises several questions for future studies. Do integrated SARS-CoV-2 sequences
express viral antigens in patients and might these influence the clinical course
of the disease? The available clinical evidence suggests that, at most, only a
small fraction of the cells in patient tissues express viral proteins at a level
that is detectable by immunohistochemistry. However, if a cell with an
integrated and expressed SARS-CoV-2 sequences survives and presents a viral- or
neo-antigen 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 (62, 63). The presence of LCMV sequences integrated in the genomes
of acutely infected cells in mice led the authors to speculate that expression
of such sequences “potentially represents a naturally produced form of DNA
vaccine” (30). It is not known how many antigen-presenting cells are needed to
elicit an antigen response, but derepressed LINE1 expression, induced by viral
infection or by exposure to cytokines (38⇓–40), may stimulate SARS-CoV-2
integration into the genome of infected cells in patients. More generally, our
results suggest that integration of viral DNA in somatic cells may represent a
consequence of a natural infection that could play a role in the effects of
other common disease-causing RNA viruses such as dengue, Zika, or influenza
virus.
Our results may also be relevant for current clinical trials of antiviral
therapies (64). 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.
MATERIALS AND METHODS
CELL CULTURE AND PLASMID TRANSFECTION.
HEK293T cells were obtained from ATCC (CRL-3216) and cultured in DMEM
supplemented with 10% heat-inactivated FBS (HyClone; SH30396.03) and 2 mM
l-glutamine (MP Biomedicals; IC10180683) following ATCC’s method. Calu3 cells
were obtained from ATCC (HTB-55) and cultured in EMEM (ATCC; 30-2003)
supplemented with 10% heat-inactivated FBS (HyClone; SH30396.03) following
ATCC’s method.
Plasmids for human LINE1 expression, pBS-L1PA1-CH-mneo (CMV-LINE-1), was a gift
from Astrid Roy-Engel, Tulane University Health Sciences Center, New Orleans, LA
(Addgene plasmid #51288 ; http://addgene.org/51288; RRID:Addgene_51288) (65);
EF06R (5′UTR-LINE-1) was a gift from Eline Luning Prak, University of
Pennsylvania, Philadelphia, PA (Addgene plasmid #42940 ;
http://addgene.org/42940; RRID:Addgene_42940) (66). Transfection was done with
Lipofectamine 3000 (Invitrogen; L3000001) following manufacturer’s protocol.
SARS-COV-2 INFECTION.
SARS-CoV-2 USA-WA1/2020 (GenBank: MN985325.1) was obtained from BEI Resources
and expanded and tittered on Vero cells. Cells were infected in DMEM plus 2% FBS
for 48 h using a multiplicity of infection (MOI) of 0.5 for infection of HEK293T
cells and an MOI of 1 or 2 for Calu3 cells. All sample processing and harvest
with infectious virus were done in the BSL3 facility at the Ragon Institute.
NUCLEIC ACIDS EXTRACTION AND PCR ASSAY.
Cellular DNA extraction was done using a published method (31). For purification
of genomic DNA, total cellular DNA was fractionated on a 0.4% (wt/vol)
agarose/1× TAE gel for 1.5 h with a 3 V/cm voltage, with λ DNA-HindIII Digest
(NEB; N3012S) as size markers. Large fragments (>23.13 kb) were cut out, frozen
in −80 °C, and then crushed with a pipette tip. Three volumes (vol/wt) of high
T-E buffer (10 mM Tris–10 mM EDTA, pH 8.0) were added, and then NaCl was added
to give a final concentration of 200 mM. The gel solution was heated at 70 °C
for 15 min with constant mixing and then extracted with
phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol/vol) (Life Technologies;
15593031) and chloroform:isoamyl alcohol 24:1 (Sigma; C0549-1PT). DNA was
precipitated by the addition of sodium acetate and isopropyl alcohol. For
samples with low DNA concentration, glycogen (Life Technologies; 10814010) was
added as a carrier to aid precipitation.
RNA extraction was done with RNeasy Plus Micro Kit (Qiagen; 74034) following
manufacturer’s protocol.
