A single mutation in the cis-acting replication element identified within the EV-A71 2C-coding region causes defects in virus production in cell culture

Emerg Microbes Infect. 2021 Dec;10(1):1988-1999. doi: 10.1080/22221751.2021.1977590.

Abstract

ABSTRACTEnterovirus A71 (EV-A71) can cause hand, foot and mouth disease with neurological and systemic complications, most frequently affecting children and infants. We describe a cis-acting replication element (cre) with a conserved stem-loop structure within the EV-A71 2C-coding region. By site-directed mutagenesis and reverse genetics using the EV-A71 full-length genome and the EV-A71 replicon containing the firefly luciferase reporter gene in place of the P1 region, the stem-loop structure and the AAACA in the loop of the cre were confirmed to be required for the EV-A71 replication phenotype. EV-A71 genomes containing a mutation at the first or third A residue of AAACA could not be recovered. Insertion of a wild-type cre from EV-A71 or poliovirus in the 5'UTR led to successful recovery of the replication of nonviable mutants. Furthermore, the cre mutants showed lower binding capacity with the host cellular factor IGF2BP2, knockdown of which resulted in a significant decrease in EV-A71 production. All the available evidence shows the location independence but functional importance of the interaction of the cre with the cellular host for efficient production of EV-A71, contributing to the growing body of knowledge regarding picornavirus cres.

Keywords: Enterovirus A71; cellular factors; cre, 2C-coding region; virus replication.

MeSH terms

  • Animals
  • Cell Line
  • Chlorocebus aethiops
  • Enterovirus A, Human / genetics*
  • Enterovirus A, Human / growth & development
  • Enterovirus Infections / virology
  • Genome, Viral / genetics*
  • Hand, Foot and Mouth Disease / virology
  • Humans
  • Integrases / genetics
  • Interspersed Repetitive Sequences / genetics*
  • Nucleic Acid Conformation*
  • RNA Interference
  • RNA, Small Interfering / genetics
  • RNA, Viral / genetics*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Vero Cells
  • Virus Replication / genetics*

Substances

  • IGF2BP2 protein, human
  • RNA, Small Interfering
  • RNA, Viral
  • RNA-Binding Proteins
  • Cre recombinase
  • Integrases

Grants and funding

This work was supported by the National Key Research and Development Programme of China (Grant Number 2016YFA0100702); the CAMS Innovation Fund for Medical Sciences (CIFMS) (Grant Number 2016-I2M-3-026); the Natural Science Foundation of Yunnan Province (Grant Number 2019FB102); the High-level Health Technology Special Talent Project of Yunnan Province (Grant Number H-2019064); the Key Project of Institute of Medical Biology, the Chinese Academy of Medical Sciences (Grant Number 2018IMBCAMS001).