Roles Of Endoplasmic Reticulum Molecular Chaperones Calnexin/Calreticulin In The Maturation Of Ebola Virus Glycoprotein

EBOV is the causative agent of Ebola haemorrhagic fever(EHF), a highly contagious disease of human and nonhuman primates with high case-fatality rates(up to 90%). EBOV is a member of the genus Filovirus in the Filoviridae family. It is an enveloped RNA virus that contains a single-stranded, negative-sense genome. EBOV glycoprotein(GP), a highly glycosylated protein, is the key molecule for viral entry and specific immune response.Endoplasmic reticulum molecular chaperone calnexin(CNX) and calreticulin(CRT) monitors the maturation of glycoprotein. To study the role of CNX,CRT in the maturation of EBOV GP, we constructed the CNX/CRT gene knockout 293 T cell lines using CRISPR/Cas9 technology. Ebola-GP(delta mucin,GP △ MLD)expressing recombinant plasmid was co-transfected with human immunodeficiency syndrome virus(HIV-1) based retroviral vector into human embryonic kidney(HEK) 293 T cells for the construction of the Ebola-GP pseudo typed lentivirus. We found that GP △ MLD protein had an less expression and lower package efficiency in the CNX KO and CRT KO cell lines especially CNX/CRT D.KO cells. The IP results showed that EBOV glycoprotein(GP △ MLD) carrying Flag tag can bind to CNX and CRT. Those results demonstrated that CNX,CRT may play an important role in the EBOV virus replication.To further study the mechanism of the CNX,CRT influence the GP △ MLD maturation, we constructed the glycosylation mutant GP △ MLD using site-specific mutation. It was found that site directed mutagenesis on the C terminal N-glycan side chains of GP protein showed significantly lower expression and infectivity of N563 and N613 missing simultaneously. Treatment with endo-Nacetylglucosaminidase H(Endo H), which removes only types of high-mannose structures and hybrid structures from glycoproteins in the endoplasmic reticulum, showed that the GP protein with mutation at N563 and N618 is mostly digested whereas little GP protein existed in the ER with mutation at other seven potential N-Glycan sites. This result demonstrated that GP with N563,N618 missing block the transport of GP from the endoplasmic reticulum to Golgi, and inhibit further processing and maturation. More interestingly, the interaction between CNX or CRT and GP△N257→N618 got stronger whereas a lower interaction with the GP removed C-linked four glycans. This result demonstrated that C-linked glycan is important for the structure and function of GP and CNX,CRT regulate protein quality control system by blocking unfolded GP protein transport.The GP containing mucin were also analysised to ensure the conclusion accuracy. The result demonstrated that there is an obvious less protein expression and virus packing in the CNX/CRT D.KO cells. Meanwhile, GP with N563,N618 glycans deletion has a lower protein processing proficiency and virus infectivity.In summary, our data demonstrated that CNX,CRT can interact with GP and CNX,CRT deletion can inhibited the expression and maturation of GP. N563,N618 glycans is essential for the GP maturation and virus packing. Furthermore, CNX,CRT monitors the intracellular steady by blocking the unfolded protein transport from ER to Golgi.