chimeric, Coronavirus, DNA, genome, HIV, human cell DNA, human DNA, human genome, lentiviral vectors, mRNA vaccine, retrovirus, Reverse transcribed SARS-CoV-2 RNA, Reverse transcription, RNA, RNA virus, SARS-CoV-2
In “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, and Rudolf Jaenisch (2021) report that they “investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture…” Supporting this hypothesis, they “found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells”. Furthermore they “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.” They “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”. They state that because they “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.” See: PNAS May 25, 2021 118 (21) e2105968118 https://doi.org/10.1073/pnas.2105968118 https://www.pnas.org/content/118/21/e2105968118
This suggests that the mRNA vaccines for Covid-19 could also be integrated into the DNA.
This research appears to challenge the current classification system-consensus and suggests that it may be faulty. While currently not all viruses made of RNA are classified as retroviruses, it appears that this classification may need to be changed, since SARS-CoV-2 appears to be sometimes integrated into the human genome. Unless, of course, SARS-CoV-2 has a retrovirus backbone, as has been suggested in a “withdrawn” paper (See abstract further below). Retroviruses could possibly come into play for either Covid-19 or the vaccine in this manner: Lentivirus are retroviruses and can be used “to introduce a gene product into in vitro systems or animal models”. https://en.wikipedia.org/wiki/Lentivirus “Most of the lentiviral vectors presently in use are HIV-derived vectors.“ https://ehs.stanford.edu/reference/lentivirus-fact-sheet
Nonetheless, according to the research paper, by Zhang et al., this is not unique: “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)”.
Since RNA viruses start with RNA instead of DNA, it appears common sense that all RNA viruses should be called retroviruses because they start with RNA instead of DNA, and that they might have the capacity to integrate into the genome. However, the Wikipedia excerpt, below, explains the current classification.
(A DNA virus has DNA as their genetic material and an RNA virus has RNA) “An RNA virus is a virus which has (ribonucleic acid) RNA as its genetic material. The nucleic acid is usually single-stranded RNA (ssRNA) but it may be double-stranded RNA (dsRNA). Notable human diseases caused by RNA viruses include the common cold, influenza, SARS, MERS, COVID-19, Dengue Virus, hepatitis C, hepatitis E, West Nile fever, Ebola virus disease, rabies, polio, mumps, and measles…. Animal RNA viruses are classified by the ICTV. There are three distinct groups of RNA viruses depending on their genome and mode of replication:
* Double-stranded RNA viruses (Group III) contain from one to a dozen different RNA molecules, each coding for one or more viral proteins.
* Positive-sense ssRNA viruses (Group IV) have their genome directly utilized as mRNA, with host ribosomes translating it into a single protein that is modified by host and viral proteins to form the various proteins needed for replication. One of these includes RNA-dependent RNA polymerase (RNA replicase), which copies the viral RNA to form a double-stranded replicative form. In turn, this dsRNA directs the formation of new viral RNA.
* Negative-sense ssRNA viruses (Group V) must have their genome copied by an RNA replicase to form positive-sense RNA. This means that the virus must bring along with it the enzyme RNA replicase. The positive-sense RNA molecule then acts as viral mRNA, which is translated into proteins by the host ribosomes.
