key: cord-0733893-s276f7s0
authors: Wang, Ruiyang; Yu, Ruisong; Chen, Bingqing; Si, Fusheng; Wang, Jian; Xie, Chunfang; Men, Chengfang; Dong, Shijuan; Li, Zhen
title: Identification of host cell proteins that interact with the M protein of porcine epidemic diarrhea virus
date: 2020-05-21
journal: Vet Microbiol
DOI: 10.1016/j.vetmic.2020.108729
sha: d5de7c2debb0c79f7ed3de6ec40a2cd052d2e381
doc_id: 733893
cord_uid: s276f7s0

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes severe diarrhea in pigs of all ages and a high fatality rate in neonates. The PEDV membrane protein (M) plays crucial roles in viral assembly, viral budding and host immune regulation, most likely by interacting with host cell proteins that have yet to be identified. In this study, co-immunoprecipitation (Co-IP) using an M-specific monoclonal antibody, coupled with LC-MS/MS, was employed to identify M protein-interacting proteins in PEDV-infected cells. Three viral proteins (S, E and ORF3) and 218 host cell proteins were identified as putative M-interacting partners. Bioinformatic analysis showed that the identified host cell proteins were related to 131 signal pathways and 10 biological processes. In addition, interaction between translation initiation factor 3(eIF3L) and M protein was validated by Co-IP. Down-regulation of eIF3L expression significantly increased viral production, which suggests that eIF3L could be a negative regulator in PEDV replication. This interactome study of the PEDV M protein will serve to clarify its function during viral replication.

Porcine epidemic diarrhea virus (PEDV) is the etiologic agent of porcine epidemic diarrhea (PED), a highly contagious disease with a high mortality rate in neonatal pigs. PED was first reported among feeder and fattening pigs in England and Belgium during the 1970s (Wood et al., 1977) . The disease was first recorded in China during the 1980s, and a more virulent form of the virus appeared in 2010 (Wang et al., 2013) . PEDV is an alphacoronavirus assigned to the family non-structural proteins (pp1a and pp1ab), and an accessory gene (ORF3) located between the S and E genes. The M protein, a component of the outer envelope of the viral particle, is categorized as a type III glycoprotein consisting of a short amino-terminal ectodomain, three successive transmembrane domains, and a long carboxy-terminal inner envelope domain (De Haan et al., 2000) . The protein J o u r n a l P r e -p r o o f participates in viral assembly through interaction with the S and N proteins, viral budding (De Haan et al., 2000; Klumperman et al.,1994; Arndt et al., 2010) , and may also mitigate the host innate immune responses. For instance, M protein is reported to induced cell cycle arrest at the S-phase via the cyclin A pathway (Xu et al., 2015) and may modulate the host innate immune responses by inhibiting the IFN-β and IRF3 promoter activities (Zhang et al., 2016; Song et al., 2012) . As obligate parasites, viruses rely on host: pathogen protein-protein interactions to modulate the ongoing biochemical activities of the host cells so that they benefit virus proliferation (Crua et al., 2017) . However, the interactome profile of PEDV M protein in host cells is still unclear although, in order to fulfill the aforementioned functions of M proteins, their interaction with a large number of host cell proteins would be expected to occur. In this study, co-immunoprecipitation (Co-IP) coupled with liquid chromatography-mass spectrometry (LC-MS/MS) was used to generate an interactome profile of PEDV M protein and to identify the viral and host cell protein interactions. M protein, the L subunit of eukaryotic translation initiation factor 3(eIF3L), and Rab11A, CDC42 and H2AFY expression plasmids containing tags were generated using the homologous recombinant method. All the primer sequences in this study will be made available upon request. M-HA gene was amplified by RT-PCR using PEDV DR13 RNA as template, and cloned into vector pCAGGS-HA with homologous recombinant reagent (one-step cloning kit, Vazyme, China) to form the recombinant plasmid pCAGGS-M-HA. RT-PCR using full-genome RNA of Vero cells as template was employed to amplify the eIF3L/Rab11A/CDC42/H2AFY-Flag genes, which were cloned into vector pCAGGS with homologous recombination reagent to obtain the recombinant plasmids pCAGGS-eIF3L/Rab11A/CDC42/H2AFY-Flag, respectively. All the plasmids were verified by sequencing.

PEDV M monoclonal antibody (mAb) was a gift from Shaobo Xiao, Huazhong Agricultural University. Anti-GAPDH mouse mAb was purchased from Abclonal Co. 

