Figure 1.
A combined computational and synthetic approach identifies TTV encoded miRNAs.
(A) Diagram of genomic locations of predicted TTV-yonKC197 (AB038624) and TTV-TWH (AFL351132) miRNAs. Reference sequence annotated ORFs are indicated in red and predicted miRNA stemloop sequences are indicated in blue. Secondary structure predictions of the putative TTV miRNAs are shown to the right with bolded sequences indicating most abundant mature miRNA sequences observed in small RNA sequencing library. (B) Northern blot analysis of RNA harvested from HEK293T cells transfected with synthetic TTV-yonKC197 miRNA vector (SG for “synthetic gene”) or empty vector (NC). Ethidium bromide stained low molecular weight RNA is shown as a load control. (C) Northern blot analysis of RNA harvested from HEK293T cells transfected with synthetic TTV-TWH miRNA vector (SG) or empty vector (NC). Ethidium bromide stained low molecular weight RNA is shown as a load control. (D) Pooled small RNA profiling of HEK293T cells transfected with TTV-yonKC197 miRNA vector. Sequences were mapped to the synthetic region of the AB038624 NCBI reference sequence. The vertical axis depicts small RNA read coverage observed; while the horizontal axis shows the relative position within the synthetic gene sequence. (E) Pooled small RNA profiling of HEK293T cells transfected with TTV-TWH miRNA vector. Sequences were mapped to the synthetic region of the AFL351132 NCBI reference sequence. The vertical axis depicts small RNA read coverage observed; while the horizontal axis shows the relative position within the synthetic gene sequence.
Figure 2.
TTV miRNAs are expressed during in vitro cell culture replication and are detectable from in vivo small RNA profiling of human PBMCs.
(A) Diagram of genomic locations of predicted TTV-tth8 (AJ260231) miRNA. Reference sequence annotated ORFS are indicated in red and predicted miRNA stemloop sequence is indicated in blue. Secondary structure prediction of the putative TTV miRNA is shown below with bolded sequences indicating most abundant mature miRNA sequences observed in small RNA sequencing library. (B) Northern blot analysis of RNA harvested from HEK293T cells transfected with synthetic TTV-tth8 miRNA vector (SG) or empty vector (NC) and HEK293TT cells transfected with viral genome prep (TTV) or mock transfection (Mock). Northern blot analysis for host miRNA hsa-miR-19a and ethidium bromide stained low molecular weight RNA serve as load controls. (C) Small RNA profiling of human PBMC small RNAs mapped to TTV-sle2057 strain. The vertical axis depicts small RNA read coverage observed; while the horizontal axis shows the relative position within the TTV-sle2057 genomic sequence.
Figure 3.
TTV tth8 miRNAs are generated by the canonical host miRNA biogenesis pathway.
(A) Northern blot analysis of RNA from HEK293 cells transfected with MHV68-miR-M1-7 expression vector, SV40-miR-S1 TTV recombinant genome preparation, or wildtype TTV-tth8 genome preparation with or without treatment with the RNA pol II inhibitor α-amanitin. Ethidium bromide stained low molecular weight RNA is shown as a load control. (B) Northern blot analysis of RNA from HEK293T cells co-transfected with MHV68-miR-M1-7, SV40-miR-S1, or TTV-tth8-miR-T1 expression vectors and either Drosha siRNA (siDrosha) or negative control siRNA (siNC). Ethidium bromide stained low molecular weight RNA is shown as a load control. (C) Northern blot analysis of RNA from either DLD1 (WT) or DLD1DicerEx5- (DicerEx5-) cells transfected with either MHV68-miR-M1-7 or TTV-tth8-miR-T1 expression vectors. Ethidium bromide stained low molecular weight RNA is shown as a load control. (D) RISC reporter assay for TTV-tth8-miR-T1. HEK293 cells were co-transfected with either empty miRNA expression vector (Vector) or TTV-tth8-miR-T1 expression vector (TTH8) and both control firefly luciferase reporter and Renilla luciferase based reporter plasmids with vector UTR (Vector), two sites perfectly complementary to TTV-tth8-miR-T1-5p (5p), two sites perfectly complementary to TTV-tth8-miR-T1-3p (3p), or control 3′UTR reporter with GAPDH 3′UTR (GAPDH). The average relative Renilla luciferase activity normalized to firefly luciferase activity of three replicates is shown. Error bars indicate SD of three replicates and p-values were calculated using Student's t-test.
Figure 4.
Generation of mutant and SV40 recombinant TTVs.
