Identification of RNase L-dependent, 3'-end-modified, viral small RNAs in Sindbis virus-infected mammalian cells

Abstract

Small RNAs play a critical role in host-pathogen interaction. Indeed, small RNA-mediated silencing or RNA interference (RNAi) is one of the earliest forms of antiviral immunity. Although it represents the main defense system against viruses in many organisms, the antiviral role of RNAi has not been clearly proven in higher vertebrates. However, it is well established that their response to viral infection relies on the recognition of viral RNAs by host pattern recognition receptors (PRRs) to trigger activation of the interferon pathway. In the present work, we report the existence of a novel small noncoding RNA population produced in mammalian cells upon RNA virus infection. Using Sindbis virus (SINV) as a prototypic arbovirus model, we profiled the small RNA population of infected cells in both human and African green monkey cell lines. Here, we provide evidence for the presence of discrete small RNAs of viral origin that are not associated with the RNA-induced silencing complex (RISC), that are highly expressed and detected by Northern blot analysis, and that accumulate as 21- to 28-nucleotide (nt) species during infection. We report that the cellular antiviral endoribonuclease RNase L cleaves the viral genome, producing in turn the small RNAs. Surprisingly, we uncovered the presence of a modification on the 3'-end nucleotide of SINV-derived viral small RNAs (SvsRNAs) that might be at the origin of their stability. Altogether, our findings show that stable modified small viral RNAs could represent a novel way to modulate host-virus interaction upon SINV infection.

Importance: In a continuous arms race, viruses have to deal with host antiviral responses in order to successfully establish an infection. In mammalian cells, the host defense mechanism relies on the recognition of viral RNAs, resulting in the activation of type I interferons (IFNs). In turn, the expression of many interferon-stimulated genes (ISGs) is induced to inhibit viral replication. Here we report that the cytoplasmic, interferon-induced, cellular endoribonuclease RNase L is involved in the accumulation of a novel small RNA population of viral origin. These small RNAs are produced upon SINV infection of mammalian cells and are stabilized by a 3'-end modification. Altogether, our findings indicate that in our system RNA silencing is not active against Sindbis virus (SINV) and might open the way to a better understanding of the antiviral response mediated by a novel class of small RNAs.

FIG 1

Identification of SINV-derived small RNAs. (A) Virus-specific small RNA reads were recovered from each library (HEK293 cells infected with SINV [HEK293-SINV] and Vero-SINV) and were mapped to both positive (+) and negative (−) strands of the viral genome. (B) Size distribution of SINV-derived small RNA populations. The proportion of viral sRNAs that were derived from the genomic sense strand in each size class is shown as a percentage of the total viral small RNA reads for this strand. (C) Distribution of viral reads mapping to the SINV genomic strand. The abundance of small RNAs was calculated and plotted as the sum of normalized reads (per 10⁵ viral reads) in each single-nucleotide sliding window along the viral genome. The small black arrows indicate the peaks chosen for Northern blot validation (see Fig. S1 in the supplemental material). A schematic diagram represents the organization of SINV genome. The two open reading frames (ORF), which encode the nonstructural (ns) and structural proteins, are shown. The nontranslated region (NTR) at the 3′ end of the virus is shown as a small black bar. (D) Northern blot validation of SINV candidates. Total RNA was isolated at the indicated time points from HEK293 cells infected with SINV at an MOI of 0.01. Noninfected cells (−) were used as a negative control. U6 snRNA was used as a loading control. (E) Quantification of the SINV genome during infection by reverse transcription-quantitative PCR (RT-qPCR). The relative quantification is normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and is represented on a logarithmic scale as a mean of three replicates. NI, not infected.

FIG 2

Effects of RNase L and Xrn1 knockdown on SvsRNA accumulation. (A and D) RNase L and Xrn1 knockdown in SINV-infected HEK293 cells was verified by Western blotting. Tubulin (Tub) was used as a normalizer. sictrl, control siRNA; siRNaseL, RNase L-specific siRNA; siXrn1, Xrn1-specific siRNA. (B and E) SINV genomic RNA was quantified by RT-qPCR, before and after RNase L or Xrn1 KD. The relative quantification is normalized to GAPDH levels and is represented on a logarithmic scale as a mean of three replicates. (C and F) Northern blot analysis on total RNA from infected HEK293 cells before and after RNase L or Xrn1 KD. U6 was used as a normalizer.

FIG 3

Characterization of 5′ and 3′ extremities of SvsRNA-1. (A) Synthetic oligonucleotides that have 5′ OH or 5′ P ends or total RNA were treated with Terminator exonuclease (+) or not treated with Terminator (−) before being analyzed by Northern blotting. (B) β-elimination on HEK293-SINV total RNA followed by Northern blotting of SvsRNA-1 and miR-16 after migration on 17.5% polyacrylamide gel. Asterisks denote putative precursors of SvsRNA-1. (C) Synthetic SvsRNA-1 molecules that have 3′ P, 3′ OH, or 3′ OH 2′-O-me ends were treated with calf intestinal phosphatase (CIP) (lanes 2, 4, 6, 8, 10, 12, 14, and 16). Dephosphorylation was followed by β-elimination (lanes 4, 8, 12, and 16). oligo, oligonucleotide; me, methyl; elim, elimination.

FIG 4

SINV genomic RNA is modified. (A) Schematic diagram of the primer design for RTL-P to detect the presence of 2′-O-methylated nucleotides (m) on the viral genome. SvsRNA-1 is indicated in red. (B) Detection of methylation by RTL-P under both high and low dNTP concentrations and PCR cycles on SINV-infected HEK293 cells. The ratio of PCR signal intensity is indicated beneath each lane. (C) Primer extension mapping of 2′-O-methylated nucleotides. Lanes U, G, C, and A represent dideoxy sequencing reactions performed on the in vitro transcript amplified for the same viral region. Asterisks indicate stops of the reverse transcriptase.

FIG 5

Deep sequencing upon periodate oxidation in SINV-infected HEK293 cells. (A) Distribution of viral reads mapping to the SINV genome. The abundance of small RNAs was calculated and plotted as the sum of normalized reads (per 10⁵ viral reads) in each single-nucleotide sliding window along the SINV RNA. The small black arrows indicate the peaks chosen for Northern blot validation. (B) Size distribution of SINV-derived small RNA populations. The percentage of viral sRNAs deriving from the genomic sense strand in each size class is shown as a percentage of the total viral small RNA reads for this strand. Data from SINV-infected HEK293 cell samples treated with NaIO₄ or not treated with NaIO₄ (NT) are shown. (C) Relative nucleotide frequency per position. The viral reads with 0 mismatch to the SINV positive strand were considered for this analysis. Only the last five nucleotides from the 3′ end of each sequence are displayed.