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

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.

Fig. 1

A second ORF of EV-A71 is efficiently translated in challenged cells. a Schematic diagram of the EV-A71 structure. The EV-A71 genome contains a long ORF flanked by a 5′UTR and 3′UTR. The ORF encodes a 250-kDa polyprotein that is processed into the P1, P2 and P3 regions, which are further cleaved into mature proteins (VP1 to VP4, 2 A to 2 C, and 3 A to 3D). Red box, the second ORF, which begins at the 3’ border of the IRES in the 5’UTR, encodes a 64-76-amino acid polypeptide in diverse sub-genotypes of EV-A71. b Amino acid and nucleic acid sequences of ORF2p from EV-A71 strain AH08/06. c HT-29 cells and isolated primary IECs were infected with EV-A71 or EV-A71ΔORF2p at an MOI of 0.1. Cells were harvested and prepared for immunoblotting at 48 hpi and 72 hpi using antibodies against EV-A71 ORF2p, VP1 and α-tubulin. Source data are provided as a Source Data file

Fig. 2

ORF2p is crucial for intestinal infection by EV-A71. a–c One-step growth curve of EV-A71 and EV-A71ΔORF2p in RD, HT-29 and isolated primary IECs at an MOI of 10. Error bars denote SEM; ANOVA test, n = 3 biologically independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001. d HT-29 cells stably expressing EV-A71 ORF2p or control HT-29-CDH cells generated by transduction with the empty lentiviral vector pCDH-CMV-MCS-EF1-Puro were challenged with EV-A71 WT or EV-A71ΔORF2p at an MOI of 10. Viral titres were determined at 0, 3, 6, 9, and 12 hpi (n = 3 biologically independent experiments). Error bars denote SEM. e HT-29 cells expressing the ORF2p protein derived from EV-A71 sub-genotypes A, B0, B1, B2, B3, B4, B5, C1, C2, C3, C4 and C5 were infected with EV-A71ΔORF2p at an MOI of 0.1. Viral titres were determined at 72 hpi (n = 3 biologically independent experiments). Error bars denote SEM. Source data are provided as a Source Data file

Fig. 3

The WIGHPV domain of ORF2p from enteric enteroviruses is important for viral infection in HT-29 cells. a HT-29 cells expressing the ORF2p protein derived from CV-A16, EV-A71, CV-B3, Echovirus 6, Echovirus 19, EV-B73, Poliovirus 1, CV-A24 and EV-C96 were infected with EV-A71ΔORF2p at an MOI of 0.1. Viral titres were determined at 72 hpi (n = 3 biologically independent experiments). Error bars denote SEM. b Alignment of amino acid sequences of the N-terminal portion of ORF2p from human enteroviruses. Residues of WIGHPV are marked with red arrows. c HT-29 cells were challenged with EV-A71 and mutant viruses EV-A71 (ORF2p WIG/AAA) and EV-A71 (ORF2p HPV/AAA) at an MOI of 0.1. Viral titres were determined at 0, 12, 24, 48, and 72 hpi (n = 3 biologically independent experiments). Error bars denote SEM. d Viral titres of HT-29 cells expressing wild-type ORF2p, WIG/AAA, or HPV/AAA; control cells were infected with EV-A71ΔORF2p. Viral titres were determined at 72 hpi. Error bars denote SEM, n = 3 biologically independent experiments. Source data are provided as a Source Data file

Fig. 4

ORF2p facilitates the release of enterovirus particles from isolated IECs and HT-29 cells. a–c Isolated primary IECs were transfected with 1 µg in vitro-synthesized RNA transcripts of EV-A71 or EV-A71ΔORF2p (n=3 biologically independent experiments). Error bars denote SEM. Viral RNA in the culture supernatant a and cell lysates b was detected by an RT-PCR assay at the indicated times. c Virus titres of the supernatant or frozen-thawed lysates were determined at 24, 36, 48 h post-transfection. d, e Isolated primary IECs were challenged with EV-A71 or EV-A71ΔORF2p at an MOI of 10. d Virus titres of EV-A71 or EV-A71ΔORF2p in the supernatant or cell lysates were determined at the indicated times (n=3 biologically independent experiments). Error bars denote SEM. e The supernatant and cells were harvested at indicated time, followed by immunoblot analysis, as described above. f HT-29 cells stably expressing EV-A71 ORF2p or control HT-29-CDH cells were challenged with EV-A71ΔORF2p at an MOI of 0.1. The supernatants and cells were harvested for immunoblotting using antibodies against EV-A71 VP1 and α-tubulin. CDH, control HT-29-CDH cells. g Detection of the EV-D68-induced cytopathic effect (CPE). HT-29-ORF2p or control HT-29-CDH cells were infected with equal amounts of EV-D68, and CPE was imaged via light microscopy. Scale bars equal 50 μm. h, i HT-29-ORF2p or control HT-29-CDH cells were infected with equal amounts of EV-D68. h The supernatant was harvested at 24 and 48 hpi for RT-PCR assays (n=3 biologically independent experiments). Error bars denote SEM. i Cells were harvested at 8, 24, and 48 hpi, followed by immunoblot analysis. Source data are provided as a Source Data file