To detect DNA copies of SARS-CoV-2 sequences, we chose four NC gene-targeting
PCR primer sets that are used in COVID-19 tests [SI Appendix, Fig. S1A, primer
source from World Health Organization (67), modified to match the genome version
of NC_045512.2]. See SI Appendix, Table S4 for PCR primer sequences used in this
study. PCR was done using AccuPrime Taq DNA Polymerase, high fidelity (Life
Technologies; 12346094). PCR products were run on 1% or 2% (wt/vol) agarose gel
to show amplifications.
NANOPORE DNA SEQUENCING AND ANALYSIS.
A total of 1.6 μg of DNA extracted from HEK293T cells transfected with the
pBS-L1PA1-CH-mneo (CMV-LINE-1) plasmid and infected with SARS-CoV-2 was used to
make a sequencing library with the SQK-LSK109 kit (Oxford Nanopore Technologies)
and sequenced on one R9 PromethION flowcell (FLO-PRO002) for 3 d and 5 min. The
sequencing data were base-called using Guppy 4.0.11 (Oxford Nanopore
Technologies) using the high-accuracy model.
Nanopore reads were mapped using minimap2 (68) (version 2.15) with parameters
“-p 0.3 -ax map-ont” and a fasta file containing the human genome sequence from
ENSEMBL release 93
(ftp://ftp.ensembl.org/pub/release-93/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz)
concatenated to the SARS-CoV-2 sequence, GenBank ID: MN988713.1, “Severe acute
respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/USA/IL-CDC-IL1/2020,
complete genome.” From the SAM file, we selected all the sequences that mapped
to the viral genome and divided them into groups based on the human chromosomes
they mapped to. We blasted the selected sequences, using blastn, against a BLAST
database made with the human and virus sequences described above. We parsed the
blast output into a text file containing one row per high-scoring segment pair
(HSP) with a custom perl script. We further filtered that file, for each
sequence, by selecting all the viral HSPs and the top three human HSPs. We
inspected those files visually to identify sequences containing
human–viral–human or human–viral junctions. For a few sequences, longer than 30
kb, we inspected the top 15 human HSPs. Additionally, we visually inspected all
the identified reads containing human and viral sequences by the University of
California, Santa Cruz (UCSC) BLAT (69) tool. Due to errors in Nanopore
sequencing and/or base-calling, artifactual “hybrid sequences” exist in a subset
of these reads, sometimes with Watson and Crick strands from the same DNA
fragment present in the same read. Therefore, we only focused on chimeric
sequences showing clear human–viral junctions and analyzed known LINE1-mediated
retroposition features such as target-site duplications and LINE1 endonuclease
recognition sequences for evidence of integration.
TN5 TAGMENTATION-MEDIATED INTEGRATION SITE ENRICHMENT.
We used a tagmentation-based method to enrich for viral integration sites (47,
48). Briefly, we used Tn5 transposase (Diagenode; C01070010) to randomly tagment
the cellular DNA with adapters (adapter A, the Illumina Nextera system).
Tagmentation was done using 100 ng of DNA for 10 min at 55 °C, followed by
stripping off the Tn5 transposase from the DNA with SDS. We used a reverse
primer targeting the near-5′ end of SARS-CoV-2 NC gene (CCA AGA CGC AGT ATT ATT
GGG TAA A) or a forward primer targeting the near-3′ end of SARS-CoV-2 genome
(CTT GTG CAG AAT GAA TTC TCG TAA CT) to linearly amplify (PCR0, 45 cycles) the
tagmented DNA fragments containing viral sequences. We took the product of PCR0
and amplified the DNA fragments containing adapter and viral sequences
(potential integration sites) using 15–20 cycles of PCR1, with a barcoded (i5)
Nextera primer (AAT GAT ACG GCG ACC ACC GAG ATC TAC ACN NNN NNN NTC GTC GGC AGC
GTC, NNNNNNNN indicates the barcode) against the adapter sequence and a viral
primer. The viral primer was designed to either target the near-5′ end of
SARS-CoV-2 NC gene (GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGCC GAC GTT GTT
TTG ATC G, viral sequence underlined) or target the near-3′ end of SRAS-CoV-2
genome (GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GCGC GGA GTA CGA TCG AGT G,
viral sequence underlined). The viral primer also contained an adapter sequence
for further PCR amplification. We amplified the PCR1 product by 15–20 cycles of
PCR2, using a short primer (AAT GAT ACG GCG ACC ACC GA) against the i5 Nextera
primer sequence and a barcoded (i7) Nextera primer (CAA GCA GAA GAC GGC ATA CGA
GAT NNN NNN NNG TCT CGT GGG CTC GG, NNNNNNNN indicates the barcode) against the
adapter sequence introduced by the viral primer in PCR1. The final product of
the PCR2 amplification was fractionated on 1.5% agarose gel (Sage Science;
HTC1510) with PippinHT (Sage Science; HTP0001) and 500- to 1,000-bp pieces were
selected for Illumina paired-end sequencing. All three PCR steps (PCR0–PCR2)
were done with KAPA HiFi HotStart ReadyMix (KAPA;KK2602).
