Physiologic RNA targets and refined sequence specificity of coronavirus EndoU

Abstract

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for U ^↓ A and C ^↓ A sequences (endoY ^↓ A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggests a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoU^mut) or 2'-5' phosphodiesterase (PDE^mut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. Coronavirus EndoU cleaves U ^↓ A and C ^↓ A sequences (endoY ^↓ A) within viral (+) strand RNA to evade dsRNA-activated host responses.

Keywords: coronavirus; dsRNA; endoribonuclease; innate immunity; mouse hepatitis virus.

FIGURE 1.

Coronavirus RNA genome and experimental approach. (A) MHV RNA genome highlighting two mutations: His to Arg mutation in the MHV phosphodiesterase domain active site (PDE^H126R), and a His to Ala mutation in the MHV EndoU domain active site (EndoU^H277A) (Roth-Cross et al. 2009; Kindler et al. 2017). MHV proteins are categorized as nonstructural (light gray), accessory (dark gray), and structural (black). Subgenomic mRNAs 2–7, produced during infection, are illustrated. (B) Bone marrow–derived macrophage (BMM) from wt, IFNAR^−/−, and RNase L^−/− mice were mock-infected or infected with wt MHV (MHV^(S) and MHV^(V)), the PDE^mut, or EndoU^mut for 9 and 12 h (Zhao et al. 2012; Kindler et al. 2017), after which total cellular RNA was isolated for cyclic phosphate sequencing. (C) Schematic of cyclic phosphate sequencing; protocol adapted from Schutz et al. (2010).

FIGURE 2.

Endoribonuclease cleavage in host and viral RNAs. (A,B) Relative amounts of endonucleolytic cleavage in host and viral RNAs. Normalized cyclic phosphate cDNA reads (reads at each position/total reads in library) mapped to host and viral RNAs at 9 and 12 hpi in wt, IFNAR^−/−, and RNase L^−/− bone marrow macrophages (BMMs). (C) Frequency and location of cleavage sites in MHV RNA. Normalized cyclic phosphate cDNA reads captured at each position along the MHV genomic RNA at 9 and 12 hpi from MHV^(S)-, MHV^(V)-, PDE^mut-, and EndoU^mut-infected wt BMM. Putative cleavage sites attributed to EndoU or RNase L were calculated from RNase L– or EndoU-dependent signal generated by subtracting signal from each captured position that occurs in the absence of either enzyme (RNase L^−/− BMM or during EndoU^mut infection). These data were then filtered for sites with reads representing at least 0.01% of total reads in the library. At each of these positions, the log₂-fold change in signal when either RNase L or EndoU were absent was calculated and sites with ≥2.5-fold change were designated putative RNase L or EndoU sites.

FIGURE 3.

Sequence specificity of cleavage sites in MHV RNA. (A,D) Dinucleotide specificity analysis for cleavage in MHV RNA by percent total cDNA reads captured at each 3′-dinucleotide in wt BMM at 9 and 12 hpi for (A) Dinucleotide analysis for positions −2:−1 and (D) dinucleotide analysis for positions −1:+1 from captured cleavage position (0 position). (B,E) Dinucleotide enrichment for dinucleotide positions from −2:−1 (B) or −1:+1 (E) for each condition of viral infection at 12 hpi in wt BMM by comparing the frequency of dinucleotide capture in experimental conditions to the frequency of occurrence for each dinucleotide in the MHV RNA sequence (control). Significant enrichment was determined by adjusted P-value (q) for fold change (log₂[experiment/control]). <0.02*, <0.0001**, <1 × 10^8***. Only dinucleotides with positive enrichment are shown. (C,F) Sequence logos for the six bases surrounding the cleavage site for position −2:−1 (C) or −1:+1 (F). Logos generated from the top 1% of either RNase L (215 sites) or EndoU-dependent cleavages (306 sites). (G) UA cleavage scoring analysis. All UA sequences in the MHV RNA with ≥30 cyclic phosphate counts in either the UA^↓ or U^↓A cleavage position were compared by calculating the ratio of normalized counts (UA^↓ counts/U^↓A counts). Ratios >1 were scored as UA^↓ (RNase L) sites and ratios <1 were scored as U^↓A sites (EndoU) and total number of scored sites for either position are shown for each condition of viral infection in wt BMM at 9 and 12 hpi. (H) Model of EndoU and RNase L interaction at UA sites in MHV RNA.

