EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication

Abstract

Transmissible gastroenteritis coronavirus (TGEV) is enteropathogenic coronavirus that causes diarrhea in pigs, and is associated with high morbidity and mortality in sucking piglets. The TGEV membrane (M) protein is a decisive protein for the proliferation of viral proteins, and is associated with virus assembly and budding. To identify the cellular proteins that interact with the TGEV M protein, yeast two-hybrid screening was employed, and seven cellular proteins were identified M-binding partners. Using the GST pull-down approach and a CO-IP assay, the M protein was found to interact with porcine intestinal cells via eukaryotic translation initiation factor 4-alpha (EIF4A2), an essential component of the cellular translational machinery. Additionally, confocal microscopy revealed that EIF4A2 and M were colocalized in the cytoplasm. Furthermore, the function of EIF4A2 in intestinal cells during TGEV infection was examined. A knockdown of EIF4A2 by siRNA markedly decreased M protein proliferation and TGEV replication in target cells. Thus demonstrating that EIF4A2 plays a significant role in TGEV replication. The present study provides mechanistic insight into the interaction between the TGEV M protein and intestinal cells which contributes to the understanding of coronavirus replication and may be useful for the development of novel therapeutic strategies for TGEV infection.

Keywords: Eukaryotic translation initiation factor 4-alpha (EIF4A2); Interaction; Membrane (M) protein; TGEV.

Fig. 1

Testing bait protein expression, autoactivation, and toxicity. (A) Yeast colony identification by PCR. M: DNA Marker DL2000; 1–2: PCR of colonies transformed with pGBKT7-M; (B) Identification of toxic of pGBKT7-M bait plasmid. Yeast harboring pGBKT7-M and control pGBKT7 were spread at two dilutions (1:10 and 1:100) on to SD/Trp plates, and the size and number colonies were compared to evaluate the toxicity of the bait plasmid; (C) Identification of autoactivation of the pGBKT7-M bait plasmid. pGBKT7-M were spread at 1/10 and 1/100 dilutions on SD/−Trp, SD/−Trp/X-α-Gal, and SD/-Trp/X-α-Gal/AbA for autoactivation testing; (D) Expression of M in yeast by Western blot. 1: pGBKT7 protein negative control; 2: pGBKT7-M protein; and 3: pGBKT7-53 protein positive control.

Fig. 2

A yeast two-hybrid system was used to screen for M-interacting proteins.(A) Diploid after yeast two-hybrid screening. The zygotes typically displayed a three-lobed structure similar to a “Mickey Mouse” face at 40× magnification under a microscope; (B) The third screening using SD/−Trp/−Leu/−Ade/-His/X-α-Gal/AbA selective medium. A total of 108 blue clones were visible and identified by PCR (D) M: DL2000 DNA Marker; 1–96: PCR amplification products of the positive clones. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

GST-pull down and CO-IP were used to analyze the interaction between the M protein and candidate protein EIF4A2 by SDS-PAGE and Western blot. (A) The results of the GST pull-down by SDS-PAGE M: Prestained protein marker; 1: purified GST-M fusion protein + purified His-EIF4A2 fusion protein; 2: purified GST-tag protein + purified His-EIF4A2 fusion protein; 3: purified His-EIF4A2 fusion protein; 4: purified GST-M fusion protein; 5: purified GST-tag protein; (B) The results of the GST pull-down by Western blot M: Prestained protein marker; 1–2: His-EIF4A2 fusion protein. (C) pIECs infected with TGEV and control cells were harvested at 24 h, and the M protein was precipitated by an antibody in TGEV-infected group but not the mock group. TGEV+ represent the pIECs infected with TGEV and TGEV- represent uninfected TGEV in pIECs.

Fig. 4

Co-localization of EIF4A2 with the M protein. Determining the location of the M protein using FITC-conjugated goat anti-rat IgG (green) as the secondary antibody after pIECs were infected with TGEV. The red colour indicates the distribution domain of EIF4A2. The nucleus was stained with DAPI (blue). A ZEISS LSM 800 with Airyscan was used to examine the triple-stained location. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

EIF4A2 gene silencing. pIECs were cultured in six-well culture plates and transfected with siRNAs (20 nM). The cell lysates were harvested for Western blotting at 48 h. The average densitometric intensity of EIF4A2 in the immunoblot analysis, with β-tubulin used as a control (A and B). The relativelevel of EIF4A2 mRNA expression was used to further evaluate the interference effect of siRNA2 by RT-qPCR at 36 h (C). *P < 0.05.

Fig. 6

Virus titration. EIF4A2-knockdown with siRNA2 cells and negative control knockdown cells were adsorbed with TGEV (MOI = 0.1) at 37 °C for 1 h. The culture supernatants of the cells and the virus-associated cells infected with TGEV were collected at different points. The viral titers are presented as the averages from three independent samples. **P < 0.01 and ***P < 0.001.

Fig. 7

Western blotting and RT-qPCR analysis following siRNA2 interference and viral infection. At 24 h after interference, the cells were incubated with TGEV Miller. After 48 h, the cells were harvested and the expression of the TGEV M protein was detected by Western blot (A and B). At 36 h, real-time RT-PCR was performed to detect the level of TGEV M mRNA expression (C). *P < 0.05.