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<p>Porcine reproductive and respiratory syndrome virus (PRRSV), an <i>Arteriviridae</i> family enveloped RNA virus, is a major swine pathogen. Using yeast transformation-associated recombination (TAR) cloning, we efficiently generated infectious PRRSV and GFP-expressing clones, identifying transcription-regulating sequences as essential for stable foreign gene expression. Screening SARS-CoV-2 antivirals showed potent inhibition by the multitarget drug ribavirin, the polymerase inhibitors remdesivir and its metabolite GS-441524. Molnupiravir, targeting the polymerase by a different mechanism, showed reduced efficacy against PRRSV, while the protease inhibitor GC376 was ineffective. The AlphaFold-predicted structure of the PRRSV polymerase revealed conserved catalytic architecture with the SARS-CoV-2 polymerases, explaining cross-family inhibitor activity. In contrast, structural divergence in proteases correlated with GC376’s inefficacy. These findings underscore the utility of the TAR cloning for arterivirus engineering, with potential applications in vector vaccine development.</p>

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Development of GFP-expressing infectious clones for PRRSV using TAR cloning for antiviral drug screening

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年9月5日

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  • Minze Zhang,
  • Bang Qian,
  • Dusan Kunec,
  • Michael Veit

摘要

Porcine reproductive and respiratory syndrome virus (PRRSV), an Arteriviridae family enveloped RNA virus, is a major swine pathogen. Using yeast transformation-associated recombination (TAR) cloning, we efficiently generated infectious PRRSV and GFP-expressing clones, identifying transcription-regulating sequences as essential for stable foreign gene expression. Screening SARS-CoV-2 antivirals showed potent inhibition by the multitarget drug ribavirin, the polymerase inhibitors remdesivir and its metabolite GS-441524. Molnupiravir, targeting the polymerase by a different mechanism, showed reduced efficacy against PRRSV, while the protease inhibitor GC376 was ineffective. The AlphaFold-predicted structure of the PRRSV polymerase revealed conserved catalytic architecture with the SARS-CoV-2 polymerases, explaining cross-family inhibitor activity. In contrast, structural divergence in proteases correlated with GC376’s inefficacy. These findings underscore the utility of the TAR cloning for arterivirus engineering, with potential applications in vector vaccine development.