Feng et al. BMC Veterinary Research (2018) 14:255 https://doi.org/10.1186/s12917-018-1553-6 RESEARCHARTICLE Open Access Co-localization of and interaction between duck enteritis virus glycoprotein H and L Daishen Feng1,2†, Min Cui1,2†, Renyong Jia1,2,3* , Siyang Liu1,2, Mingshu Wang1,2,3, Dekang Zhu1,3, Shun Chen1,2,3, Mafeng Liu1,2,3, Xinxin Zhao1,2,3, Yin Wu1,2,3, Qiao Yang1,2,3, Zhongqiong Yin3 and Anchun Cheng1,2,3* Abstract Background: Duck Enteritis Virus (DEV), belonging to the α-herpesvirus subfamily, is a linear double-stranded DNA virus. Glycoprotein H and L (gH and gL), encoded by UL22 and UL1, are conserved in the family of herpesviruses. They play important roles as gH/gL dimers during viral entry into host cells through cell-cell fusion. The interaction between gH and gL has been confirmed in several human herpesviruses, such as Herpes Simplex Virus (HSV), Epstein-Barr virus (EBV) and Human Cytomegalovirus (HCMV). In this paper, we studied the interaction between DEV gH and gL. Results: Recombinant plasmids pEGFP-N-gH and pDsRED-N-gL were constructed successfully. Expressions of both DEV gH and gL were observed after incubation of COS-7 cells transfected with pEGFP-N-gH and pDsRED-N-gL plasmids after 12 h, respectively. Also, the co-localization of a proportion of the gH and gL was detected in the cytoplasm of COS-7 cells after co-transfection for 24 h. Then, pCMV-Flag-gL and pCMV-Myc-gH recombinant plasmids were constructed and co-transfected into COS-7 cells. It was showed that both gH and gL were tested with positive results through co-immunoprecipitation and Western-blotting. Conclusions: Our results demonstrated not only the co-localization of DEV gH and gL in COS-7 cells, but also the interaction between them. It will provide an insight for the further studies in terms of protein-protein interaction in DEV. Keywords: DEV, Glycoprotein H , Glycoprotein L, Interaction, Co-localization, Co-immunoprecipitation Background [5, 19, 32]. DEV, also known as Duck Plague Virus Duck Enteritis Virus (DEV) is a linear double-stranded (DPV) and Anatid Herpesvirus 1 (AnHV-1), mainly DNA virus, of the α-herpesvirus subfamily [34]. Herpes- causes acute and contagious infection to waterfowl. Until viruses, consisting of core, capsid, tegument and enve- now, the duck enteritis virus is one of the most severe lope, are a large family of the linear double-stranded diseases that seriously affect economy in the worldwide DNA group of viruses [27, 28]. It has three subfamilies duck industry [23, 39]. including α-, β- and γ- herpesvirus. The α-herpesvirus Glycoprotein H is highly conserved in the herpes- contains: Herpes Simplex Virus 1 (HSV-1), Herpes Sim- viruses. Heterodimer gH/gL, commonly formed plex Virus 2 (HSV-2), Varicella-zoster Virus (VZV), and through non-covalent linkages, is of great import- Pseudorabies Virus (PRV); β-herpesvirus: Human Cyto- ance to membrane fusion of the enveloped viruses megalovirus (HCMV), Human Herpesvirus 6 and 7 and host cells [6]. It has been studied that in the (HHV-6 and HHV-7); and γ-herpesvirus: Epstein-Barr process of HSV-1 and HCMV entry into host cells, virus (EBV) and Kaposi’s sarcoma-associated herpesvirus gB, gH and gL are three essential glycoproteins for (KSHV, also known as Human Herpesvirus 8 (HHV-8)) the cell-cell fusion [1, 2, 24, 26]. Firstly, glycopro- teins of herpesviruse bind with their specifically cel- * Correspondence: [email protected]; [email protected] lular receptors, in order to attaching to the † Daishen Feng and Min Cui contributed equally to this work. membrane of host cells [10]. Then, gB, as a fusogen, 1Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China is activated by gH/gL through interaction, therefore Full list of author information is available at the end of the article the fusogenic function of gB is promoted [23, 24, 30]. The © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Feng et al. BMC Veterinary Research (2018) 14:255 Page 2 of 10 function of connection between gB and gH/gL, also Methods known as “core fusion machinery,” is essential for herpes- Virus and DNA extraction viruses to initiate membrane fusion [15]. For example, The DEV CHv strain (GenBank No. JQ647509.1) was the first step of EBV entry into Epithelial cells is teth- precured from Avian Disease Research Center of Si- ering gH/gL to its cellular membrane receptors, in- chuan Agricultural University. Monolayer