Vero cells seeded in 10-cm-diameter culture dishes were infected with PEDV DR13 att at an MOI of 0.1. After 36 hpi, the infected cells were lysed in RIPA lysis buffer containing 1mM phenylmethylsulfonyl fluoride (PMSF) and 1% protease inhibitor cocktail by incubation at 4 ℃ on a shaker for 30 min, followed by centrifugation at 14,000×g for 10 min. Supernatants were precipitated with anti-M protein mAb and incubated with gentle rocking overnight at 4 ℃. Protein A/G beads washed with cell lysate were added to supernatant fractions and incubated with gentle rocking for 12 h at 4 ℃. The beads were washed four times with cold cell lysate and boiled with 1×SDS loading buffer for 10 min, followed by SDS-PAGE and silver staining. 

Hela cells, seeded in 10 cm diameter culture dishes, were transfected with pCAGGS-M-HA and cellular target gene expression plasmids. Transfected cells were harvested 36 h post-transfection (hpt), and lysis buffer containing 1 mM protease inhibitor was added. After incubation for 30 min on ice, the lysed cell solutions were centrifuged at 14000×g for 10 min and the lysate supernatants, containing 1-2 mg total protein, were incubated overnight at 4 ℃ with anti-Flag mouse mAb. Protein A/G beads washed with cell lysate were added to the supernatants and incubated with gentle rocking for 12 h at 4 ℃. The beads were washed four times with cold cell lysate J o u r n a l P r e -p r o o f and boiled with 1×SDS loading buffer for 10 min, followed by SDS-PAGE.

Whole cell extracts and immunoprecipitated (IP) samples were separated by 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 10% non-fat milk in Tris-buffered saline-Tween (TBST), the membranes were incubated with the specified primary antibodies at room temperature for 1 h, washed with TBST, and then treated with HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG.

Specific protein bands were visualized by enhanced chemiluminescence using ECL plus western blot detection reagents (GE Healthcare, Chalfont St Giles, UK) according to the manufacturer's instructions.

Vero cells were transfected with plasmids as specified using lipofectamine 2000 according to the manufacturer's instructions. After fixing with 5% paraformaldehyde 24 hpt, cells were permeabilized with Triton X-100, followed by incubation with anti-HA or anti-Flag antibodies and then with HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibodies. Cells were stained with DAPI and observed with a Zeiss Scope A1 microscope (Zeiss Microsytems, Germany). s. Each reaction was performed in triplicate using GAPDH as the internal control, and the qRT-PCR data was analyzed using the 2 -△△ CT method.

Statistical analyses (one-way ANOVA) were performed using SPSS software (SPSS 21.0 for Windows). Data are expressed as mean values ± standard error (SEM).

The t-test was employed to determine the statistical significance (P<0.05) of the differences between the means. Data relating to viral RNA copies and virus titer levels were converted to log10 to maintain a normal distribution.

Vero cells were infected with PEDV DR13 att and M protein synthesis determined by Western blot analysis with anti-M protein mAb between 24 and 60 hpi using GAPDH as the internal reference. M protein synthesis increased during PEDV infection and reached a peak between 36 and 48 hpi (Fig.1A) . Samples were collected at 36 hpi for interaction analyses.

Vero cells were infected with PEDV DR13 att at an MOI of 0.1, and cell samples were harvested after 36 hpi and immune-precipitated with anti-M protein mAb. The immune-precipitated proteins were separated by 10% SDS-PAGE and visualized by silver staining (Fig. 1B) and Western blot (Fig. 1C) . Immunoprecipitated samples of PEDV-infected cells and mock-infected cells were checked for protein composition using SDS-PAGE electrophoresis and Coomassie brilliant blue staining, and these two lanes of the gel were subjected to MS analysis. Comparisons of the LC-MS/MS data to the chlorocebusuniprot database identified a total of 670 host cell proteins, among which 452 could also be found in the mock-infected cell samples. Therefore, the remaining 218 host cell proteins were designated putative M protein interacting host cell proteins, of which 62 shared a high confidence level (Unique Peptide≥2) ( Table   2 ). A search of the PEDV proteins database also identified PEDV S, ORF3 and E proteins among the MS data pool (Table 3) Functions) delineated at the Gene Ontology (GO) website ( Fig. 2A) . Among the proteins clustered in Biological Processes, 34 (15.6%) were involved in organonitrogen compound biosynthetic process, 27 (12.4%) in peptide metabolic processes, 28 (12.8%) in cellular amide metabolic processes, 27 (12.4%) in amide biosynthetic processes, 26 (11.9%) in translation, and 17 (7.8%) in protein transport.

The most enriched subclasses in cellular components were macromolecular complex (26.1%), cell (56.0%) and cell part (93.0%). Enrichments based on molecular function were mainly in small molecule binding, structural constituent of ribosome and nucleoside phosphate binding (Fig.2B) . A KEGG database search assigned the putative M protein-interacting host cell proteins to 11 pathways including translation (9 proteins), signal transduction (7 proteins) and transportation and catabolism (8 proteins) (Fig. 2C) . and Rab11A but not in the Co-IP sample of H2AFY (Fig. 3B ) further confirming that M protein was able to interact with CDC42, eIF3L and Rab11A proteins. In addition, IFA analysis indicated that CDC42, eIF3L and Rab11A all co-localized with M protein (Fig. 3C ).