ILLUMINA DNA SEQUENCING AND ANALYSIS.
We constructed libraries for HEK293T cell whole-genome sequencing using the
Tn5-based Illumina DNA Prep kit (Illumina; 20018704). The whole-genome
sequencing libraries or the libraries from Tn5-mediated integration site
enrichment after sizing (described above) were subjected to Illumina sequencing.
qPCR was used to measure the concentrations of each library using KAPA qPCR
library quant kit according to the manufacturer’s protocol. Libraries were then
pooled at equimolar concentrations, for each lane, based on qPCR concentrations.
The pooled libraries were denatured using the Illumina protocol. The denatured
libraries were loaded onto an SP flowcell on an Illumina NovaSeq 6000 and run
for 2 × 150 cycles according to the manufacturer’s instructions. Fastq files
were generated and demultiplexed with the bcl2fastq Conversion Software
(Illumina).
To identify human–SARS-CoV-2 chimeric DNA reads, raw sequencing reads were
aligned with STAR (70) (version 2.7.1a) to a human plus SARS-CoV-2 genome made
with a fasta file containing the human genome sequence version hg38 with no
alternative chromosomes concatenated to the SARS-CoV-2 sequence from National
Center for Biotechnology Information (NCBI) reference sequence NC_045512.2. The
following STAR parameters were used to call chimeric reads: –alignIntronMax 1
\–chimOutType Junctions SeparateSAMold WithinBAM HardClip
\–chimScoreJunctionNonGTAG 0 \–alignSJstitchMismatchNmax -1–1 -1–1
\–chimSegmentMin 25 \–chimJunctionOverhangMin 25 \–outSAMtype BAM
SortedByCoordinate. We extracted viral reads from the generated BAM file by
samtools (71) (version 1.11) using command: samtools view -b
Aligned.sortedByCoord.out.bam NC_045512v2 > NC_Aligned.sortedByCoord.out.bam. We
extracted human–viral chimeric reads by using the read names from the STAR
generated Chimeric.out.junction file to get the read alignments from the STAR
generated Chimeric.out.sam file by Picard
(http://broadinstitute.github.io/picard), using command: java -jar picard.jar
FilterSamReads I = Chimeric.out.sam O = hv-Chimeric.out.sam READ_LIST_FILE =
hv-Chimeric.out.junction.ids FILTER = includeReadList. We further confirmed each
of the chimeric reads and filtered out any unconvincing reads (too short or
aligned to multiple sites of the human genome) by visual inspection with the
UCSC BLAT (69) tool. We also loaded the STAR generated
Aligned.sortedByCoord.out.bam file or the NC_Aligned.sortedByCoord.out.bam file
containing extracted viral reads to the UCSC browser SARS-CoV-2 genome
(NC_045512.2) to search for additional chimeric reads that were missed by the
STAR chimeric calling method. To generate genome coverage file, we used the
bamCoverage from the deepTools suite (72) (version 3.5.0) to convert the STAR
generated Aligned.sortedByCoord.out.bam file to a bigwig file binned at 10 bp,
using command: bamCoverage -b Aligned.sortedByCoord.out.bam -o
Aligned.sortedByCoord.out.bw–binSize 10.
RNA-SEQ AND ANALYSIS.
