FIG 1.

Design and generation of the group II intron-based infectious clone of ZIKV. (A) Construction strategy of the infectious clone of ZIKV isolate GZ01. The SP6 promoter (blue triangle) was placed upstream of the 5′ end of ZIKV genome. The positions of the S1 to S4 fragments and endonucleases used for fragment assembly are labeled, and the numbering was calculated by setting the first nucleotide of ZIKV genome as “+1.” Note that the length of the intron sequence was calculated for the numbering. The artificially introduced KpnI site is shown in cyan. The cDNA sequence of P.li.LSUI2 group II intron was inserted near the border of the E and NS1 genes in the ZIKV genome. The EBS sequences of the inserted intron were modified to recognize the flanking ZIKV sequences. The intron-containing viral RNA transcripts were purified and subjected to in vitro splicing, which leads to the self-splicing of the intron from the viral genome and the generation of an intact viral genome. The binding locations of the primer pair used for RT-PCR detection in panel E and Fig. 2A are indicated by black arrows. (B) Secondary structure of the modified P.li.LSUI2 intron, which was inserted between position 2472 and 2473 in ZIKV genome. The EBS and IBS regions are highlighted in different colors. The EBS1 to -3 of the inserted intron was engineered to become complementary with positions 2467 to 2472, 2460 to 2464, and 2473, respectively, in the ZIKV genome. The gray circle indicates the original position of the IEP-coding region. The secondary structure was generated by the VARNA software (63). (C) 3-D structural model based on the original intron (PDB 4R0D) was generated by PyMol (pymol.org). The EBS and IBS regions were highlighted as in panel B. (D) Characterization of the in vitro splicing reaction. The electrophoresis patterns of the unspliced and spliced RNA transcripts were analyzed using a 1% agarose–TAE gel, and RNA, which was in vitro transcribed from the intron cDNA (lane “I”) and ran in parallel. Four hundred nanograms of each sample was loaded per well. The two slowly migrating bands correspond to different conformers of full-length RNA. Lane M, DNA marker DL 15,000. (E) RT-PCR was performed using the unspliced or spliced RNA transcripts as templates. Portions (5 μl) of PCR products were loaded onto a 1% agarose–TAE gel. Lane M, DNA marker DL 2,000. (F) Viral protein expression in transfected BHK-21 cells. A total of 500 ng of unspliced and spliced in vitro-transcribed RNA was transfected into BHK-21 cells, and viral E protein expression was detected by IFA at 72 h posttransfection.