Noncanonical Poly(A) Polymerase TENT4 Drives Expression of Subgenomic Hepatitis A Virus RNAs in Infected Cells

Figures

• 

  Figure 1. Reconstituting TENT4 expression in TENT4-depleted 293T cells partially rescues HAV replication. (A) Genome organization of the 18f-NLuc reporter virus [20]. (B) 293T cells were depleted of endogenous TENT4A/B by siRNAs, and recombinant TENT4A, TENT4B, or catalytic mutants TENT4A-352A and TENT4B-326A were expressed from vectors containing synonymous nucleotide changes conferring siRNA resistance. Cells were infected with 18f-NLuc virus for 48 h. Corresponding immunoblots are shown in the bottom. p-values determined by one-way ANOVA with Dunnett correction for multiple comparisons.

• 

  Figure 2. HAV 3′ poly(A) tail length distribution in infected Huh-7.5 cells. (A) Experimental scheme showing RG7834 (500 nM) or vehicle-only (DMSO) treatment of cells between 48 and 120 h after infection. (B) 3′RACE assay for HAV genomic RNA. IVT, in vitro transcribed RNA. (C) 3′ poly(A) tail lengths determined by nanopore sequencing. Data shown represent combined results from two independent experiments. The total numbers of HAV reads were 5106 for control cells and 909 for RG7834-treated cells. Vertical lines indicate quartiles: DMSO control cell median = 51 nts, RG7834-treated cells = 52 nts.

• 

  Figure 3. 3′ poly(A) tail modifications in HAV RNA and host mRNA extracted from infected cells. (A) Percentage of mono-uridylated, oligo-uridylated, and guanylated HAV (left) or cellular RNAs (right) in the nanopore experiments. (B) Immunoblots showing shRNA knockdowns of TUT4 and TUT7 in Huh-7.5 cells. (C) Replication of 18f-NLuc virus in TUT4/7 knockdown cells. Error bars denote standard deviation of 3 technical replicates.

• 

  Figure 4. Subgenomic hsRNAs are expressed in HAV-infected Huh-7.5 cells. (A) (top) Alignment plot of long-read nanopore sequences of HAV RNA isolated from RG7834 and DMSO-treated Huh-7.5 cells. 3′ poly-adenylated subgenomic hsRNAs (red band) align with the 5′ untranslated RNA (5′UTR) segment of the genome in cells treated with the DMSO vehicle. hsRNAs are not detected among RNAs isolated from cells treated with the TENT4 inhibitor RG7834. hsRNAs are 5′ co-terminal with the genome and overlap the internal ribosome entry site (IRES, black band). (bottom) Similar nanopore sequencing of poliovirus-infected cells. (B) Predicted secondary RNA structure of domain V within the 5′ UTR [32], showing stem-loops Va, Vb (to which ZCCHC14 is recruited) [4], and Vc, and the position at which hsRNAs terminate, nts 672–3 (numbering is for wildtype HM175 virus, GenBank M14707.1). pY2, second pyrimidine-rich track. (C) hsRNA abundance relative to HAV RNA transcripts in two independent experiments. HAV reads in DMSO control cells ranged from 3266 to 3701, and those in RG7834-treated cells ranged from 611 to 639. p-values < 0.003 by X2 contingency test in both experiments. (D) hsRNA 3′ poly(A) tail length.…

• 

  Figure 5. Quantitative RT-PCR assay for hsRNA. (A) Diagram demonstrating the qPCR primer sets for HAV genomic RNA and hsRNA. The reverse primer for hsRNA contains 20nt oligo-dT followed by 6nts complementary to nts 667–672 of the wildtype HAV genome. (B) Threshold cycles (Ct values) from qPCR assays spiked with synthetic HAV or hsRNA RNA containing 30 nt poly(A) using the HAV or hsRNA primer sets. (C) Ct values from qPCR assays of RNAs samples extracted from HAV-infected Huh-7.5 cells. RG7834 treatment was as in Figure 2A. (D) Huh-7.5 cells were infected with 18f virus for 2 days and treated with 500 nM RG7834. HAV and hsRNA levels at indicated time points were determined by qRT-PCR and normalized to β-actin mRNA. The RNA levels at time 0 h were arbitrarily set to 100. Error bars denote standard deviation (n = 3).

• 

  Figure 6. hsRNAs are present in vivo in infected mice but do not rescue viral replication in ZCCHC14-TENT4-depleted hepatoma cells. (A) (top) Outline of HAV infection in Ifnar1−/− mice inoculated intravenously with mouse-passaged mp6 virus and treated with RG7834 or DMSO vehicle only, administered by oral gavage at 10 µg/kg twice daily between days 4 and 7 post-infection. (bottom) qRT-PCR quantitation of genomic HAV RNA and hsRNA in infected mouse livers 7 days post-infection, with or without RG7834 treatment. RNA levels were normalized to β-actin mRNA. HAV RNA levels in infected mice without treatment were arbitrarily set to 100 (n = 3). (B) Wildtype Huh-7.5, ZCCHC14-KO, and TENT4A/B-DKO cells were transfected with synthetic hsRNA for 2 h and infected with the 18f-NLuc reporter virus. NLuc activities were measured at 72 h post-infection (n = 3).