Molecular characterization of duck enteritis virus UL41 protein

Abstract

BackgroundDuck enteritis virus (DEV) belongs to the subfamily Alphaherpesvirinae, and information on the DEV UL41 gene is limited.MethodsThe DEV UL41 gene was cloned into the pET32a(+) vector and expressed in a prokaryotic expression system. Antiserum was raised against a bacterially expressed UL41-His fusion protein for further experiments. Transcription was quantified and UL41 protein expression levels were determined in DEV-infected cells at different time points by RT-qPCR and western blotting, respectively. DEV virions were purified by sucrose gradient centrifugation and analyzed by mass spectrometry to identify protein content. We confirmed the DEV UL41 gene kinetic class using a pharmacological test. IFA was used to analyze the intracellular localization of pUL41.ResultsThe recombinant expression plasmid, pET-32a(+)-UL41, which highly expresses a 76.0 kDa fusion protein, was constructed and expressed in E. coli BL21 (DE3) after induction with 0.2 mM IPTG at 30 °C for 10 h, generating a specific mouse anti-UL41 protein polyclonal antibody. RT-qPCR and western blot analyses revealed that the UL41 transcript number peaked at 36 hpi, and peak protein expression occurred at 48 hpi. The pharmacological test showed that UL41 was a γ2 gene. Mass spectrometry analysis showed that pUL41 was a virion component. IFA results revealed that pUL41 was localized throughout DEV-infected cells but only localized to the cytoplasm of transfected cells. DEV pUL47 translocated pUL41 to the nuclei of DEF cells; this translocation was dependent on predicted pUL47 NLS signals (40–50 aa and 768–777 aa).ConclusionsDEV UL41 is a γ2 gene that encodes a virion structural protein, pUL41 localizes throughout DEV-infected cells but only localizes to the cytoplasm of transfected cells. pUL41 cannot autonomously localize to the nucleus, as this nuclear localization is dependent on predicted DEV pUL47 NLS signals (40–50 aa and 768–777 aa).

Duck enteritis virus pUL47, as a late structural protein localized in the nucleus, mainly depends on residues 40 to 50 and 768 to 777 and inhibits IFN-β signalling by interacting with STAT1

“…The expressed product was a UL47-his fusion protein (approximately 55 kDa) that was largely present in insoluble form but not in the vector control culture ( Figure 1A). Next, the fusion protein was purified by gel and electric elusion ( Figure 1B), detected with the rabbit anti-DEV antibody by western blotting [37] ( Figure 1C), and then immunized in rabbit with Freund's adjuvant to prepare a specific anti-UL47 polyclonal antibody. The pUL47 in the DEV-infected cells reacted strongly with this specific polyclonal antibody ( Figure 2B).…”

“…According to previous results [37], we further predicted the putative basic region of DEV pUL47 using software at https ://www.predi ctpro tein.org/. The corresponding sequences (residues 40 to 50 and 768 to 777 of pUL47) were fused to pEGFP-C2 and transfected into DEF cells.…”

“…The properties of some DEV genes have been preliminary characterized [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]. We previously reported that DEV UL47-encoded protein (pUL47) modulates the distribution of UL41-encoded protein (pUL41) [37]. Together with the findings of previous studies [38,39], we speculated that a close connection exists between pUL41 and pUL47, but the role of DEV pUL47 has not yet been defined.…”

The Role of VP16 in the Life Cycle of Alphaherpesviruses

“…For all herpesviruses, a complete virion consists of four parts: a core that contains a double-stranded DNA genome, a capsid, a tegument, and an envelope (Guiping et al, 2007;Boštíková et al, 2014). The alphaherpesvirinae subfamily includes herpes simplex virus type-1/2 (HSV-1/2), pseudorabies virus (PRV), duck enteritis virus (DEV) (Qi et al, 2008;Zhao et al, 2008;Chang et al, 2009;Guo et al, 2009;Jia et al, 2009), varicella-zoster virus (VZV), equine herpesviruses (EHV), bovine herpesvirus (BHV), canine herpesvirus (CHV), and Marek's disease virus (MDV) (Kobty, 2015;Smith, 2017;You et al, 2017;He et al, 2018;Lefkowitz et al, 2018).…”

Duck Enteritis Virus VP16 Antagonizes IFN-β-Mediated Antiviral Innate Immunity

“…The characteristics of some genes from DEV had been reported [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64]. However, the function of VP16 of duck enteritis virus encoded by the UL48 gene was hardly studied.…”