Profiling Selective Packaging of Host RNA and Viral RNA Modification in SARS-CoV-2 Viral Preparations

Abstract

Viruses package host RNAs in their virions which are associated with a range of functions in the viral life cycle. Previous transcriptomic profiling of host RNA packaging mostly focused on retroviruses. Which host RNAs are packaged in other viruses at the transcriptome level has not been thoroughly examined. Here we perform proof-of-concept studies using both small RNA and large RNA sequencing of six different SARS-CoV-2 viral isolates grown on VeroE6 cells to profile host RNAs present in cell free viral preparations and to explore SARS-CoV-2 genomic RNA modifications. We find selective enrichment of specific host transfer RNAs (tRNAs), tRNA fragments and signal recognition particle (SRP) RNA in SARS-CoV-2 viral preparations. Different viral preparations contain the same set of host RNAs, suggesting a common mechanism of packaging. We estimate that a single SARS-CoV-2 particle likely contains up to one SRP RNA and four tRNA molecules. We identify tRNA modification differences between the tRNAs present in viral preparations and those in the uninfected VeroE6 host cells. Furthermore, we find uncharacterized candidate modifications in the SARS-CoV-2 genomic RNA. Our results reveal an under-studied aspect of viral-host interactions that may be explored for viral therapeutics.

Keywords: SARS-CoV-2; SRP RNA; modification; packaging; tRNA.

FIGURE 1

Selective enrichment of small RNAs in SARS-CoV-2 viral preparations. (A) Experimental scheme. Vero E6 cells were either infected with SARS-CoV-2 virus isolates from infected individuals (n = 6 biological isolates) or uninfected cultures (n = 3 biological replicates). Total RNA was extracted from the cells (blue boxes) or only from the cell free viral preparations (green boxes). Small RNA-seq was carried out using total RNA with and without demethylase treatment. Large RNA-seq was carried out with the RNA fraction after the removal of small RNAs of <200 nt, and chemical fragmentation. (B) Small RNA-seq results. Vero cell data are mostly tRNA and 5S/5.8S rRNA. Aside from SARS-CoV-2 RNA, virions contain significant portions of tRNA, rRNA, and signal recognition particle (SRP) RNA. (C) Enrichment and depletion of specific tRNAs in the cell free viral samples. Shown are the combined reads from all tRNA isodecoders that share the same anticodon. Heatmap shows the abundance of tRNAs for each anticodon subtracted from the mean of control cultures. Subtraction emphasizes the differences among abundant tRNAs. Enriched tRNAs are in red, depleted tRNAs in blue. Top 3 enriched tRNAs are tRNA^Lys(TTT), tRNA^Glu(TTC), and tRNA^Ser(GCT). Top 3 depleted tRNAs are tRNA^Ile(AAT), tRNA^Tyr(GTA), and tRNA^Asn(GTT).

FIGURE 2

Selective enrichment of tRNA isodecoders in SARS-CoV-2 viral preparations. tRNA isodecoder fractions from uninfected Vero cell (n = 3, red) or cell free viral preparations (n = 6, blue) are shown. Mean values are shown as a horizontal bar. Isodecoder nomenclature is according to the tRNAScan score of the Chlorocebus sabaeus tRNA genes identified in Rfam database. (A) tRNA^Glu(TTC). (B) tRNA^Leu(AAG). (C) tRNA^Lys(TTT). (D) tRNA^Ser(AGA). (E) tRNA^Ser(GCT). (F) tRNA^Ser(TGA).

FIGURE 3

Read pileup of the enriched tRNA isodecoder in SARS-CoV-2 viral preparations. Shown are read pileups of the most abundant tRNA isodecoders in viral isolates (n = 6, blue) and their counterparts in uninfected Vero cell (n = 3, red). Isodecoder nomenclature is according to the tRNAScan score of the Chlorocebus sabaeus tRNA genes identified in Rfam database. (A) tRNA^Glu(TTC). This result is consistent with 3′ tRNA fragment being the dominant form in the viral preparations. (B) tRNA^Leu(AAG). This result is consistent with full-length tRNA in the viral preparations. (C) tRNA^Lys(TTT). This result is consistent with full-length or 3′ tRNA fragment with 5′ end exactly at position 39 in the viral preparations. (D) tRNA^Ser(AGA). The tRNA^Ser results are consistent with full-length tRNA in the viral preparations. (E) tRNA^Ser(GCT). (F) tRNA^Ser(TGA).

FIGURE 4

Selective enrichment of tRNA with m¹A modification profiles. Mutation fractions from uninfected Vero cell (n = 3, red) or cell free viral preparations (n = 6, blue) are shown. (A) Mutation fractions of tRNA^Leu(AAG) residues around the wobble anticodon position (35 for this tRNA) without (DM-) and with (DM+) demethylase treatment showing the I34 modification. (B) Mutation fractions of tRNA^Leu(AAG) around the residues at position 67 which corresponds to m¹A58 in the tRNA nomenclature. tRNA^Leu(AAG) shows higher mutation fraction in the viral preparations, consistent with SARS-CoV-2 selectively packaging m¹A modified tRNA^Leu(AAG). (C) Mutation fractions of the top five abundant tRNA^Glu(TTC) isodecoders at position 57 (DM-) which is validated as m¹A in the T loop upon removal by demethylase treatment (DM+). Isodecoder nomenclature is according to the tRNAScan score of each tRNA^Glu(TTC) gene. The two isodecoders enriched in the viral preparations are nearly unmodified, corresponding to their counterparts in the Vero cells.

FIGURE 5

Large RNA sequencing identifies viral sequence variants, subgenomic viral RNAs, and signal recognition particle RNA. (A) Single nucleotide polymorphisms (SNPs) for each viral isolate identified by >90% mutation fraction from the Wuhan SARS-CoV-2 reference genome. (B) Mapped read count ratio of SARS-CoV2 genomic RNA to large ribosomal RNA (18S and 28S) in the viral preparations. (C) Normalized ratio of SRP RNA reads to SARS-CoV-2 genomic RNA reads in the viral preparations using the transcript size of 300 nucleotides for SRP, and 29,903 nucleotides for SARS-CoV-2. (D) Relative fraction of reads that bridge the junction between the 5′ leader region and the genomic RNA (set at 1) and between individual subgenomic RNA. Box and Whisker plot for n = 6 individual isolates.

FIGURE 6

Large RNA sequencing identifies candidate SARS-CoV-2 modifications. (A) Scheme of modification detection. An enzyme treatment (DM) and a chemical treatment (CMC) are added before library construction, producing four combinations for each sample. (B) Candidate modifications from comparing four treatment conditions. Site locations are indicated in a dashed line, nucleotide identity indicated on top of the SARS-CoV-2 gene map. Data from top to bottom: with and without demethylase only (±DM, -CMC); with and without CMC only (±CMC, -DM), with and without demethylase, also CMC (±DM, +CMC); with and without CMC, also DM (±CMC, +DM). Positions with mutations >5% in at least 4 of the 6 isolates but excluding the SNP positions in Figure 5A are shown.