Interaction between endogenous microRNAs and virus-derived small RNAs controls viral replication in insect vectors

Abstract

MicroRNAs (miRNAs) play an important role in resisting virus infection in insects. Viruses are recognized by insect RNA interference systems, which generate virus-derived small RNAs (vsRNAs). To date, it is unclear whether viruses employ vsRNAs to regulate the expression of endogenous miRNAs. We previously found that miR-263a facilitated the proliferation of rice stripe virus (RSV) in the insect vector small brown planthopper. However, miR-263a was significantly downregulated by RSV. Here, we deciphered the regulatory mechanisms of RSV on miR-263a expression. The promoter region of miR-263a was characterized, and the transcription factor YY1 was found to negatively regulate the transcription of miR-263a. The nucleocapsid protein of RSV promoted the inhibitory effect of YY1 on miR-263a transcription by reducing the binding ability of RNA polymerase II to the promoter of miR-263a. Moreover, an RSV-derived small RNA, vsR-3397, downregulated miR-263a transcription by directly targeting the promoter region with partial sequence complementarity. The reduction in miR-263a suppressed RSV replication and was beneficial for maintaining a tolerable accumulation level of RSV in insect vectors. This dual regulation mechanism reflects an ingenious adaptation strategy of viruses to their insect vectors.

Fig 1. Determination of miR-263a promoter region.

(A) Relative transcript levels of tubulin polyglutamylase (TTLL4) and miR-263a in the gut of nonviruliferous (N) and viruliferous planthoppers (V) (n = 8). The planthopper EF2 gene and U6 snRNA were used as endogenous controls. (B) Promoter activity of recombinant pGL4.10 plasmids containing one of two putative promoter regions of miR-263a in human 293T cells in the dual luciferase reporter assay (n = 6). For (A) and (B), comparison was analyzed by Student’s t test. NS, no significant difference. *, P<0.05. ***, P<0.001. The relative activity of firefly luciferase (Fluc) to Renilla luciferase (Rluc) is presented. Empty pGL4.10 was used as the negative control (NC). The schematic diagram shows two candidate transcription start sites (TSSs), from which putative promoter regions were cloned. The 5’ terminal nucleotide of mature miR-263a is designated as position +1. NS, no significant difference. Data in (A) and (B) show the mean values and standard errors and were compared by Student’s t test. (C) Western blot analysis of the nuclear and cytoplasmic extracts from viruliferous planthoppers. (D) Reverse transcription-PCR of the putative transcripts starting exactly from TSS1 or from 96 bp upstream of TSS1 with the cDNA template of the nuclear extracts. Two replicates are shown. Schematic diagram shows the positions of PCR primers. M, marker.

Fig 2. Transcription factor YY1 inhibits miR-263a transcription.

(A) Schematic diagram showing two putative YY1 binding sites, TFBS1 and TFBS2 (black rectangles), in the promoter region of miR-263a. The transcription start site (TSS) is designated position +1. pri-miR-263a, primary miR-263a. (B) Relative enrichment of the two binding sites of YY1 measured by ChIP-qPCR (n = 8). IgG was used as the negative control. (C) EMSA assay on the binding of recombinant YY1-His to the DNA sequences containing TFBS1 (left image) or TFBS2 (right image) using the biotin-labeled probes. (D) Relative transcription level of YY1 in the nonviruliferous planthoppers after injection with dsRNAs for YY1 (dsYY1) or GFP (dsGFP) for 3 d (n = 8). (E) Relative transcript levels of miR-263a, precursor miR-263a (pre-miR-263a) and pri-miR-263a in planthopper whole bodies after injection with dsYY1 or dsGFP (n = 8). (F) Relative transcript levels of pre-miR-263a and pri-miR-263a in the nuclear extracts after injection with dsYY1 or dsGFP (n = 8). (G) Relative enrichment of the putative RNA polymerase II (Pol II) binding sequence precipitated by the anti-Pol II monoclonal antibody to that precipitated by IgG in the dsYY1- or dsGFP-injected nonviruliferous planthopper measured by ChIP-qPCR (n = 6). Comparable amounts of Pol II in the two groups were detected by western blot. Data information: Graphs show mean values and standard errors. For (B), (D), (E), (F), and (G) comparison was analyzed by Student’s t test. **, P<0.01. ***, P<0.001.

Fig 3. Binding with RSV NP promotes the inhibitory effect of YY1 on miR-263a transcription.

