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Viral hijacking of the TENT4–ZCCHC14 complex protects viral RNAs via mixed tailing

Abstract

TENT4 enzymes generate ‘mixed tails’ of diverse nucleotides at 3′ ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4–ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for mixed tailing and protection of viral poly(A) tails. We find that the HBV post-transcriptional regulatory element (PRE), specifically the CNGGN-type pentaloop, is critical for TENT4-dependent regulation. HCMV uses a similar pentaloop, an interesting example of convergent evolution. This pentaloop is recognized by the sterile alpha motif domain–containing ZCCHC14 protein, which in turn recruits TENT4. Overall, our study reveals the mechanism of action of PRE, which has been widely used to enhance gene expression, and identifies the TENT4–ZCCHC14 complex as a potential target for antiviral therapeutics.

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Fig. 1: Extensive mixed tailing of viral RNAs.
Fig. 2: HBV RNAs are the major mRNA substrates of TENT4 via the PRE of HBV.
Fig. 3: Stem-loop structure of PRE is necessary but may not be sufficient for TENT4-dependent RNA tailing of HBV mRNA.
Fig. 4: Stem-loop structure of HCMV RNA2.7 is the cis-acting RNA element responsible for TENT4-dependent regulation.
Fig. 5: SAM domain–containing proteins, including ZCCHC14, bind to the stem-loop structure and regulate HBV mRNAs.
Fig. 6: Cytoplasmic ZCCHC14 recruits TENT4 to protect RNAs with the stem-loop structure.

Data availability

TAIL-seq and fCLIP-seq data are available at the GEO with accession number GSE146602. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD018061. Source data for Figs. 1 and 36 and Extended Data Figs. 16 are available online.

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Acknowledgements

We thank members of our laboratory for discussion and technical help, especially B. Kim for fCLIP-seq experiment, S. Lee for RNA pulldown, S.-C. Kwon for antibody generation and technical advice and E. Kim for technical assistance. We also thank J. Kim and Y. Choi for support with the mass spectrometry, E. Ko and H.-W. Seo for their advice about handling HepG2.2.15, and T. Shenk (Princeton University) for providing HCMV Toledo BAC DNAs. This work was supported by grant no. IBS-R008-D1 from the Institute for Basic Science from the Ministry of Science, ICT and Future Planning of Korea (D.K., Y.-s.L., S.-J.J., J.Y., J.J.S., Y.-Y.L., J.L., H.C., J.S., J.h.Y., J.-S.K., K.A., and V.N.K.), BK21 Research Fellowships from the Ministry of Education of Korea (D.K., J.J.S., Y.-Y.L., and J.S.) and the NRF-2018-Global PhD Fellowship Program from the National Research Foundation of Korea (Y.-Y.L.).

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D.K., Y.-s.L., S.-J.J., J.Y. and V.N.K. designed the experiments. D.K., S.-J.J., J.Y., J.J.S., Y.-Y.L. and J.L. performed the biochemical and cell biological experiments. Y.-s.L. carried out computational analyses. H.C. supported TAIL-seq analysis. J.S. and K.A. provided HCMV-infected primary HFF cells. J.h.Y. made the TENT4A and TENT4B knockout cells and laboratory-made TENT4A and TENT4B antibodies. J.-S.K. helped with the LC–MS/MS experiment. G.J. provided the HepG2.2.15 cell line as well as valuable comments and facilities. D.K., Y.-s.L., S.-J.J., J.Y. and V.N.K. wrote the manuscript.

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Correspondence to V. Narry Kim.

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Extended data

Extended Data Fig. 1 Extensive 3′ end tail modification of viral RNAs.

a, HBV mRNAs are highly mixed tailed (G:guanylation, U:uridylation, C:cytidylation) in HepG2.2.15 cells. Fraction of each modification includes their respective terminal and internal modifications. Fraction of cytidylated and uridylated tails of cellular and viral mRNAs were calculated for those with poly(A) tail length ≥25 nt (n = 2 TAIL-seq experiments). b, HCMV RNAs are also highly mixed tailed in HCMV-infected HFF cells. HCMV RNA2.7 (VRNA2.7) is substantially mixed tailed in particular. (n = 1 TAIL-seq experiment) c, Gene-level analysis of guanylated tails of cellular (grey) and viral (yellow) mRNAs in HCMV-infected HFF cells. d, siRNA knockdown confirmation by RT-qPCR. e, Decrease in the fraction of mixed tail of HBV mRNAs in TENT4-depleted HepG2.2.15 cells. f, TENT4A/B expression is induced in HCMV-infected HFF cells. TENT4A and TENT4B RNA levels in HCMV-infected HFF cells were measured by RT-qPCR (n = 1). g, Decrease in the fraction of mixed tail of HCMV RNA2.7 in TENT4-depleted HCMV-infected HFF cells. h, HBV mRNAs exhibit shorter poly(A) tail length in TENT4-depleted HepG2.2.15 cells. The medians are marked by dashed vertical lines and shown in parentheses. i, Global poly(A) tail length distribution of viral mRNAs after TENT4 depletion in HCMV-infected HFF cells. j, Poly(A) tail length distribution of HCMV RNA2.7 after TENT4 depletion. k, HBV transcripts and their respective CDS regions are shown. Bars indicate the position of RT-qPCR amplicons (#1–3). l, Tail modifications of HBV mRNAs across poly(A) tail length in TENT4-depleted HepG2.2.15 cells. m, Half-life of HCMV RNA2.7 was measured by RT-qPCR (n = 3 independent experiments) in HCMV-infected HFF cells after TENT4 depletion. GAPDH mRNA was used for normalization. Mean was calculated. Data for graphs in a-b,d-g,m are available as source data. Source data

