Identification of a Functional Small Noncoding RNA of African Swine Fever Virus

Figures

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  FIG 1. Infection with ASFV does not induce miRNA polyadenylation or decay. (a) Northern blot of RNA extracted from Vero cells and porcine alveolar macrophages (PAMs) that were infected with either ASFV or VACV. The blot was probed for miR-27b-3p and 5s and imaged on a Phosphorimager. Arrow, mature miRNA; asterisk, polyadenylated miRNA. (b) Phosphorimager quantification of miR-27b-3p expression normalized to 5S rRNA. The image and quantification is a representation of two biological repeats.

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  FIG 2. ASFV does not induce widespread polyadenylation or decay of cellular miRNAs. (a and b) The proportion of sequencing reads that mapped to known host miRNAs that were PAM infected or mock infected with ASFV (a) and in HeLa cells infected or mock infected with VACV at early and late time points (b). (c and d) The proportion of 3′ modified miRNA that contained at least 1 nontemplated nucleotide after position 19 as a proportion of total sequencing reads, in ASFV mock-infected and infected PAMs (c) and VACV mock-infected and infected HeLa cells (d). (e) Proportion of miRNA containing 3 or more 3′ nontemplated adenosine residues after nucleotide position 19 in ASFV mock-infected and infected PAMs. Data represent the means from three biological repeats, and error bars represent standard errors of the means (SEM). Data were statistically analyzed using Student’s t test. ns, P > 0.05; ***, P < 0.001; ****, P < 0.0001. Data from panels b and d were taken from supporting information from reference 18 and represent means from three biological replicates.

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  FIG 3. ASFV infection induces rapid changes in the abundance of a select number of miRNAs in PAMs. Volcano plots showing the differential expression of host miRNAs in ASFV-infected cells relative to mock-infected cells at 0 hpi (a), 6 hpi (b), and 16 hpi (c). The miRNAs that were differentially expressed with a significant adjusted P value (≤0.05) are detailed in the table below each volcano plot.

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  FIG 4. Differential expression of miR-10b and miR-144 in ASFV Benin 97/1-infected PAMs but not ASFV Ba71v-infected Vero cells. (a to d) Changes in expression of miR-10b (a), miR-144 (b), miR-27b-5p (c), or miR-486-1 (d) in PAMs infected with ASFV Benin 97/1 or after the addition of a mock virus preparation at 0, 6, and 16 hpi were measured by RT-qPCR. Fold changes from miRNA sequencing are provided for comparison. (e to h) Changes in expression of miR-10b (e), miR-144 (f), miR-27b-5p (g), or miR-486-1 (h) in Vero cells infected with ASFV Ba71v at 0, 6, and 16 hpi were measured by RT-qPCR. miRNA expression was normalized to U6 small RNA, and fold change was calculated by the Pfaffl method. Data represent means from 3 biological repeats, and error bars indicate SEMs. Black bars, sequencing results; pink bars, Benin 97/1-infected PAM RT-qPCR results; teal bars, mock virus-treated PAM RT-qPCR results.

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  FIG 5. Mapping of unaligned small RNA reads to ASFV genome reveals peaks of small RNA sequences. (a) Plot showing small RNA sequencing reads mapping to the ASFV Benin 97/1 genome. The position along the genome, in base pairs, is along the x axis, and the number of reads is on the y axis. (b) Plot of reads mapping to the Ba71v ASFV genome (this sequence includes the inverted terminal repeats). (c) Table detailing the location and sequence of the peaks in reads. The average (mean) number of reads per sample at each time point is shown for ASFVsRNA1 (d), ASFVsRNA2 (e), and ASFVsRNA3 (f). Error bars indicate SEM.

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  FIG 6. Alignment of ASFVsRNA2 to the ASFV genome. (a) Location of the ASFVsRNA2 sequence in the C147L coding region. Arrows represent coding direction. (b) Alignment of this region in multiple strains of ASFV. The boxed region highlights the ASFVsRNA2 sequence (alignment performed using Clustal Omega). Numbering is the genome position in ASFV Benin 97/1. (c) The proportion of reads and the number of 3′ uridine residues of ASFVsRNA2.

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  FIG 7. ASFVsRNA2 is expressed in vivo. Expression of ASFVsRNA2 was determined in ASFV-infected porcine macrophages and Vero cells at 0, 6, and 16 hpi by RT-qPCR (a) or Northern blotting (b). 5S rRNA served as a loading control. (c) RT-qPCR analysis of ASFVsRNA2 expression in tissue samples from pigs infected with ASFV OUR88/1. SMLN, submandibular lymph node; MELN, mesenteric lymph node; GHLN, gastrohepatic lymph node; RLN, renal lymph node. In panels a and c, data represent means from 3 biological replicates and error bars indicate SEMs.

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  FIG 8. ASFVsRNA2 is not produced through an Ago2-dependent pathway. (a) Western blot of ASFV-infected PAM lysates before and after Ago2 immunoprecipitation using anti-Ago2 and anti-actin antibodies. Pre-IP, lysate before immunoprecipitation; ago2-IP, Ago2 immunoprecipitation lysate. Rabbit-IP, preimmune rabbit serum control immunoprecipitation. (b) The log2 fold enrichment after RT-qPCR for miR-21 and ASFVsRNA2 in the Ago2 immunoprecipitation. Expression was normalized to U6 small RNA and fold change calculated by the Pfaffl method. Data represent means from 3 technical replicates, and error bars indicate SEM.

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  FIG 9. Expression of a mimic of ASFVsRNA2 leads to a reduction in ASFV replication. (a) Vero cells transfected with miRIDIAN mimic transfection control with Dy547 for 24 h were subsequently infected with ASFV Ba71v (MOI, 5). After 24 h, cells were fixed and labeled to confirm cells can be successfully transfected and infected. (b) Vero cells were transfected with RNA mimics of ASFVsRNA2, with and without poly(U) sequence. After 24 h, cells were infected with ASFV Ba71v (MOI, 5), and supernatants were taken at 0, 4, 8, 12, and 24 hpi and titrated on Vero cells. TCID50 was calculated by the Spearman-Karber method. Error bars indicate SEM. *, P = 0.0111; ***, P = 0.001; determined by repeated-measures two-way ANOVA. Results are representative of two biological repeats. (c to f) RT-qPCR analysis of C147L expression during the growth curve at 12 hpi (c) and 24 hpi (d) and ASFV p72 expression at 12 hpi (e) and 24 hpi (f). Expression is shown relative to mock transfection (set to 0). Data represent means from 3 technical replicates. Error bars indicate SEM.