Study On The Interaction Of Pseudorabies Virus Glycoprotein D With Nectin-1 Receptors

Pseudorabies virus(PRV)belongs to a herpesvirus subfamily in the Herpesoviridae family,as with human simples herpesviruses(HSV).PRV is a member of type 1 swine herpesviruses and widely prevalent in pig herd.Pseudorabies is one of the main diseases threatening the pig industry,the sow usually display the reproductive disorders such as miscarriage,fetal death and mummy foetus,boar fertility,breath with difficulty and the piglet's mortality even up to 100%.The entry process of viruses is an important target of antiviral drugs,so the key to cure the disease is preventing virus from invading the host cell.However,presently no inhibitors of pseudorabies virus entry are used clinically,owning to the limited knowledge of their complicated entry process.Thus,understanding the entry mechanism of pseudorabies virus is a great value for the development of small molecules to prevent herpesviruses entry into host cell.PRV entry to the host cell is a multi-event requiring cooperative interactions of viral glycoproteins with host surface receptors.There are at least four glycoproteins(gB,gD,gH,gL)of PRV involved in this process.As with HSV-1,gD is the receptor-binding protein of PRV.Thus far,six gD receptors have been identified.These include 3-O-sulfonated-heparan sulfate(3-O-S-HS);the herpes virus entry mediator A(HVEM)and three immunoglobulin superfamily members:nectin-1,nectin-2 and CD 155.Nectin-1 is a member of the immunoglobulin(Ig)-like(three Ig-like domains)cell adhesion molecules and is believed to form a homodimer or heterdimer with other nectin members to exert its functions.Although previous reports showing that nectin-1,but not HVEM or 3-O-S-HS,could mediate the PRV infection,the structural and functional features of PRV gD remain poorly characterized.These are interesting scientific issues awaiting answers in the herpesvirus field,because gD homologs of PRV and HSV only share very low(22%)amino acid sequence identities.As a step towards understanding the basis of receptor recognition by the virus,in this study,we first reconfirmed the nectin-1 mediated viral entry using the PRV vaccine strain Barth K61 in CHO-K1 cells.CHO-K1 cell lacks any of the known alphaherpesvirus receptors and is therefore resistant to PRV infection.Transient expression of HU-and SW-nectin-1 in CHO-K1,however,suffices the cells for PRV entry.In the presence of either the human or the swine receptors,PRV infection with significant cytopathic effects was observed;whereas HU-HVEM failed to promote the entry of the virus.To quantitatively compare the cell entry mediated by HU-and SW-nectin-1,we further set up a cell based fusion assay.With CHO-K1 cells,remarkable cell fusion could be observed when the nectin-1-expressing cells are mixed with cells expressing PRV gD along with the viral fusion executor of gB,gH and gL.On the whole,the cell fusion mediated by HU-and SW-nectin-1 is quantitatively equivalent,indicating similar capacities of gD engagement by the two receptors.In order to evaluate the binding affinity between PRV gD and Nectin-1 receptor,we expressed and purified gD and Nectin-1 proteins and carried out a gel filtration expression.The complex peak was observed after loading necctin-1 and PRV gD mixture and elution.To gain further insight into the PRV-gD/nectin-1 interaction,we set out to characterize their binding features using the real-time surface plasmon resonance(SPR)assays.As expected,the gD protein potently interacts with both receptors,exhibiting typical slow-on/slow-off binding kinetics.The equilibrium dissociation constants(Kd)were determined to be 16.1 nM with HU-nectin-1 and 18.4 nM with SW-nectin-1,respectively.In the current study,we only found that the affinity of short PRV gD(284aa)for nectin-1 is also dramatically higher than that of long gD(337aa,containing the whole ectodomain).We believe this phenomenon echoes what has been observed with HSV-gD PFD,indicating a role of the PRV-gD membrane-proximal loop in membrane fusion.In order to explore the structure characteristics and interactive model of gD binding Nectin-1,we determined the structure of gD from PRV Becker strain and it's complex with swine Nectin-1 IgV domain by X-ray crystallography.The overall PRV gD structure is expectedly composed of an IgV-like core and the long N-and C-terminal extensions,as observed in its HSV homologs.The nectin-1 binding mode of PRV gD resembles its HSV counterparts.All the three viral ligands utilize the terminal extensions to contact a side of the receptor IgV domain which form nectin-1 homodimer.A detailed superimposition between the current structure and the previously reported HSV gD/nectin-1 complex structures,however,revealed an obvious difference in their steric position(relative to the receptor)for the bound gD proteins.By the structure-based sequence alignment between PRV and HSV gDs,it is clear that the N-loop of the PRV protein is only about half size of that in HSV gD.We believe this dramatically shortened N-loop in the PRV ligand can not support HVEM-binding,representing the structural basis of its inertness in HVEM:recognition.To further confirm the binding features observed in our complex structure,the key interface residues in nectin-1,including N77,M85,180 and F129 were mutated.As expected,the F129A mutation almost completely abrogated the gD/nectin-1 binding and therefore the gD/nectin-1 dependent cell fusion.The results indicate that hydrophobic interaction has contributed greatly to gD/Nectin-1 binding.Therefore,the hydrophobic pockets of gD which interact with key residues of Nectin-1 might be used as potential targets for antiviral-drug design.In conclusion,in this study,we have revealed the interaction modes of PRV gD with Nectin-1 and identified the key amino acids of Nectin-1 involved in their interaction.All these findings together not only could contribute to the unraveling of the fusion mechanism of herpesviruses,but also would direct us to a proposal for rational design of inhibitors of pseudorabies virus.