A second open reading frame in human enterovirus determines viral replication in intestinal epithelial cells

Haoran Guo,Siyu Shen,Yunhe Jiang,Chunxi Wang,Yan Li,Wei Wei,Honglan Huang,Ran Bi,Tong Cheng,Guanchen Liu

doi:10.1038/s41467-019-12040-9

Abstract

Human enteroviruses (HEVs) of the family Picornaviridae, which comprises non-enveloped RNA viruses, are ubiquitous worldwide. The majority of EV proteins are derived from viral polyproteins encoded by a single open reading frame (ORF). Here, we characterize a second ORF in HEVs that is crucial for viral intestinal infection. Disruption of ORF2p expression decreases the replication capacity of EV-A71 in human intestinal epithelial cells (IECs). Ectopic expression of ORF2p proteins derived from diverse enteric enteroviruses sensitizes intestinal cells to the replication of ORF2p-defective EV-A71 and respiratory enterovirus EV-D68. We show that the highly conserved WIGHPV domain of ORF2p is important for ORF2p-dependent viral intestinal infection. ORF2p expression is required for EV-A71 particle release from IECs and can support productive EV-D68 infection in IECs by facilitating virus release. Our results indicate that ORF2p is a determining factor for enteric enterovirus replication in IECs.

Highlights

Human enteroviruses (HEVs) of the family Picornaviridae, which comprises non-enveloped RNA viruses, are ubiquitous worldwide
These findings suggest that all preceding steps in EV-D68 replication were unaffected and that virus release from HT-29 cells was blocked (Supplementary Fig. 1d–f)
When we examined EV-A71 replication in a panel of intestinal epithelial cell lines, we found that deletion of orf2p caused a marked reduction of virus titers in several cell lines, and smaller, but still significant reductions in the others (Supplementary Fig. 3a)

Summary

Introduction

Human enteroviruses (HEVs) of the family Picornaviridae, which comprises non-enveloped RNA viruses, are ubiquitous worldwide. The majority of EV proteins are derived from viral polyproteins encoded by a single open reading frame (ORF). Disruption of ORF2p expression decreases the replication capacity of EV-A71 in human intestinal epithelial cells (IECs). Ectopic expression of ORF2p proteins derived from diverse enteric enteroviruses sensitizes intestinal cells to the replication of ORF2p-defective EV-A71 and respiratory enterovirus EVD68. ORF2pdefective EV-A71 has decreased viral infectivity in both transformed and freshly isolated primary human IECs. Diverse enterovirus ORF2p proteins show an ability to sensitize human intestinal HT-29 cells to the replication of ORF2p-defective EVA71 and respiratory enterovirus EV-D68. Mutation of the conserved WIGHPV domain of ORF2p destroys its intestinal infection capacity, and ORF2p enhances the viral replication capability of EV-A71 and EV-D68 by facilitating virus release from gut cells. Our findings shed light on an determinant for enterovirus replication at the primary site of replication, namely, intestinal cells

Methods

Results

Conclusion