FIGURE 4.

RNase L–dependent and EndoU-dependent cleavage sites in MHV RNA. (A) Schematic outline of analysis to identify EndoU/RNase L–dependent cyclic phosphate reads. (B,C) Fold change values for the top 100 RNase L–dependent or EndoU-dependent cleavage sites. Fold change in cyclic phosphate signal when comparing wt or IFNAR^(−/−) BMM infected with MHV^(S), MHV^(V), PDE^mut, and EndoU^mut virus to RNase L^−/− BMM (B) or MHV^(S), MHV^(V), PDE^mut virus to infection with EndoU^mut virus across all cell types (C) displayed as a violin scatterplot. Log₂-fold change in the absence of RNase L activity (B) or in the absence of EndoU activity (C) was calculated for each position in the MHV RNA. Fold change values for the top 100 RNase L–dependent or EndoU-dependent sites were compared in wt and IFNAR^−/− BMM under conditions of infection with MHV^(S), MHV^(V), PDE^mut, and EndoU^mut virus at 12 hpi (B) or in all cell types across conditions of infection with MHV^(S), MHV^(V), PDE^mut virus at 12 hpi. (D) Frequency and location of RNase L–dependent cleavage sites in MHV RNA. Cyclic phosphate counts at each position in the viral genome were normalized by removing signal that occurred in the absence of RNase L, which emphasizes sites that are RNase L–dependent in wt BMM infected with MHV^(S), and PDE^mut at 9 and 12 hpi. Labeled positions and dinucleotides (−2 base:−1 base) on the graph of PDE^mut represent the top 15 RNase L–dependent cleavage sites (from B) with the greatest fold change in RNase L activity (*site with robust cleavage without canonical RNase L dinucleotide preference and independent of EndoU activity; not identified as top site by RNase L–fold-change analysis). (E) Frequency and location of EndoU-dependent cleavage sites in MHV RNA. Cyclic phosphate counts at each position in the viral genome were normalized by removing signal that occurred in the absence of EndoU, which emphasizes sites that are EndoU-dependent and RNase L–independent in RNase L^−/− BMM infected with wt MHV^(V) at 9 and 12 hpi. Labeled positions and dinucleotides (−1 base:+1 base) represent the top 15 EndoU-dependent cleavage sites with the greatest fold change in EndoU activity (from C). (F) Cumulative distribution of normalized counts by position of MHV genome for every position with ≥10 cyclic phosphate counts across all cell types and infection conditions.

FIGURE 5.

Abundance of cyclic phosphate ends by MHV genomic region and MHV mRNA abundance. (A) Normalized cyclic phosphate counts per MHV genomic region in wt, IFNAR^−/−, and RNase L^−/− BMM across all conditions of viral infection at 12 hpi. Transcriptional regulatory sequences (TRSs) are numbered by their associated mRNA (2–7). Other MHV genomic regions are labeled as shown in Figure 1A. (B) Frequency and location of cleavage in the MHV TRS elements in wt BMM during infection with MHV^(V) and EndoU^mut at 12 hpi. The x-axis includes the sequence and position of the six-base MHV TRS elements. (C) Normalized RNA-seq counts (sum of MHV sg mRNA/sum of all MHV mRNAs) of MHV sg mRNAs detected in wt, IFNAR^−/−, and RNase L^−/− BMM across all conditions of viral infection at 9 and 12 hpi.

FIGURE 6.