Retroviruses (Group VI) have a single-stranded RNA genome but, in general, are not considered RNA viruses because they use DNA intermediates to replicate. Reverse transcriptase, a viral enzyme that comes from the virus itself after it is uncoated, converts the viral RNA into a complementary strand of DNA, which is copied to produce a double-stranded molecule of viral DNA. After this DNA is integrated into the host genome using the viral enzyme integrase, expression of the encoded genes may lead to the formation of new virions… Numerous RNA viruses are capable of genetic recombination when at least two viral genomes are present in the same host cell. RNA recombination appears to be a major driving force in determining genome architecture and the course of viral evolution among Picornaviridae ((+)ssRNA) (e.g. poliovirus). In the Retroviridae [retroviruses] ((+)ssRNA)(e.g. HIV), damage in the RNA genome appears to be avoided during reverse transcription by strand switching, a form of recombination. Recombination also occurs in the Reoviridae (dsRNA)(e.g. reovirus), Orthomyxoviridae ((-)ssRNA)(e.g. influenza virus) and Coronaviridae ((+)ssRNA) (e.g. SARS). Recombination in RNA viruses appears to be an adaptation for coping with genome damage. Recombination can occur infrequently between animal viruses of the same species but of divergent lineages. The resulting recombinant viruses may sometimes cause an outbreak of infection in humans… ” https://en.wikipedia.org/wiki/RNA_virus So, not all viruses made of RNA are classified as retroviruses. https://www.lehigh.edu/~jas0/V07.html
However, it appears that this classification may get turned on its head, since SARS-CoV-2 appears to be sometimes integrated into the human genome. Unless, of course, SARS-CoV-2 has a retrovirus backbone, as has been alleged.
Original research paper:
“Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues” by Liguo Zhang, Alexsia Richards, M. Inmaculada Barrasa, Stephen H. Hughes, Richard A. Young, and Rudolf Jaenisch aWhitehead Institute for Biomedical Research, Cambridge, MA 02142; bHIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702; cDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142 PNAS May 25, 2021 118 (21) e2105968118; https://doi.org/10.1073/pnas.2105968118 Contributed by Rudolf Jaenisch, April 19, 2021 (sent for review March 29, 2021; reviewed by Anton Berns and Anna Marie Skalka) https://www.pnas.org/content/118/21/e2105968118
Response to Parry et al., who attacked the research-paper: “Strong evidence for genomic integration of SARS-CoV-2 sequences and expression in patient tissues” by Liguo Zhang, Alexsia Richards, M. Inmaculada Barrasa, Stephen H. Hughes, Richard A. Young, and Rudolf Jaenisch PNAS August 17, 2021 118 (33) e2109497118; https://doi.org/10.1073/pnas.2109497118
“Our paper (1) draws two conclusions:
1) SARS-CoV-2 sequences can integrate into the genome of infected cells that either overexpress (tables 1 and 2 of ref. 1) or do not overexpress (figure 2F of ref. 1) LINE1 by a LINE1-mediated retroposition mechanism.
2) We have identified large fractions of chimeric RNAs derived from negative-strand viral RNA in patients. Chimeric sequences containing negative-strand viral RNA are unlikely caused by artifacts associated with sequencing technology and likely derive from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences integrated into genomic DNA.
We summarize our responses to Parry et al. (2).
1) Parry et al. state that “SARS-CoV-2 integration into the host genome is unlikely.” Our response is that the “percent of library” calculation is not an estimate of integration frequency, which requires consideration of whole-genome sequencing (WGS) coverage. We identified integration events and observed two to five integrations per 10,000 cells at the current sequencing depth (Table 1). This is similar to the estimated integration frequency of lymphocytic choriomeningitis virus after acute infection (3). “Low frequency” of integration events cannot be interpreted as “unlikely.”
2) Parry et al. state that, because “‘insertions’ are found preferentially in protein-coding exons, a bias unknown to L1 endonuclease insertions . . ., these findings are likely spurious.” Our response is that we are not measuring L1 integrations but L1-mediated retroposition of SARS-CoV-2 RNA. Our data suggest that L1-mediated retroposition of RNA may have a different integration preference than L1.
3) Parry et al. state that “2 of the identified 61 chimeric nanopore genomic DNA (gDNA) reads contains human DNA from separate chromosomes.” Our response is that it is well known that nanopore sequencing occasionally ligates fragments from different chromosomes. Our demonstration of LINE1-mediated retroposition is based on sequence features of the integrated DNA and is not affected by these rare artifacts.