To identify human–SARS-CoV-2 chimeric reads, published RNA-seq data were
downloaded from Gene Expression Omnibus (GEO) with the accession numbers
GSE147507 (50), GSE153277 (51), GSE156754 (52), GSE157852 (53), GSE153684 (54),
and GSE154998 (55) (summarized in SI Appendix, Fig. S5C). Raw sequencing reads
were aligned with STAR (70) (version 2.7.1a) to human plus SARS-CoV-2 genome and
transcriptome made with a fasta file containing the human genome sequence
version hg38 with no alternative chromosomes concatenated to the SARS-CoV-2
sequence from NCBI reference sequence NC_045512.2, and a gtf file containing the
human gene annotations from ENSEMBL version GRCh38.97 concatenated to the
SARS-CoV-2 gene annotations from NCBI
(http://hgdownload.soe.ucsc.edu/goldenPath/wuhCor1/bigZips/genes/). The
following STAR parameters (56) were used to call chimeric reads unless otherwise
specified (SI Appendix, Fig. S5C):–chimOutType Junctions SeparateSAMold
WithinBAM HardClip \–chimScoreJunctionNonGTAG 0 \–alignSJstitchMismatchNmax -1–1
-1–1 \–chimSegmentMin 50 \–chimJunctionOverhangMin 50.
For RNA-seq strandedness analysis, we generated RNA-seq data using RNA from
SARS-CoV-2–infected Calu3 cells. Stranded libraries were constructed with the
Kapa mRNA HyperPrep kit (Roche; 08098115702). Libraries were qPCR'ed using a
KAPA qPCR library quant kit as per manufacturer’s protocol. Libraries were then
pooled at equimolar concentrations, for each lane, based on qPCR concentrations.
The pooled libraries were denatured using the Illumina protocol. The denatured
libraries were loaded onto an HiSeq 2500 (Illumina) and sequenced for 120 cycles
from one end of the fragments. Basecalls were performed using Illumina offline
basecaller (OLB) and then demultiplexed. We downloaded published RNA-seq data
(stranded libraries) from GEO with the accession numbers GSE147507 (50) (Calu3,
SI Appendix, Table S1), GSE148697 (58) (lung organoids, SI Appendix, Table S1),
and GSE150316 (60) (patient FFPE tissues, SI Appendix, Table S2). Raw RNA-seq
reads were aligned as described above, using parameters–chimSegmentMin 30
\–chimJunctionOverhangMin 30 to call chimeric reads. We extracted total viral
reads and human–viral chimeric reads as described above. We convert the viral
read BAM files into Bed files using the bamToBed utility in BEDTools (73). We
then counted the total and stranded read numbers in the converted BED files.
Published single-cell RNA-seq data were downloaded from GEO with the accession
number GSE145926 (61) (patient BALF samples, SI Appendix, Table S3). For bulk
analysis, duplicate reads with the same read1 (UMI) and read2 sequences in raw
fastq files were removed by dedup_hash (https://github.com/mvdbeek/dedup_hash).
Then the pool of read2 were aligned as described above, using parameters
–chimSegmentMin 30 \–chimJunctionOverhangMin 30 to call chimeric reads. Read
strandedness was analyzed as described above. For single-cell analysis, we
generated a custom genome by Cell Ranger (10× Genomics Cell Ranger 3.0.2) (74)
mkref, using a fasta file containing the human genome sequence from ENSEMBL
release 93
(ftp://ftp.ensembl.org/pub/release-93/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz)
concatenated to the SARS-CoV-2 sequence, GenBank ID: MN988713.1, and a gtf file
containing human and viral annotations. Read mapping, assigning reads to cell
barcodes and removing PCR duplicates were done with Cell Ranger (10× Genomics
Cell Ranger 4.0.0) (74) count, using the custom genome described above. We
processed the counts using Seurat (version 3.2.2) (75). We removed cells that
had less than 200 genes detected or more than 20% of transcript counts deriving
from the mitochondria. For each cell, we counted the number of reads mapping to
either the positive or negative viral strand.
DATA AVAILABILITY
All data supporting the findings of this study are available within the article
and supporting information. All sequencing data generated in this study have
been deposited to the Sequence Read Archive, https://www.ncbi.nlm.nih.gov/sra
(accession no. PRJNA721333). All published data analyzed in this study are cited
in this article with accession methods provided in Materials and Methods.
CHANGE HISTORY
July 14, 2021: The SI appendix has been updated.