MHV secondary structures associated with RNase L–dependent and EndoU-dependent cleavage sites. (A,C) Nucleotide resolution graphs displaying normalized counts by position for the regions encompassing secondary structure predictions. (B,D) Secondary structures of frameshift stimulation element (B) and MHV 3′-UTR pseudoknot (D), generated using available consensus alignment and the R-scape program (Rivas et al. 2017). MHV A59 sequence mapped to consensus secondary structures using available covariation model and the Infernal program (Nawrocki and Eddy 2013). Base coloring of MHV A59 sequence based on normalized cDNA reads as indicated in key for 12 hpi in wt BMM infected with MHV^(V). *Base RNase L–dependent cleavage activity is increased in PDE^mut or EndoU^mut infection as compared to MHV^(V) infection.

FIGURE 7.

Endoribonuclease cleavage of cellular RNAs and changes in host gene expression. (A,B) Gene ontology (GO) analysis of host gene expression during MHV infection. Categories of biological processes enriched with significantly up-regulated genes (P < 0.01, log₂FC > 2) from (A) MHV^(s)-infected or (B) EndoU^mut-infected wt BMM. The top five significantly enriched categories (weightFisher < 0.01) are shown. (C) Expression of host genes in GO category “response to exogenous dsRNA.” Expression (log₁₀-normalized counts) of genes in the GO category “response to exogenous dsRNA” for wt BMM at 12 hpi. (D,E) Volcano plots of changes in host gene expression during MHV infection. (C) Plot comparing MHV^(s)-infected and mock-infected wt BMM and (D) comparing EndoU^mut-infected and MHV^(s)-infected wt BMM. Host genes were considered significantly differentially expressed at FDR < 0.05 and logFC > 2 (up-regulated) or logFC < −2 (down-regulated). (F,G) Relationship between cyclic phosphate and RNA-seq enrichment scores. An enrichment ratio was calculated for all mRNAs using the total sum of cyclic phosphate or RNA-seq normalized counts in MHV^(S) infected samples/total sum of cyclic phosphate or RNA-seq normalized counts in EndoU^mut-infected samples at 9 and 12 hpi in wt and RNase L^−/− BMM (enrichment score = [MHV^(S)/[EndoU^mut]). Genes were assigned to bins as follows: bin 1 = cyclic phosphate and RNA-seq enrichment ratio <1, bin 2 = cyclic phosphate and RNA-seq enrichment ratio ≥1, bin 3 = cyclic phosphate ratio <1 and RNA-seq ratio ≥1, bin 4 = cyclic phosphate ratio ≥1 and RNA-seq ratio <1. Each bin includes genes assigned from 9 and 12 hpi and highlighted genes represent those identified in the dsRNA response GO category (Fig. 7C). (H,I) Dinucleotide specificity analysis for cleavage of transcripts involved in the dsRNA response (Fig. 7C) during infection with MHV^(s) and PDE^mut for positions −2:−1 (H) or MHV^(s) and EndoU^mut for positions −1:+1 (I). Percent of cleavage at each 3′-dinucleotide calculated relative to the total cDNA reads aligned to the mm10 transcriptome per library in wt and RNase L^−/− BMM at 9 and 12 hpi.

FIGURE 8.

EndoU targets in MHV RNA. MHV RNA was targeted for cleavage by EndoU within infected BMM. MHV RNA was cleaved by EndoU in all regions of the genome, at C^↓A and U^↓A sequences. Because MHV RNA is a template for both viral mRNA translation and viral RNA replication, cleavage by EndoU could inhibit both of these biosynthetic processes. Intriguingly, MHV TRS sequences contain EndoU target sequences (C^↓A and U^↓A sequences). TRS6, which was targeted more frequently by EndoU than other TRS elements, contains a C^↓A target sequence rather than a U^↓A sequence. We postulate that EndoU cleaves MHV RNA in a regulated manner, to inhibit negative-strand RNA synthesis, thereby inhibiting the accumulation of viral dsRNA. Nsp16 (2′-O-MT) could regulate EndoU-mediated cleavage of MHV RNA by methylating C^↓A and U^↓A sequences. EndoU and RNase L cleave an overlapping set of UA sequences within MHV, suggesting a functional interplay between host and viral endoribonucleases.