Expression, Purification, Crystallization And Structural Analysis Of Glycoprotein D From Herpes Simplex Virus Type2

Herpes Virus is a common pathogenic agent which can infect many hosts in the world. Herpes simplex virus (HSV) is a member of the alphaherpesvirus subfamily, and the speed of the virus’applification in this subfamily is very fast. According to the difference in the serotype, Herpes simplex virus has two members: HSV-1and HSV-2. HSV-1infection in oral Epithelium cell and the eye results in herpes of mouth, herpetic keratitis and so on. However, HSV-2infects genital cell and causes genital herpes. After initial infection, both HSV-1and HSV-2remain latent in neurons.The process of HSV infection could be divided into three steps: attachment, receptor-binding and fusion. Glycoproteins of the enveloped virus play a key role in the viral entry process. The initial attachment is mediated via interaction of glycoprotein C(gC) and/or glycoprotein B(gB) with heparan sulfate（HS）. Later,gB bind to its receptors （PILRαor Non-muscle myosin ⅡA）and glycoprotein D（gD）bind to receptors（HVEM， nectin-1，nectin-2or3-O sulfated heparan sulfate）. In this step， conformational changes in gD that mobilize a fusion active multi-glycoprotein complex involving gB, gD, gH and gL. The complex initiates fusion of viral envelope with a cellular membrane.HSV-1and HSV-2infect different cells，and they prefer different receptors during the binding process. In order to research the receptor Receptor preference and usage，we need to learn about the detail of receptor-binding. Recently，only the structure of HSV-1gD was resoluted.The resolution of the crystal structure of a soluble form of gD is initially up to amino acid residue259, and later on up to residue306. Meanwhile, the structure of HSV-1gD and HVEM complex was resoluted.In this study, we expressed HSV-2strain333gD (residues1to285) by Baculovirus-Infected Insect Cells as a secreted soluble protein with His tag. The protein was then purified with Ni-NTA and molecular sieve chromatography. It was crystallized using the hanging-drop vapor-diffusion method at18oC in a condition consisting of0.1M Hepes pH7.2,5%(v/v) MPD and10%PEG10000. The crystals diffracted to1.8resolution and belonged to space group P21, with unit-cell parameters a=63.6, b=55.4, c=65.3, β=96.3°.