cultures of cluding integrin αγβ5, αγβ6andαγβ8, which will duck embryo fibroblasts (DEFs) were infected with DEV change the conformation of gH/gL delicately due to CHv strain at a multiplicity of infection (MOI) of 0.1, receptor-binding. Then, the heterodimer will interact which was incubated in Dulbecco’s modified Eagle with gB and trigger its fusogenic function [5, 10, 11]. medium (DMEM) containing 10% fetal bovine serum Therefore, gH/gL is more likely a regulator in the (FBS). The use of duck embryos was approved by the process of cellular membrane fusion. Animal Ethics Committee of Sichuan Agricultural Uni- Recently, the three-dimensional crystal structures and versity (approval No. XF2014–18). Then, the cells were interaction domains of gH/gL of HSV-2 and EBV gH/gL incubated at 37 °C until a cytopathic effect (CPE) ap- have been solved [8, 26], which helped immensely in un- peared in about 80% of the cells. DNA of DEV was ex- derstanding the structure and function of gH/gL. How- tracted from the infected cell lysis using DNA Extraction ever, the functional mechanisms of gH/gL are complicated Kit for Virus (TianGen Biotech Co., LTD.). and diverse in different herpesviruses. For instance, al- though the structural conformation of gH/gL is generally Primer design and PCR amplification homologous among HSV-2, EBV and HCMV, some differ- A bioinformatics analysis of DEV CHv genome sequence ences still exist, including divided domains, interdomain (GenBank No. JQ647509.1) was performed and a pair of packing angles and fragment antigen-binding regions of primers was designed to amplify the truncate UL1 gene the neutralizing antibody [8, 10]. When gH binds gL at (UL1t). The forward primer UL1t-1F and reverse primer the N-terminal domain (H1) of HSV-2, gH/gL therefore UL1t-1R were showed in Table 1 (the underlined se- forms a boot-like heterodimer with a ∼ 60° kink between quences was restriction enzyme HindIII site). domain H2 and H3 at the C terminus [8]. Similarly, com- Two pairs of primer were designed to amplify the UL1 prising a kink between domains III and IV, the structure and UL22 gene respectively, which encode the gL and of HCMV gH/gL has the semblable boot-like heterodimer gH of DEV respectively. The two pairs of primer for as HSV-2 [8–10, 26]. Furthermore, in most human her- amplifying UL1 gene are seen in Table 1 as UL1-1F/1R pesviruses, gL requires correct enfoldment to bind with and UL1-2F/2R respectively. The other two pairs of pri- gH. HSV-1 gL is more likely a scaffolding protein than a mer for amplifying UL22 gene are also shown in Table 1 chaperone to traffic gH and promotes its surface expres- as UL22-1F/1R and UL22-2F/2R respectively. The DEV sion, then gH anchors gL to the cell surface [12, 20, 33]. CHv DNA was used as template. The PCR was per- During membrane fusion, the fusogenic potential of formed with 10 μL mixture as reactions containing 5 μL HSV-1 gB is activated through interaction with gH/gL, be- PrimeSTAR (premix) DNA polymerase, 0.2 μL each of fore the alteration of gH/gL is initiated by gD [1, 4]. In primer at the concentration of around 5 nmol/OD, short, the main function of HSV gH/gL is activating gB 0.4 μL DNA template at the concentration of 280 ng/μL, through direct interaction during entry of host cells. and 4.2 μLH2O. Then, the PCR amplification was per- Although the structure and function of gH/gL in some formed in pre-denaturation at 98 °C for 2 min, denatur- human herpesviruses have been studied to some extent, ation at 98 °C for 10s, annealing at 55 °C for 30s, for example the interaction regions of gH and gL of extension at 72 °C for 30s, and final extension at 72 °C HSV-2 and EBV have been studied and published already for 10 min after 30 cycle repeats. [8, 26], there is insignificant research data in the study of DEV gH and gL until now. According to the existence of Plasmid construction gH/gL dimers in other herpesviruses, the hypothesis that Expression vectors pET-32a(+), pEGFP-C, pDsRED-N, DEV gH would interact with gL was proposed in this pCMV-Flag and pCMV-Myc were available in the la- paper. In this study, the two expression plasmids, boratory. After digested with both EcoRI and HindIII re- pEGFP-N-gH and pDsRED-N-gL, were co-transfected striction enzymes respectively, the truncate DEV UL1 into COS-7 cells to analyze the co-localization of DEV gH gene and vector pET-32a(+) were both linked to the and gL.