(A) RNA secondary structure predictions of miRNA region from TTV-tth8 wildtype, TTV-tth8 miRNA mutant, and TTV-tth8 SV40 recombinant viruses. (B) Northern blot analysis of total RNA harvested from HEK293TT cells transfected with either pUC plasmid (Vector), wildtype TTV-tth8 (TTH8 WT), miRNA mutant TTV-tth8 (TTH8 MUT), or SV40 miRNA recombinant TTV-tth8 (TTH8 SV40). Northern blot analysis for host miRNA hsa-miR-19a and ethidium bromide stained low molecular weight RNA serve as load controls.
Figure 5.
Characterization of wildtype and miRNA mutant TTV-tth8.
Small RNA profiling of HEK293TT cells transfected with either wildtype TTV-tth8 (WT) or miRNA mutant TTV-tth8 (MUT). At top, ORF map derived from NCBI reference sequence AJ260231 annotations in red. The newly identified miRNA is indicated in blue. The vertical axis depicts small RNA read coverage observed; while the horizontal axis shows the relative position within the TTV-tth8 genomic sequence.
Figure 6.
Host antiviral gene NMI is a direct target of TTV-tth8-miR-T1.
(A) Diagram of predicted TTV-tth8-miR-T1-5p docking site in the 3′UTR of NMI transcripts (top) with complementary base pairing to the TTV-tth8-miR-T1-5p (middle) seed indicated (“|” for predicted base pairing and “·” for wobble pairing). At bottom is diagrammed the seed mutant (NMI MUT) sequence with the introduced two nucleotide mismatch in the seed complementary region (underlined sequence). (B) NMI 3′UTR miRNA docking site reporter assay. HEK293 cells were co-transfected with either empty miRNA expression vector (Vector), TTV-tth8-miR-T1 expression vector (TTH8), or SV40-miR-S1 expression vector (SV40) and both control firefly luciferase control and Renilla luciferase based reporter plasmids with vector UTR (Vector), full length NMI 3′UTR (NMI), full length NMI 3′UTR with 2 nt mutation in predicted docking site (NMI MUT), or control 3′UTR reporter with GAPDH 3′UTR (GAPDH). The average relative Renilla luciferase activity normalized to firefly luciferase activity of three replicates is shown. Error bars indicate SD of three replicates and p-values were calculated using Student's t-test. (C) Immunoblot analysis of total protein harvested from HeLa cells transfected with either TTV-tth8-miR-T1 miRNA mimic (miTTH8) or negative control miRNA mimic (miNC). Shown is a representative blot. (D) Quantification of the average of three independent experiments as in panel C. Error bars indicate SD of three independent experiments and p-values were calculated using Student's t-test.
Figure 7.
TTV-tth8-miR-T1 or knockdown of NMI inhibits interferon signaling.
(A) Schematic of the interferon signaling assay. Target cells are co-transfected with dual luciferase plasmids (firefly luciferase with interferon-stimulated response element (ISRE) based promoter and transfections control Renilla luciferase with constitutive promoter) and either re-circularized viral genome, miRNA mimic, or siRNA. Forty-eight hours later, cells are treated with 100 U/mL Type I interferon. Sixteen hours after interferon treatment, dual luciferase assay is performed. (B) Interferon signaling assay performed as outlined in Fig. 7A with TTV-tth8 wildtype (WT) and miRNA mutant (MUT) genomic preparations in HEK293T cells. Relative luciferase activity is normalized to pUC19 vector control (VEC). Error bars indicate SD of three replicates and p-values were calculated using Student's t-test. (C) Interferon signaling assay performed as outlined in Fig. 7A with miRNA mimics and siRNAs in HEK293T cells. TTV-tth8 miRNA mimic (miTTH8) was normalized to negative control miRNA mimic (miNC). NMI siRNA was normalized to negative control siRNA (siNC). Co-transfection of both a miRNA mimic and siRNA is indicated by a “/” and was normalized to co-transfection of both siNC and miNC. Error bars indicate SD of three replicates and p-values were calculated using Student's t-test. Not significant (n.s.) indicates p-value greater than 0.05. (D) Interferon signaling assay performed as outlined in Fig. 7A with miRNA mimics in HEK293T and ISG15 expression assayed by qPCR following interferon treatment. TTV-tth8 miRNA mimic (miTTH8) treatment was normalized to negative control miRNA mimic (miNC). NMI siRNA (siNMI) treatment was normalized to negative control siRNA (siNC). Relative ISG15 expression was determined by comparative Ct method to GAPDH. Error bars indicate SD of three replicates and p-values were calculated using Student's t-test. (E) Growth curves of interferon treated B cell lines that stably overexpress the TTV tth8 miRNA (TTH8) or a negative control irrelevant miRNA (NC) or vector alone (VEC). Viable cells as determined by trypan blue staining were counted each day and then either mock-treated with BSA or treated with universal type I IFN (+IFN). Data points represent averages of three independent experiments (each performed in triplicate) on different days and error bars indicate SD of the three experiments.