4) Parry et al. state that “larger pools of negative-sense SARS-CoV-2 chimeric reads may be due to differences in RNA extraction, ….” Our response is that there are no “larger pools of negative-sense chimeric reads.” The fraction of negative-strand RNAs produced during virus replication is 1,000- to 10,000-fold lower than that of positive-strand RNAs (figure 3 C and D in ref. 1). The low abundance of negative-strand RNA makes artifactual template fusion unlikely. The fraction of negative-strand viral RNAs detected in some patient tissues, which show no evidence of virus replication, are orders of magnitude higher than in cells with replicating virus (figure 3 E−G in ref. 1).
5) Parry et al. state that “there is no evidence of coronaviruses ever having integrated into the germline of host species.” Our response is that the lack of prior evidence is not an argument against new evidence.
* Whether coronavirus sequences are found in the germline of different species is irrelevant, as our experiments have focused on SARS-CoV2 integration into the genome of somatic cells and not into the germline.
1To whom correspondence may be addressed. Email: email@example.com.
Author contributions: 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. 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. Copyright © 2021 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).
1. L. Zhang et al., Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues. Proc. Natl. Acad. Sci. U.S.A. 118, e2105968118 (2021).Google Scholar
2. R. Parry, R. J. Gifford, S. Lytras, S. C. Ray, L. Coin, No evidence of SARS-CoV-2 reverse transcription and integration as the origin of chimeric transcripts in patient tissues. Proc. Natl. Acad. Sci. U.S.A. 118, doi:10.1073/pnas.2109066118 (2021).FREE Full TextGoogle Scholar
3. P. Klenerman, H. Hengartner, R. M. Zinkernagel, A non-retroviral RNA virus persists in DNA form. Nature 390, 298–301 (1997).CrossRefPubMedGoogle Scholar”
“Key to the unique attributes of a retrovirus is the pol region, which encodes a reverse trancriptase (RT), RT is the enzyme which takes the RNA form of the retrovirus genome and converts into DNA, the DNA form of which can integrate into the host genome”. “Retroviral RNA Genome”. (2021, January 3). Retrieved August 8, 2021, from https://bio.libretexts.org/@go/page/9914
Abstract of paper which was withdrawn:
“Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp120 and Gag” By Prashant Pradhan$1,2, Ashutosh Kumar Pandey$1, Akhilesh Mishra$1, Parul Gupta1, Praveen Kumar Tripathi1, Manoj Balakrishnan Menon1, James Gomes1, Perumal Vivekanandan*1and Bishwajit Kundu*1
1Kusuma School of biological sciences, Indian institute of technology, New Delhi-110016, India.
2Acharya Narendra Dev College, University of Delhi, New Delhi-110019, India $Equal contribution
We are currently witnessing a major epidemic caused by the 2019 novel coronavirus (2019- nCoV). The evolution of 2019-nCoV remains elusive. We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have identity or similarity to those in the HIV-1 gp120 or HIV-1 Gag. Interestingly, despite the inserts being discontinuous on the primary amino acid sequence, 3D-modelling of the 2019-nCoV suggests that they converge to constitute the receptor binding site. The finding of 4 unique inserts in the 2019-nCoV, all of which have identity /similarity to amino acid residues in key structural proteins of HIV-1 is unlikely to be fortuitous in nature. This work provides yet unknown insights on 2019-nCoV and sheds light on the evolution and pathogenicity of this virus with important implications for diagnosis of this virus.” https://www.biorxiv.org/content/10.1101/2020.01.30.927871v1.full.pdf+html https://www.biorxiv.org/content/10.1101/2020.01.30.927871v1.full.pdf
“Lentivirus is a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in the human and other mammalian species.… Lentiviruses can become endogenous (ERV), integrating their genome into the host germline genome, so that the virus is henceforth inherited by the host’s descendants.” https://en.wikipedia.org/wiki/Lentivirus Lentivirus can be used “to introduce a gene product into in vitro systems or animal models”. https://en.wikipedia.org/wiki/Lentivirus “Most of the lentiviral vectors presently in use are HIV-derived vectors.” https://ehs.stanford.edu/reference/lentivirus-fact-sheet