(A) Dual luciferase reporter assay on the promoter activity of miR-263a after expression of planthopper YY1 or both YY1 and RSV NP in 293T cells with the addition of siYY1. The relative activity of firefly luciferase (Fluc) to Renilla luciferase (Rluc) is presented. Empty pGL4.10 and pcDNA3.1 were used in the negative control groups. Western blot assays show the protein expression. Different letters indicate significant differences in Tukey’s multiple comparison test. (B) Enrichment of YY1 binding sites TFBS1 and TFBS2 precipitated by the anti-YY1 polyclonal antibody relative to that precipitated by IgG in nonviruliferous (N) and viruliferous (V) planthoppers measured by ChIP-qPCR (n = 8). Comparable amounts of YY1 in the two groups were detected by western blot. (C) Enrichment of the putative RNA polymerase II (Pol II) binding sequence precipitated by the anti-Pol II monoclonal antibody relative to that precipitated by IgG in N and V planthoppers measured by ChIP-qPCR (n = 6). Comparable amounts of Pol II in the two groups were detected by western blot. (D) Relative transcript levels of miR-263a, YY1 and Pol II in the guts of nonviruliferous and viruliferous planthoppers. Data information: Graphs show mean values and standard errors. NS, no significant difference. **, P < 0.01. ***, P < 0.001.

Fig 4. Identification of RSV vsRNAs putatively targeting primary miR-263a.

(A) Relative RNA levels of the top ten most abundant vsRNAs in nonviruliferous (N) and viruliferous (V) fourth-instar planthoppers (n = 8). NS, no significant difference. **, P<0.01. ***, P<0.001. (B) Identification of vsR-1524 and vsR-3397 in N and V planthoppers by northern blot using biotin-labeled LNA oligonucleotide probes. (C) and (D) Relative RNA levels of vsR-1524, vsR-3397, and miR-263a in six organs of viruliferous adult planthoppers (C) and in the planthoppers at different days post-injection with RSV crude extractions (D) (n = 8). Different letters indicate significant differences in Tukey’s multiple comparison test. Planthopper U6 snRNA was used as an endogenous control for vsRNA in each experiment. Data information: Graphs show mean values and standard errors.

Fig 5. vsR-3397 suppresses miR-263a transcription by targeting the promoter region.

(A) Sequence alignment of vsR-3397 and vsR-1524 with their predicted target sites (Tar-WT) in the promoter region of miR-263a. The seed sequences are in red. Mutated nucleotides in the target (Tar-MT) are in blue. TSS, transcription start site. pri-miR-263a, primary miR-263a. (B) Dual luciferase reporter assays in S2 cells cotransfected with recombinant psiCHECK2 plasmids containing Tar-WT or Tar-MT of vsR-3397 and 100 nM vsR-3397 mimics (n = 6). (C) and (D) Relative RNA levels of vsR-3397 and miR-263a in the whole body (C) and relative transcript levels of pri-miR-263a and precursor miR-263a (pre-miR-263a) in the nuclei (D) of nonviruliferous planthoppers after injection with the vsR-3397 activator for 4 d (n = 8). (E) and (F) Relative RNA levels of vsR-3397 and miR-263a in the whole body (E) and relative transcript levels of pri- and pre-miR-263a in the nuclei (F) of viruliferous planthoppers after injection with the vsR-3397 inhibitor for 4 d (n = 8). (G) Western blot analysis of Ago1 in the nuclear and cytoplasmic extracts from viruliferous planthoppers. (H) Relative enrichment of vsR-3397 and its promoter region in the nucleus fraction of viruliferous planthoppers measured by RIP and ChIP combined with qPCR after injection with vsR-3397 activator for 4 d (n = 6). Mouse IgG instead of the anti-Ago1 monoclonal antibody was used as a negative control. Data information: Graphs show mean values and standard errors. NS, no significant difference. *, P<0.05. **, P<0.01. ***, P<0.001.

Fig 6. vsR-3397 inhibits RSV replication by affecting miR-263a.

(A) and (B) Relative RNA levels of vsR-3397 and RSV NP (n = 8) and the protein level of NP (n = 4) in the viruliferous planthoppers after injection with the vsR-3397 activator or inhibitor for 4 d (A) and in the nonviruliferous planthoppers after injection with the mixture of RSV and the activator or inhibitor of vsR-3397 for 6 d (B). (C) and (D) Relative RNA levels of miR-263a, vsR-3397 and RSV NP (n = 8) and protein level of NP (n = 4) in the viruliferous planthoppers after injection with the mixture of vsR-3397 activator and miR-263a agomir (C) or vsR-3397 inhibitor and miR-263a antagomir (D) for 4 d. NC, negative control. The NC sequences for vsRNA activator and miR-263a agomir are 5’-UUCUCCGAACGUGUCACGUTT-3’ (sense) and 5’-ACGUGACACGUUCGGAGAATT-3’ (antisense). The NC sequence for vsRNA inhibitor and miR-263a antagomir is 5’-ACGUGACACGUUCGGAGAATT-3’. Data information: Graphs show mean values and standard errors. NS, no significant difference. *, P < 0.05. **, P < 0.01. ***, P < 0.001.

Fig 7. Model of the regulatory mechanisms for miR-263a by RSV.

The NP of RSV binds the transcription factor YY1 to promote its inhibitory effect on miR-263a transcription probably due to enlargement of the steric hindrance to the binding of Pol II to the promoter with more YY1. At the same time, RSV-derived small RNA, vsR-3397, directly targets the promoter region of miR-263a to downregulate miR-263a transcription. TFBS, transcription factor binding site.