The Role And Mechanism Of N-linked Glycosylation On Fibroblasts In The Process Of HCMV Infection

Objective: Protein glycosylation is one of the main post-translational modification methods of organisms,which plays an important role in various physiological and biochemical processes of life activities.N-linked glycosylation,as the main modification of glycosylation,has been widely recognized to be involved in cell-cell,cell-virus interactions.At the same time,the synthesis of viral N-linked glycoproteins also depends on the protein synthesis and post-translational modification system of host cells.Therefore,N-linked glycosylation is assoiated with all parts of the viral infection process.Human cytomegalovirus(hCMV)is a ubiquitous ? herpesvirus,with high seroprevalence worldwide.It causes significant morbidity and mortality in immunocompromized patients,such as transplant recipients,neonates,and patients with acquired immune deficiency syndrome(AIDS).HCMV infection has also been increasingly recognized for its association with vascular pathology and immunosenescence during aging.Despite considerable research efforts,there is no effective hCMV vaccine.Currently available antiviral therapy is suboptimal because of its significant side effects and emerging drug resistance.HCMV is known to have a wide range of cell tropism.It can infect many cell types,including fibroblasts,epithelial and endothelial cells and macrophages.Since higher viral titer could be obtained in fibroblasts and hCMV intracellular life cycle is also well studied,fibroblasts are good cell model for studying hCMV infection,replication and release.1.The fibroblast surface protein is highly N-glycosylated.The role and mechanism of these N-glycans in the early infection process of hCMV is still unclear.Stable virus-host binding and invasion processes are considered a critical step in the early stages of viral infection and may be a key event for the prevention of hCMV infection.Host cell glycosylation involves a variety of viral infections.The structure of the different glycans determines the type of interaction with virus and the susceptibility of the host cell to virus.N-and O-linked glycosylation is the two major glycosylation forms of cells.N-glycosylation is a widely glycosylated form of cell surface membrane proteins,which is thought to play an important role in binding and entering into host cells.The hCMV binding and entry process involves complex interaction with fibroblast surface receptors.Studies have shown that epidermal growth factor receptor(EGFR),platelet-derived growth factor-? receptor(PDGFR-?)and members of the integrin family act as hCMV receptors to mediate viral entry.At the same time,these protein receptors that mediate hCMV entry into fibroblasts are also highly modified by N-glycosylation.Therefore,by studying the N-glycosylation of fibroblast surface proteins and their effects on the early infection process of hCMV,it is possible to find a biological target for early prevention of hCMV infection.2.Meanwhile,fibroblasts as one of the main host cells of hCMV,it is necessary to study the effects of N-glycan processing system on the replication and release of hCMV.Tunicamycin(TM)is a nucleotide antibiotic produced by actinomycetes.The reaction between UDP-N-acetylglucosamine and polysterol phosphate in the first step of glycoprotein synthesis can be blocked,thereby inhibiting the synthesis of all N-linked glycoproteins.It can eventually cause endoplasmic reticulum stress response(ER stress)at higher concentrations.It has been demonstrated that TM can inhibit the proliferation of a virus having an envelope(glycoprotein).This mechanism of action is caused by the damage of the sugar chain processing system of the host cell,leading to the inhibition of the synthesis of the N-linked glycoprotein sugar chain on enveloped virus and can not produce the protein with the correct sugar chain structure to form viral proteins.Since hCMV is a typical enveloped virus,and its membrane glycoprotein synthesis depends on the glycoprotein synthesis system of the host cell.Therefore,theoretically,TM will affect the whole life cycle of hCMV,but currently there is no report on the effect of TM on hCMV replication and release.The other objective of this study was to investigate the effects of TM-induced damage to the host N-glycan synthesis system on hCMV replication and release.TM or a TM derivative with less toxic side effects could be considered as a potential drug candidate for the treatment of hCMV infection.Our study combines virology and glycobiology to form multidisciplinary research methods and system and ensures a certain feasibility and innovation.This study will be the first time to study the relationship between the N-linked glycosylation level and the entry of hCMV into host processes in human fibroblasts.It is also the first study of the replication and release of TM on the intervention of hCMV-infected fibroblasts.Through the