Frontiers in Microbiology (Jun 2021)

Substitutions at Loop Regions of TMUV E Protein Domain III Differentially Impair Viral Entry and Assembly

  • Tao Hu,
  • Zhen Wu,
  • Shaoxiong Wu,
  • Mingshu Wang,
  • Mingshu Wang,
  • Mingshu Wang,
  • Renyong Jia,
  • Renyong Jia,
  • Renyong Jia,
  • Dekang Zhu,
  • Dekang Zhu,
  • Dekang Zhu,
  • Mafeng Liu,
  • Mafeng Liu,
  • Mafeng Liu,
  • Xinxin Zhao,
  • Xinxin Zhao,
  • Xinxin Zhao,
  • Qiao Yang,
  • Qiao Yang,
  • Qiao Yang,
  • Ying Wu,
  • Ying Wu,
  • Ying Wu,
  • Shaqiu Zhang,
  • Shaqiu Zhang,
  • Shaqiu Zhang,
  • Juan Huang,
  • Juan Huang,
  • Juan Huang,
  • Sai Mao,
  • Sai Mao,
  • Sai Mao,
  • Xumin Ou,
  • Xumin Ou,
  • Xumin Ou,
  • Qun Gao,
  • Qun Gao,
  • Qun Gao,
  • Di Sun,
  • Di Sun,
  • Di Sun,
  • Yunya Liu,
  • Ling Zhang,
  • YanLing Yu,
  • Shun Chen,
  • Shun Chen,
  • Shun Chen,
  • Anchun Cheng,
  • Anchun Cheng,
  • Anchun Cheng

DOI
https://doi.org/10.3389/fmicb.2021.688172
Journal volume & issue
Vol. 12

Abstract

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Flavivirus envelope protein (E) plays an important role in cellular infection, especially in virulence and antigenicity. E domain III of Tembusu virus (TMUV) is highly conserved among flaviviruses and contains four loop regions. However, the functions of the loop regions of TMUV E domain III in the viral life cycle have not yet been discovered. In this study, using a reverse genetics system, we performed site-directed mutagenesis on loops I, II, III, and IV of TMUV E domain III. Mutant 6 (S388A.G389A.K390A) showed better proliferation than the wild-type virus, while mutants 1–5 exhibited decreased in vitro infectivity, as determined by immunofluorescence assay (IFA). Based on a TMUV replicon system, the mutations exhibited no apparent effect on TMUV RNA replication. Subcellular fractionation assays and packaging system assays indicated that mutations in loops II–IV (T332A, T332S, S365A.S366A.T367A, and S388A.G389A.K390A, respectively) disrupted virion assembly. Moreover, loops I–IV played an important role in virus binding and entry, while mutant 6 (S388A.G389A.K390A) exhibited robust activity in virus entry. Taken together, our findings indicated the critical role of the loop regions in TMUV E domain III in the virus entry and assembly process.

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