Cryo-EM Structure Of Pseudorabies Virus Capsid With Portal Vertex

Pseudorabies virus(PRV),belonging to the subfamily of alpha herpesvirus,can cause Pseudorabies(PR)in pigs.PR has the characteristics of wide spread and high mortality,causing huge economic losses to the pig industry.Pseudorabies virus can cross infect cattle,sheep,cats and other animals.Thera are also a small number of human infection cases,the main symptoms of infected patients are endophthalmitis and encephalitis.Although live attenuated vaccines for PR have been widely used,PRV has neuro-latent properties that may reactivate infection after genetic mutations,causing new epidemics.PRV has a complex structure mainly consisting of four parts:an outermost glycoprotein-rich capsule,an interlayer tegument protein,an icosahedral nucleocapsid,and inner genome.There are three types of capsids during PRV maturation:A-capsids are hollow;B-capsid contains scaffold protein.C-capsids contain the entire genome and have the potential to mature into infective virions.PRV is a model virus for the study of herpes virus and has great potential in nerve tracer and oncolytic viruses.It is of great significance to study the structure of PRV in depth for the prevention and control of virus and the modification and utilization of PR.In this study,the structure and internal interaction mechanism of PRV A-capsid and PRV C-capsid were studied by means of cryo-EM combined with structural biology analysis.First of all,high purity PRV virus samples were obtained in this study,and PRV A-capsid and C-capsid particle forms were observed under cryo-EM.Furthermore,the structures of 4.53A A-capsid and 4.43A C-capsid were obtained by single-particle cryo-EM 3D reconstruction.The EM density map showed that PRV genomes were arranged in concentric circles within the C-capsid.It was found that the structures of two kinds of PRV capsids were highly similar,but only C-capsid was covered with capsid associated tegument complex(CATC).Due to more than 120 nm in diameter,the conventional single-particle reconstruction may lead to inaccurate evaluation of local defocus values.In order to resolve this problem,the localized reconstruction method was adopted in this study to extract the local region signals of two PRV capsids within two-,three-and five-fold axes respectively,and the PRV capsid structures have been successfully improved to near-atomic resolutions(<4?).Then,based on the high-resolution structures of cryo-EM,the atomic models of two kinds of PRV capsids were built.PRV capsids are mainly composed of 955 major capsid proteins(MCP),900 small capsid proteins,960 triplex proteins and other subunit proteins.The MCP assembles into pentons and hexons,forming extensive inter-capsomer interaction networks.The SCP covering the top of the Hexon and the Triplex embedded between the Hexon and Penton further stabilize the entire capsid.At the same time,we observed suspected N-terminal anchoring structures beneath the Triplex,anchoring the genome to the entire capsid.These observations have enhanced our understanding of the capsid structure of PRV and even herpes virus.Finally,this study focuses on the structural analysis of PRV capsid specific portal vertices.The portal only exists in one of the twelve vertices of PRV capsid,and with a twelve-fold symmetrical structure.The symmetry mismatch occurs between the PRV capsid and the whole capsid.The signal of portal structure is averaged out by the traditional icosahedral construction method.In this study,by symmetry relaxation and localized reconstruction,we have determined the structures of the PRV capsid with an asymmetrically attached DNA-translocating portal.Finally,the portal structure with 6.8? resolution and the complete PRV C-type capsid structure with portal at 6.9?were obtained.The structure shows that there are obviously differences between portal vertices and ordinary penton vertices.The portal vertex consists of a twelve-fold symmetrical portal at the bottom and a five-fold symmetrical tentacle helice at the top.The tail DNA density in the middle of the portal vertex also demonstrated the role of portals in the packaging and releasing of genomes,suggesting the important role of portal structure in PRV capsid assembly.In summary,this study analyzed the high resolution cryo-EM structures of PRV A-capsid and C-capsid by means of localized reconstruction,illustrated the interaction mechanism between PRV capsid subunits at the molecular level,and for the first time,analyzed the portal structure of PRV capsid.These findings provide important references for more comprehensive understanding of PRV and its capsid assembly mechanism,and enrich the structural studies of herpes virus family.