Zika virus produces noncoding RNAs using a multi-pseudoknot structure that confounds a cellular exonuclease

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Science  02 Dec 2016:
Vol. 354, Issue 6316, pp. 1148-1152
DOI: 10.1126/science.aah3963
  • Fig. 1 ZIKV produces sfRNAs by exonuclease resistance.

    (A) Northern blot of purified RNA from mock-infected (m) and ZIKV-infected (zv) human A549, Vero, mosquito C6/36, and cultured primary mouse neuron cells. Probe was complementary to the putative dumbbell sequence in the ZIKV 3′UTR. gRNA, genomic RNA; sfRNA1, band corresponding to largest sfRNA. Inset shows a high-contrast view of the region in the dashed box. Infections and Northern blots were confirmed in three independent experiments. (B) Secondary structure diagram with sequence conservation in known stem-loop (SL)–type xrRNAs from MbFVs. Universally conserved nucleotides (yellow) and positions of tertiary interactions (red, blue) are shown. The P2, P3, and P4 stems and associated loops contain variable sequences (var); N represents any nucleotide. (C) Sequence alignment of several known or putative MbFV xrRNA sequences with regions colored as in (B). DENV, dengue virus; JEV, Japanese encephalitis virus; WNV, West Nile virus. (D) Cartoon of the predicted ZIKV 3′UTR secondary structure, with SL and dumbbell (DB) elements. In the Kunjin strain of WNV (WNVKun), the SL1 and SL2 RNAs are xrRNAs (4, 19, 21). (E) Ethidium-stained gel of in vitro transcribed ZIKV full-length 3′UTR (FL UTR) or a 3′UTR lacking the first putative xrRNA1 element (ΔxrRNA1) treated with recombinant pyrophosphohydrolase RppH (to generate a 5′ monophosphate) and recombinant exonuclease Xrn1. The smaller band indicates Xrn1 resistance. The presence of Xrn1 activity in the absence of RppH is due to spontaneous loss of the 5′ pyrophosphate moiety that occurs at some level even in the absence of the RppH enzyme, as previously observed (19).

  • Fig. 2 Structure of the ZIKV xrRNA1.

    (A) Secondary structure diagram of the crystallized RNA. Lowercase letters represent sequences altered to facilitate RNA expression and crystallization. Colored lines indicate interactions discussed in the text. (B) Xrn1 resistance assays of wild-type (Wt) ZIKV xrRNA1 and the crystallized RNA (Cryst) using 3′ end-labeled RNA (yellow circle). The percentage of the total RNA that formed an Xrn1-resistant band (listed below the gel) was quantified and is reported as the average ± SD of three independent experiments. (C) Ribbon representation of the structure of ZIKV xrRNA1, colored to match (A). Magnesium ions are shown as yellow spheres. (D) Detail of interactions at the 5′ end of the RNA. Residue C22 (cyan) contacts the phosphate backbone of neighboring residues, setting up a kink in the RNA critical for folding. Residues U4, A24, and U42 (green) form a base triple interaction orienting the 5′ end. Residue G3 forms a long-range base-pairing interaction with residue C44. Residue G2 was mutated from a U to promote transcription; the wild-type sequence is predicted to form a base pair with residue A45 (predicted position change indicated by arrow) (21).

  • Fig. 3 Details of the ZIKV xrRNA1 structure.

    (A) Detailed view of the A37-U51 base pair (red) and intervening nucleotides (blue), which circle the 5′ end of the RNA. Other nucleotides discussed in the text are labeled. (B) The A37-U51 base pair (red) and intervening nucleotides (blue) are highlighted. The box shows the previously predicted secondary structure of the P3-L3 stem-loop. Leontis-Westhof nomenclature is used to indicate noncanonical pairing (30). Inset displays details of all three noncanonical base pairs; electron density is displayed at the 2σ contour level. (C) The L3-S4 pseudoknot with the P4 stem coaxially stacked. Colors are as in Fig. 2, A and C. (D) Xrn1 resistance assays of pseudoknot mutants and a mutant known to disrupt xrRNA folding (C22G) (19, 21). Quantitation of resistance from three experiments is shown, determined as in Fig. 2B. (E) Northern blot of viral RNA isolated from viral infection with wild-type virus and virus mutated in the xrRNA1 structure. The mutants are labeled to match the analogous mutants in (D) and fig. S4C; corresponding positions in the viral RNA are provided below.

  • Fig. 4 Model of ZIKV xrRNA–Xrn1 interaction.

    (A) Comparison of the S4 region (orange) and adjacent RNA in the partially folded MVE (left) and fully folded ZIKV (right) xrRNAs. (B) Overlay of the MVE (cyan) and ZIKV (yellow) structures, showing the change in the position of the P4-L4 hairpin. (C) Models of the MVE (top) and ZIKV (bottom) xrRNAs docked onto the surface of Xrn1, colored according to electrostatic potential (blue, positive; red, negative). Structural features are labeled.

Supplementary Materials

  • Zika virus produces noncoding RNAs using a multi-pseudoknot structure that confounds a cellular exonuclease

    Benjamin M. Akiyama, Hannah M. Laurence, Aaron R. Massey, David A. Costantino, Xuping Xie, Yujiao Yang, Pei-Yong Shi, Jay C. Nix, J. David Beckham, Jeffrey S. Kieft

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S8
    • Table S1
    • References

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