Fig 1.
Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA.
XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
Fig 2.
Determine the region of 3’ UTR involved in the accumulation of sfRNA constructed in the context of a full-length infectious JEV clone.
(A) Predicted secondary structures within the 585-nt 3’ UTR of JEV genome. Nucleotides are numbered from the first base of the genome. The deletion or base substitution regions made in this study are marked or color-coded. (B) Schematic diagram of the 3’ UTR mutants and the results of Northern hybridization for detecting the genome and sfRNA are summarized on the right. (C) BHK-21 cells were infected with recombinant mutant viruses at an MOI of 0.1. RNAs were extracted at 48 h post-infection and Northern hybridization was done as described in Fig 1A. (D) HEK293T cells (WT) or XRN1-knockdown cells (KD) were infected with a PK disrupted mutant (PK1”), and a PK compensatory changed mutant (PK1’1”) viruses at an MOI of 1. Cytoplasmic RNAs were extracted at 60 h post-infection and analyzed by Northern blot. (E) Plaque assays of WT and all mutant viruses were done on BHK-21 cells. Cells were fixed and stained with naphthol blue–black dye at 4 days post-infection. (F) Viral growth kinetics of the WT infectious clone and the recombinant mutant viruses. BHK-21 cells were infected at an MOI of 0.1, and the supernatant fluid of the infected cells was sampled at the indicated times post infection. Titers were determined by plaque assays on BHK-21 cells.
Table 1.
Virus titera in supernatant fluids collected at 5 days post-transfection (dpt) and at the time of post-transfection showed a strong CPE for each cDNA construct.
Fig 3.
Replication of the viral genome is essential for abundant accumulation of the sfRNA.
BHK-21 cells were mock-infected (lane 1) or infected with JEV at an MOI of 0.01, and the RNA was extracted at 36 h post-infection (lane 2). Total RNAs were isolated from BHK-21 cells transfected with a WT infectious clone or the NS5mt at the indicated hours post-transfection (hpt). RNAs were measured by Northern hybridization with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’ UTR.
Fig 4.
The (-)10431-10566 RNA fragment is a functional template for the initiation of RNA synthesis.
(A) EMSA showing the interaction between the (-)10431-10566 RNA with the purified recombinant RdRp protein. Uniformly DIG-labeled (-)10431-10566 RNA (0.25 pmol) or nonspecific RNA (nsRNA) from 5’ 152-nt of bovine coronavirus genome were titrated with increasing concentrations of RdRp (or BSA) from 0.25, 0.5, 1 to 2.5 μM. The complexes were resolved on a 5% native polyacrylamide gel. Position of the free probe and RNA-protein complexes (RPC) are indicated. (B) Specific interaction of RdRp with the (-)10431-10566 RNA. All reactions contained the same amount of DIG-labeled (-)10431-10566 RNA (0.25 pmol). (Lane 1) free probe; (lanes 2–11) 2.5 μM of RdRp were used; (lane 2) no competitor; (lanes 3–11) unlabeled RNA competitors, representing either (-)10431-10566, tRNA, or nsRNA (10-, 50-, 100-fold molar excess) as indicated, were added to the gel-shift assay to compete against complex formation. The complexes were resolved on a 5% native polyacrylamide gel. (C) Template specificity of the purified RdRp. Templates tested were nsRNA and (+)1-160 RNA (the SLA-containing promoter element) as indicated on the top. (D) Transcripts driven by T7 RNA polymerase used as size markers (lanes 1–5) or RNA templates used for in vitro RdRp assays (lanes 6–10) are shown on the top. The products were resolved on 6% polyacrylamide gel containing 6M urea. (E) Schematic diagram of the minus RNA fragments used as templates for the in vitro RdRp assays. The numbers correspond to the nucleotide positions in the JEV genome. The products are depicted as de novo synthesis or transcription products with size (nts) indicated. (F) Diagram of the secondary structure of the 3’-UTR up to nt 10566 with relative position marked for the templates used by in vitro RdRp assays.
Fig 5.
Schematic model depicting the mechanism of JEV sfRNA formation.
The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.