Development of a Universal Cloning System for Reverse Genetics of Human Enteroviruses

Figures

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  FIG 1. Construction of the pUC19-CMV/T7 dual-promoter vector for universal enterovirus (EV) cloning. (A) Configuration of the dual-promoter system. The optimized T7 promoter sequence was extended after the cytomegalovirus (CMV) promoter sequence. Two dotted arrows indicate the transcription initiation site of the CMV and T7 promoters. RNAs transcribed by the promoters are cleaved to correct ends of the EV RNA genome by hammerhead ribozyme (Hh-Rbz) and hepatitis delta virus ribozyme (HDV-Rbz) as indicated by thin arrows and scissors, with estimated secondary structure of Hh-Rbz universally available for EV in the box. (B) Schematic of the mechanism of EV infectious clones.

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  FIG 2. Generation of enterovirus whole-genome cDNA for universal cloning and RACE-PCR. (A) Reverse transcription was performed using a designed primer with a dT-rich-extended sequence (CS-1) coupled with a template-switching (CS-2) primer used to generate EV whole-genome cDNA with 5′- and 3′-end tags. (B) Schematic diagram of the design principle for comparison of EV-specific template-switching and typical template-switching efficiencies. The Tag2-GGG primer is generally a template-switching primer. The Tag2-GGG-TTAAA primer is an EV-specific template switching primer for improved EV specificity. The template switching induction period was controlled either simultaneously with reverse transcription or 30 min later. (C) Gel electrophoresis analysis of 5′ UTR RACE-PCR (CV-B5) to compare the two strategies. The arrow shows the impact of the late addition of TS primer in minimizing the nonspecific band amplification during cDNA synthesis. Lane M, 1-kb DNA size ladder.

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  FIG 3. Primer design and preparation of Universal enterovirus vector and insert. Primers were designed to universally amplify EV whole-genome inserts. A total of 2,349 whole-genome sequences from the 5′ UTR to the 3′ UTR of EV-A (n = 737), EV-B (n = 260), EV-C (n = 804), and EV-D (n = 548) were downloaded from the Virus Pathogen Database and Analysis Resource (ViPR) research database site (https://www.viprbrc.org) and aligned to verify the conservation of EV 5′ and 3′ ends. Identified regions with more than 88% conservation were used for designing the universal EV insert primers (UC-3 containing the flexible region in bold italic and UC4). The 5′-end insert primer is extended to the Hh-Rbz (light purple) and the 3′-end insert primer is extended to HDV-Rbz (Tag1). Both the 5′ and 3′ ends of insert primer regions are overlapped with the 3′ end (sequence in light purple located in Hh-Rbz) and 5′ end (sequence in blue sequences located at the ends of the universal EV vector). The designed cloning primers UC-1 and UC-2 were utilized to prepare the linearized pUC19-CMV/T7 vector for universal EV cloning. The cloning vector construct had a dual-promoter system equipped with two autocleaving…

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  FIG 4. Schematic illustration and amplicon validation of the universal enterovirus RACE-PCR strategy. (A) EV universal RACE-PCR method and universal primer design principle schematic diagram. The dotted box indicates the primers’ targeted region in the 5′ UTR and 3′ UTR of the EV whole-genome cDNA. (B and C) Gel electrophoresis analysis of EV universal 5′ UTR RACE-PCR and 3′ UTR applied to EV-A (EV-A71), EV-B (CV-B5, ECHO6, and ECHO30), EV-C (CV-A24), and EV-D (EV-D68). All reaction mixtures were separated on 0.9% agarose gels and visualized using gel red staining. Lane M, 1-kb DNA size ladder.

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  FIG 5. Characterization of the recovered viruses from enterovirus A to D infectious clones. (A) Plaque morphologies of recovered EV-A (EV-A71), EV-B (CV-B5, ECHO6, and ECHO30), EV-C (CV-A24), and EV-D (EV-D68) were compared to their respective parental strains (WT) 72 h postinfection. (B to G) Multistep growth curves of WT viruses and generated recombinant viruses grown in cells. Error bars represent standard deviations of the means from three independent experiments.

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  FIG 6. Schematic diagram of universal enterovirus cloning and comparison with the other cloning methods. The diagram shows the simplification and convenience of the universal enterovirus cloning when the devised strategies are applied compared to other cloning methods.