Published in:
Open Access 01-12-2018 | Research
Molecular characterization of duck enteritis virus UL41 protein
Published in: Virology Journal | Issue 1/2018
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Background
Duck enteritis virus (DEV) belongs to the subfamily Alphaherpesvirinae, and information on the DEV UL41 gene is limited.
Methods
The 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.
Results
The 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).
Conclusions
DEV 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).