LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells

Abstract

SARS-CoV-2 sequences can be reverse-transcribed and integrated into the genomes of virus-infected cells by a LINE1-mediated retrotransposition mechanism. Whole-genome sequencing (WGS) methods detected retrotransposed SARS-CoV-2 subgenomic sequences in virus-infected cells overexpressing LINE1, while an enrichment method (TagMap) identified retrotranspositions in cells that did not overexpress LINE1. LINE1 overexpression increased retrotranspositions about 1000-fold as compared to non-overexpressing cells. Nanopore WGS can directly recover retrotransposed viral and flanking host sequences, but its sensitivity depends on the depth of sequencing (a typical 20-fold sequencing depth would only examine 10 diploid cell equivalents). In contrast, TagMap enriches the host-virus junctions and can interrogate up to 20,000 cells and is able to detect rare viral retrotranspositions in LINE1 non-overexpressing cells. Although Nanopore WGS is 10-20-fold more sensitive per tested cell, TagMap can interrogate 1000-2000-fold more cells and, therefore, can identify infrequent retrotranspositions. When comparing SARS-CoV-2 infection and viral nucleocapsid mRNA transfection by TagMap, retrotransposed SARS-CoV-2 sequences were only detected in infected but not in transfected cells. Retrotransposition in virus-infected cells, in contrast to transfected cells, may be facilitated because virus infection, in contrast to viral RNA transfection, results in significantly higher viral RNA levels and stimulates LINE1 expression by causing cellular stress.

Keywords: LINE1; RNA transfection; SARS-CoV-2; WGS; enrichment sequencing; retrotransposition.

Figure 1

TagMap method for the enrichment and sequencing of a junction of retrotransposed viral cDNA sequence with its flanking human chromosome sequence. Schematic and an example sequencing read-pair showing the enrichment of one junction of retrotransposed viral cDNA. This method is based on random Tn5 tagmentation on cellular genomic DNA. A primer targeting the inserted adapter sequence (brown arrow) and a primer targeting a SARS-CoV-2 sequence (green arrow) are used to enrich the retrotransposition junction. In this example, the left read mapped to human chromosome 1 (blue). The right read mapped to the 3′-end of a SARS-CoV-2 RNA sequence (pink) starting from the enrichment primer sequence (green arrow), covering a poly-A tract (orange) and ending with a human sequence (blue) at the retrotransposition site containing a LINE1 endonuclease recognition sequence (TTTT/A, purple).

Figure 2

No retrotransposition events were detected by TagMap in cells transfected by viral nucleocapsid mRNA alone. (A,B) Retrotranspositions detected by TagMap in 293T cells transfected with SARS-CoV-2 nucleocapsid (NC) subgenomic mRNA (A) with the leader sequence or (B) without the leader sequence, with or without LINE1 overexpression. 293T cells were transfected by the listed NC mRNA with a concentration of 1 μg RNA per 1mL cell culture medium. For LINE1 overexpression, a CMV-LINE1 plasmid was co-transfected with the NC mRNA, with a concentration of 0.5 μg or 1 μg plasmid per 1 mL cell culture medium. The in vitro transcribed NC mRNA can either express or not express the encoded NC protein depending on whether or not the mRNA was 5′-capped, as listed in the tables. (C) Retrotransposition detected by TagMap in cells infected with SARS-CoV-2 or transfected with NC mRNA, with or without LINE1 overexpression. CoV2 infection + LINE1 overexpression: 678 or 1110 retrotransposition events were detected in ~4000 293T cells (Table 1). CoV2 NC mRNA (5′-capped) transfection + LINE1 overexpression: 14–92 retrotransposition events were detected in ~2000 293T cells (A). CoV2 infection: 1–2 retrotransposition events were detected in ~12,000–20,000 hESC-ACE2 or Calu3 cells (Table 1). CoV2 NC mRNA (5′-capped) transfection: No retrotransposition events were detected in ~20,000 293T cells (Figure 2A). (D) Fractions of viral poly-A RNA relative to total cellular poly-A RNA in cells infected with SARS-CoV-2 or in cells transfected with SARS-CoV-2 NC mRNA. For SARS-CoV-2 infected cells, Calu3 cell RNA was harvested 2 days post-infection; RNA-seq data were from a previous publication [27]. For cells transfected with NC mRNA, 293T cells cultured in 24-well plates were transfected by 0.5 μg (red dot), 1 μg (magenta dot), or 2 μg (blue dot) RNA per 1mL cell culture medium for 6 h and then lysed for extraction of cellular RNA and poly-A RNA-seq using the same method that was used for infected cells.

Figure 3

SARS-CoV-2 infection and related cellular stress, but not SARS-CoV-2 nucleocapsid mRNA transfection, can induce endogenous LINE1 expression. (A–C) LINE1 mRNA level fold-change detected by RT-qPCR in (A) Calu3 cells infected by SARS-CoV-2 for 2 days, (B) 293T cells transfected by nucleocapsid (NC) mRNA (~1.8 kb with polyA), with 0.5 μg RNA per 1mL cell culture medium for 24 h, or (C) 293T cells transfected by a synthetic analog of double-stranded RNA, Poly(I:C) (HMW, 1.5–8 kb), with 2 μg per 1mL cell culture medium for 24 h. RT-qPCR was performed using L1HS/L1PA(2-6) specific primers targeting LINE1 5′-UTR or ORF1, and L1HS specific primers targeting LINE1 ORF2, on purified cellular poly-A RNA (method and primer sequences following the protocols in a previous publication [2], see Materials and Methods). n = 3 independent experiments (biological replicates). Data are the mean ± standard deviation (SD). One-tailed t-test for LINE1 mRNA upregulation in infected/transfected cells: (A) p = 0.098 (L1-5′UTR), p = 0.021 (L1-ORF1), p = 0.303 (L1-ORF2); (B) p = 0.194 (L1-5′UTR), p = 0.379 (L1-ORF1), p = 0.315 (L1-ORF2); (C) p = 0.143 (L1-5′UTR), p = 0.121 (L1-ORF1), p = 0.020 (L1-ORF2). (D) Immunofluorescent staining of L1ORF1p (red) and G3BP1 (green) and merged channels with DAPI staining (blue) in mock-transfected or Poly(I:C) (HMW, 1.5–8 kb) transfected 293T cells. Cells were transfected by Poly(I:C) (HMW, 1.5–8 kb), with 2 μg per 1mL cell culture medium for 24 h. (E) A proposed model for mechanisms involved in LINE1-mediated viral RNA retrotransposition in infected cells. In this model, double-stranded viral RNAs (formed by viral replication and transcription) and reactive oxygen species produced upon SARS-CoV-2 infection can stimulate cell endogenous LINE1 expression. The expressed LINE1 proteins (L1ORF1p and L1ORF2p) can interact with single-stranded viral polyadenylated mRNAs to form ribonucleoprotein complex in trans, which enter the cell nucleus and retrotranspose through a well-known TPRT mechanism. The high level of viral mRNAs in host cells during viral infection increases the chance of their interaction with LINE1 proteins. Cell stress granules, a cell antiviral/anti-retrotransposition defense mechanism, can be attenuated/cleared by SARS-CoV-2 infection. dsRNA: double-stranded RNA. ssRNA: single-stranded RNA. ROS: reactive oxygen species. RNP: ribonucleoprotein. TPRT: target-primed reverse transcription.