The knockdown efficiencies of CDC42, eIF3L and Rab11A siRNAs proteins on PEDV replication in Vero cells were validated by real time RT-PCR (Fig. 4A ) and western blot (Fig. 4B) . Viral titers in cell suspensions with eIF3L knockdown were significantly higher compared with negative controls (Fig. 4C) , demonstrating that knockdown of eIF3L increased PEDV multiplication. However, there were no significant differences between the viral titers recorded in the CDC42 and Rab11A knockdown groups and negative controls. previously shown to be highly susceptible and permissive to PEDV infection (Hofmann and Wyler., 1988) , to investigate the M protein interactome. Co-IP and LC-MS/MS identified three viral proteins (Spike-S, Envelope-E and ORF3) and 218 host cell proteins that appeared to interact with M protein in PEDV DR13 att -infected Vero cells. Coronavirus (CoV) studies have shown that the M protein interacts with E protein and S proteins during the assembly of the CoV envelope (Nguyen and Hogue., 1997; Opstelten et al.,1995) . In the present study, both the PEDV S and E proteins were immunoprecipitated together with the M protein, indicating an interaction with the latter. Our data also showed that anti-M mAb immunoprecipitated truncated ORF3 protein in PEDV DR13 att -infected cells, which suggested an interaction between the ORF3 and M proteins. Accessory proteins from other coronaviruses such as SARS-CoV and hCoV-NL63 have also been shown to interact with M, S and E proteins (Tan et al., 2004; Muller et al., 2010) . hCoV-NL63 ORF3 is a structural viral protein incorporated into viral particles (Muller et al., 2010) while PEDV ORF3 is involved in many host cell processes and in viral replication (Si et al., 2020; Ye et al., 2015; Kaewborisuth et al., 2018; Wongthida et al., 2017) . The likelihood of J o u r n a l P r e -p r o o f interaction between PEDV M and ORF3 proteins suggests that the latter cooperates with M to take part in the process of PEDV assembly and budding.

It was notable that 6 Rab family members (Rab2A, Rab5C, Rab6B, Rab7A, Rab9 and Rab11A) were among the 218 host cell proteins identified. Rab proteins are a subfamily of the Ras superfamily of small GTPases that are known to regulate specific intracellular membrane trafficking pathways (Spearman 2018) . Among the six subfamily members, Rab11A has been studied most extensively in terms of its role in viral budding and release. Rab11 is reported to be essential for viral budding and release in RSV and HIV-1 (Brock et al., 2003; Varthakavi et al., 2006) . Rab11A also played a role in influenza virus morphogenesis and budding, and depletion of Rab11 decreased viral titers by up to 100-fold (Bruce et al., 2010) . Furthermore, Rab11A promoted porcine reproductive and respiratory syndrome virus (PRRSV) replication in a manner that correlated with autophagy (Wang et al., 2017) . Rab11A may serve a similar function in the proliferation of PEDV, PRRSV and influenza viruses. However, our siRNA studies failed to reveal any correlation between Rab11A expression levels and PEDV production although the knockdown of the Rab11A gene may not have been sufficiently strong enough to interfere with the Rab11A protein-M protein interaction. Alternatively, other routes may exist that replace the function of Rab11A, and future studies will further investigate the role of Rab11A in PEDV infection.

In the present study, we also confirmed interaction between the M protein and translation factor eIF3L. A reduction in the expression level of eIF3L significantly increased PEDV production compared to negative controls, indicating that the factor acted as a negative regulator in viral replication. eIF3L is one of the subunits of the eukaryotic translation initiation factor eIF3 (Morris- Desbois et al., 2001) and has been shown to inhibited yellow fever virus (YFV) replication by binding to the viral NS5 protein (Morais et al., 2013) . Other eIFs, such as eIF3F, play an inhibiting role in HIV-1 replication by interfering with the 3' end processing of viral mRNAs (Valente et al., 2009 ).

CDC42, another host cell protein identified as interacting with M protein, belongs to the Rho family, an important subgroup of small RAS-like GTPases (Ha et al., 2018) . Studies have shown that many RNA viruses such as respiratory syncytial virus (RSV) (San-Juan-Vergara et al., 2012), rotavirus (RV) (Diaz-Salinas et al., 2014) and Ebola virus (EBOV) (Quinn et al., 2009 ) depend on CDC42 signaling pathway to infect target cells. However, in this study, interference with CDC42 expression did not influence the proliferation of PEDV, and the significance of the interaction between M protein and CDC42 needs to be further explored.

In conclusion, a total of 218 host cell proteins that potentially interact with 

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