above,we will investigate the effects and mechanisms of N-linked glycosylation and intracellular N-glycan synthesis on the host binding to entry,replication and release of hCMV after or before infection in fibroblasts.It will improve the understanding of pathogenesis of hCMV infection and provide a scientific strategy and potential biological targets for finding more effective prevention and treatment of hCMV.Experimental method: Two fibroblasts,MRC-5 cells and WI-38 cells were used as experimental subjects.Cell surface N-sugar inhibitors such as Tunicamycin(TM),glycosidase PNGase F,lectin and N-acetylglu cosaminyltransferase I(GnT I)gene silencing were used to remove or block N-sugar chains on the cell surface,respectively.The effects of N-linked glycosylation on the surface of fibroblasts on viral infection rate were verified by morphological observation,viral gene copy number and immediate early protein expression detection at different time of infection of hCMV.The antibody was labeled with hCMV tegument protein,and the attachment and entry of the virus before and after the change of N-glycosylation level were detected by quantitative PCR,cellular immunofluorescence and flow cytometry experiments.Then we used western blot to detect related signaling pathway expression of hCMV entry.Co-immunoprecipitation experiments were performed to verify the interactions of hCMV cellular surface receptor and ligand.The level of total protein N-linked glycosylation treated with TM was detected in hCMV-infected fibroblasts at different time.Morphological,QPCR and western blot were used to detect the morphological changes,and gene copy number and immediate early,early,and late protein expression of hCMV treated with TM in hCMV infected fibroblasts at different time.At the same time,the hCMV gene copy number and the virulence titer of the released virus particles in the extracellular culture medium of each group were detected to study the effect of TM on the assembly and release of hCMV virus particles.Results:(1)Pretreatment of fibroblasts with different de-N-linked glycosylation methods can significantly reduce the expression level of N-linked glycosylation in cells.(2)HCMV plaque formation,gene copy number and immediate early protein expression were significantly decreased after infection of hCMV in human fibroblasts.(3)The defect of N-linked glycosylation in human fibroblasts significantly inhibited hCMV entry,but did not affect the binding of virus to fibroblasts.(4)The decrease of N-glycans on the surface of hCMV receptor EGFR and integrin ?3 in human fibroblasts significantly inhibited the interaction with hCMV surface glycoproteins gB and gH,and the ?3-src signaling pathway was also significantly inhibited.(5)After infection of fibroblasts by hCMV,the addition of TM reduced the level of total N-linked glycosylation in the cells and significantly inhibited the expression of hCMV gene copy and the immediate early protein IE,early protein UL44 and late protein UL99.(6)Reduction of N-linked glycosylation of fibroblasts induced by TM treatment inhibited the release process of hCMV.Conclusion: Fibroblast N-glycans are essential for hCMV infection,and decreased levels of cellular N-glycans significantly reduce hCMV infection.N-glycans on the surface of fibroblasts do not affect hCMV attachment,but are required for hCMV entry.The hCMV receptor EGFR and ?3 surface N-glycan mediates the activation of itself and downstream entry signaling pathways,which mediate the entry of hCMV into fibroblasts by interacting with viral ligands via surface N-glycans.Tunicamycin may inhibit the replication and release of the virus by inhibiting host cell N-glycan synthesis,leading the abnormal expression of hCMV proteins.From the above experimental results,we found that fibroblast N-glycan is essential for hCMV infection,and cell surface N-glycan may be involved in the early infection of fibroblasts in hCMV.Subsequently,we found that N-glycans on the surface of fibroblasts did not affect hCMV binding to fibroblasts,but significantly inhibited the invasion of hCMV.The mechanism of this phenomenon may be that fibroblasts' hCMV receptors EGFR and ?3 interact with viral ligands through their surface N-glycans,thereby mediating activation of the receptor itself and downstream entry-related signaling pathways,thereby triggering cell membrane fusion and cell skeletal structure rearrangement,promotes the entry of hCMV into fibroblasts.Finally,we found that TM treated with hCMV-infected fibroblasts significantly inhibited viral replication and release.The mechanism may be caused by TM inhibiting the function of the host cell N-glycan synthesis system,leading to hCMV replication and abnormal synthesis of new viral proteins.In summary,cell surface N-glycans could be considered as an important biological target for the prevention of early infection of hCMV.Moreover,TM and its derivatives may become a novel drug for the treatment of hCMV infection.