ACKNOWLEDGMENTS
We thank members in the laboratories of R.J. and R.A.Y. and other colleagues
from Whitehead Institute and Massachusetts Institute of Technology (MIT) for
helpful discussions and resources. We thank Thomas Volkert and staff from the
Whitehead genomics core, and Stuart Levine from the MIT/Koch Institute BioMicro
center for sequencing support. We thank Lorenzo Bombardelli for sharing protocol
and advice for Tn5 tagmentation-mediated integration enrichment sequencing. We
thank Jerold Chun, Inder Verma, Joseph Ecker, and Daniel W. Bellott for
discussion and suggestions. This work was supported by grants from the NIH to
R.J. (1U19AI131135-01; 5R01MH104610-21) and by a generous gift from Dewpoint
Therapeutics and from Jim Stone. S.H.H. was supported by the Intramural Research
Program of the Center for Cancer Research of the National Cancer Institute.
Finally, we thank Nathans Island for inspiration.
FOOTNOTES
* ↵1To whom correspondence may be addressed. Email: jaenisch@wi.mit.edu.
* Author contributions: L.Z., R.A.Y., and R.J. designed research; L.Z. and A.R.
performed experiments; L.Z., A.R., M.I.B., S.H.H., R.A.Y., and R.J. analyzed
data; and L.Z. and R.J. wrote the paper with input from all authors.
* Reviewers: A.B., Netherlands Cancer Institute; and A.M.S., Fox Chase Cancer
Center.
* Competing interest statement: R.J. is an advisor/co-founder of Fate
Therapeutics, Fulcrum Therapeutics, Omega Therapeutics, and Dewpoint
Therapeutics. R.A.Y. is a founder and shareholder of Syros Pharmaceuticals,
Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics.
* This article contains supporting information online at
https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2105968118/-/DCSupplemental.
* Copyright © 2021 the Author(s). Published by PNAS.
This open access article is distributed under Creative Commons Attribution
License 4.0 (CC BY).
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WE RECOMMEND
1. Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured
human cells and can be expressed in patient-derived tissues
Liguo Zhang et al., Proc Natl Acad Sci U S A, 2021
2. No evidence of SARS-CoV-2 reverse transcription and integration as the
origin of chimeric transcripts in patient tissues
Rhys Parry et al., Proc Natl Acad Sci U S A, 2021
3. Response to Parry et al.: Strong evidence for genomic integration of
SARS-CoV-2 sequences and expression in patient tissues
Liguo Zhang et al., Proc Natl Acad Sci U S A, 2021
4. Assessment of potential SARS-CoV-2 virus integration into human genome
reveals no significant impact on RT-qPCR COVID-19 testing
Erica Briggs et al., Proc Natl Acad Sci U S A
5. SARS-CoV-2 expresses a microRNA-like small RNA able to selectively repress
host genes
Paulina Pawlica et al., Proc Natl Acad Sci U S A, 2021
1. Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and
artifactual | Journal of Virology
J Virol, 2021
2. The NF-κB Transcriptional Footprint Is Essential for SARS-CoV-2 Replication
J Virol, 2021
3. Depicting SARS-CoV-2 faecal viral activity in association with gut
microbiota composition in patients with COVID-19
Tao Zuo et al., Gut, 2021
4. New coronavirus (SARS-CoV-2) mapped out
Phys.org, 2020
5. The COVID-19 virus may not insert genetic material into human DNA, research
shows
MedicalXpress, 2021
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Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured
human cells and can be expressed in patient-derived tissues
Liguo Zhang, Alexsia Richards, M. Inmaculada Barrasa, Stephen H. Hughes, Richard
A. Young, Rudolf Jaenisch
Proceedings of the National Academy of Sciences May 2021, 118 (21) e2105968118;
DOI: 10.1073/pnas.2105968118
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* Response to Parry et al.: Strong evidence for genomic integration of
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Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured
human cells and can be expressed in patient-derived tissues
Liguo Zhang, Alexsia Richards, M. Inmaculada Barrasa, Stephen H. Hughes, Richard
A. Young, Rudolf Jaenisch
Proceedings of the National Academy of Sciences May 2021, 118 (21) e2105968118;
DOI: 10.1073/pnas.2105968118
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