Identification Of The Key Golgi-retained Functional Domain Of Envelope Protein GD Encoded By Equine Herpes Virus Type 1

Equine herpesvirus type 1(EHV-1),as a class II animal epidemic in China and a legally reported infectious disease by the World Organization for Animal Health,infects horses and causes acute respiratory disease,neurologic and abortion disease,leading to a huge economic loss to the horse industry.Controversy erupted for the secondary envelopment of EHV-1 and other herpesvirus due to the complexity,which hinders the development of anti-herpesvirus drugs and effective vaccines.Therefore,studying the role of envelope glycoproteins on the envelopment is important to understand the mechanism of secondary envelopment.In this paper,we determined the key Golgi-retained functional domain of glycoprotein D encoded by EHV-1 to provide a theoretical basis for studying the function of gD on the secondary envelopment.The physicochemical properties,secondary structure,tertiary structure and post-translational modification of homologous proteins,EHV-1 gD(gDEHV-1)and HSV-1 gD(gDHSV-1),were analyzed using bioinformatics software.Using genetic engineering technology,a series of chimeric plasmids encoding gD mutant protein which is substituted with single functional region,double functional region or triple functional region were constructed templated with gDEHV-1 and gDHSV-1 gene.The plasmids verified by sequencing were transfected into BHK-21 cells and the target proteins was detected by Western Blot.The key of domain of gDEHV-1 Golgi-retained was identified through observing the subcellular localization of the 14 recombinant proteins using a laser confocal microscope.The bioinformatics results showed that both gDEHV-1 and gDHSV-1 were type I transmembrane proteins with molecular weights of 45 k Da and 43 k Da,respectively.Each protein has similar function domains,which are signal peptide(SP),extracellular domain(ECD),transmembrane domain(TM),and cytoplasmic domain(CT).The 14 chimeric plasmids were successfully constructed identified by PCR and sequencing.All the plasmids were expressed successfully in BHK-21 cells because of the target bands were detected near 100 k Da using Western blot.Subcellular localization showed that the majority of fusion proteins with TM-CT of gDEHV-1 resided in the Golgi apparatus,while the fusion proteins of gDEHV-1 replaced TM/CT/TM-CT was more likely to remain in the endoplasmic reticulum.In this experiment,14 chimeric plasmids were successfully constructed by genetic engineering technology and homologous functional region replacement,all of which can be normally expressed in BHK-21 cells.It was identified that TM-CT of gDEHV-1 is the key functional region of EHV-1 gD envelope protein Golgi-retained.These results provide an experimental basis for studying the Golgi-retained molecular mechanism of EHV-1 gD and are expected to provide a theoretical basis for investigating the secondary envelopment of herpesvirus.