Extended Data Fig. 2 Analysis and confirmation of TENT4A/B and HBV mRNA interaction.

a, TENT4B-bound RNA fragments are again enriched within the PRE region of HBV (highlighted in yellow). Standardized read coverage of fCLIP-seq libraries across the HBV genome (NCBI:U95551.1). X-axis scale is the same as in Fig. 2a. b, Immunoprecipitation with anti-TENT4A and anti-TENT4B followed by RT-qPCR was used to measure the enrichment of TENT4-bound RNA in HepG2.2.15 cells (n = 3 independent experiments). Immunoprecipitation with normal mouse IgG was used for normalization. Mean was calculated and Error bars represent SEM. **P < 0.01, ****P < 0.0001; two-sided t test. Data for graphs in b is available as source data. Source data

Extended Data Fig. 3 Confirmation of TENT4-dependent regulation of PRE reporters.

a, Knockout confirmation by western blotting. GAPDH was used as a loading control. b, RNA levels of firefly reporters harboring subregions of PRE were measured by RT-qPCR (PRE:PREα:PREβ, n = 4; αΔ1, n = 2; αΔ2, n = 1 independent experiment). mRNA levels of both renilla and control vector were used for normalization. Mean was calculated and Error bars represent SEM. *P < 0.05; two-sided t test. c, siRNA knockdown confirmation by western blotting in HEK293T cells. GAPDH was used as a loading control. d, Firefly luciferase activity of PRE reporter constructs in TENT4-depleted HEK293T cells (PRE:PREα:PREβ, n = 7; αΔ1, n = 6; αΔ2, n = 5 independent experiments). Luciferase activities of both renilla and control vector were used for normalization. Mean was calculated and Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001; two-sided t test. e, Over-expression confirmation by western blotting in KO cells. GAPDH was used as a loading control. f, RNA levels of firefly reporters used in rescue experiments in TENT4 KO cells with ectopically expressed TENT4 wild-type or mutant (n = 3 independent experiments) were measured by RT-qPCR (n = 3 independent experiments). mRNA levels of both renilla and control vector were used for normalization. Mean was calculated and Error bars represent SEM. *P < 0.05, **P < 0.01; two-sided t test. Uncropped blots for panel a,c,e and data for graphs in b,d,f are available as source data. Source data

Extended Data Fig. 4 Confirmation of TENT4-dependent regulation of RNA2.7 reporters.

a, Immunoprecipitation with anti-TENT4A was confirmed by western blotting in RNA2.7-transfected HEK293T cells. Normal mouse IgG (NMG) was used as a negative control. RNA2.7 transcript is shown with bar indicating the position of RT-qPCR amplicon. b-e, RNA levels of firefly reporters harboring b, subregions of RNA2.7 (n = 3 independent experiments), c, deletion constructs of fragment 1 (1–513) of RNA2.7 (n = 3 independent experiments), d, construct 1D (314-413), 1E (414–513) and stem-loop mutant 1E (n = 3 independent experiments) and e, construct SL2.7 (414–463) and controls (n = 3 independent experiments) in parental and TENT4 KO cells. mRNA levels of both renilla and control vector were used for normalization. Mean was calculated and Error bars represent SEM. *P < 0.05, **P < 0.01; two-sided t test. Uncropped blots for panel a and data for graphs in b-e are available as source data. Source data

Extended Data Fig. 5 Confirmation of ZCCHC14 and SAMD4A/B knockdown.

siRNA knockdown confirmation by RT-qPCR (n = 4 independent experiments) and western blotting in HepG2.2.15 cells. Mean was calculated and Error bars represent SEM. ****P < 0.0001; two-sided t test. GAPDH was used as a loading control. Uncropped blots and data for graphs are available as source data. Source data

Extended Data Fig. 6 Confirmation of ZCCHC14 knockdown, stem-loop-dependent tail regulation, and immunoprecipitation.

a, siRNA knockdown confirmation by western blotting in ZCCHC14-depleted HEK293T cells. GAPDH was used as a loading control. The dashed line indicates discontinuous lanes from the same gel. b, Stem-loop structures of WPREα and their respective mutants. c, Distributions of poly(A) tail length of reporter RNAs measured by Hire-PAT assay. The reporter constructs were transfected into HEK293T cells after ZCCHC14 depletion. d, Immunoprecipitation with anti-ZCCHC14 was confirmed by western blotting in HepG2.2.15 cells. Normal rabbit IgG (NRG) was used as a negative control. e, Immunoprecipitation with anti-TENT4A and anti-TENT4B followed by western blotting was used to measure the physical interaction between TENT4A/B and ZCCHC14 in HepG2.2.15 and primary HFF cells. Cell extracts were treated with RNase A. Normal mouse IgG (NMG) was used as a negative control. The asterisks indicate cross-reacting bands. Uncropped blots for panel a,d-e are available as source data. Source data

Supplementary information

Reporting Summary

Supplementary Table 1

The information about genomic sequences of TENT4 and oligonucleotide sequences used in this study.

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Kim, D., Lee, Ys., Jung, SJ. et al. Viral hijacking of the TENT4–ZCCHC14 complex protects viral RNAs via mixed tailing. Nat Struct Mol Biol 27, 581–588 (2020). https://doi.org/10.1038/s41594-020-0427-3

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