Real-Time Tracing Of Pseudorabies Virus Infection In Vitro And Molecular Mechanism Underlying The Viral Reactivation From Latency Regulated By The UL54 Protein

Pseudorabies（PR）is an acute infectious disease caused by the Pseudorabies virus（PRV）.It can infect various mammals,including pigs,cattle,and sheep.Domestic pigs and wild boars are natural hosts of PRV.Infection with PRV can cause illness in pigs of all ages and can result in a mortality rate of up to 100%in piglets.PRV is a neurotropicα-herpesvirus that typically invades its host through the oral and nasal mucosa.The virus replicates and produces progeny viruses that can establish latent infections in the host’s peripheral nervous system.Latently infected PRV can persist within the host’s body,and factors such as stress can reactivate the latent virus,making it a source of infection.Currently,there are no effective antiviral drugs or vaccines that can completely eliminate PRV latent within the host’s body.Additionally,there is a phenomenon where wild and vaccine viruses coexist.Establishing simple and stable in vivo and in vitro latent infection models,screening for virus proteins or host molecules related to latent infection,and studying the infection process of PRV in neurogenic cells can provide a theoretical basis and technical platform for researching PRV latent infection mechanisms.To establish an easy-to-operate in vitro latent infection model of PRV,this study optimized experimental conditions and determined that infecting N2a cells with PRV（MOI=0.01）and incubating them at 37°C for 2 hours before transferring them to a higher temperature of 42°C can establish a stable latent infection of the virus.After returning to the normal incubation temperature（37°C）,the latent virus can be reactivated.To establish a stable in vivo latent infection model of PRV,we administered astragalus polysaccharides to mice through drinking water to enhance their immunity and established a stable latent infection model in mice by infecting them with 10³ TCID₅₀ of PRV through the eye.After treatment with the immunosuppressant dexamethasone,the PRV latent within the mice’s bodies can be reactivated,with a reactivation rate as high as 80%.Due to technical limitations,the lytic and latent infection processes of PRV in neurogenic cells are not fully understood.To address this,we used a novel ANCHOR DNA labeling system to label the PRV genome.This system is easy to operate,has good compatibility,and does not affect the virus’s biological characteristics.Using the ANCHOR DNA labeling system and gene fusion technology,this study constructed a recombinant PRV（r PRV-Anchor3-m Cherry）with dual fluorescence labeling of the viral genome and envelope.The replication efficiency and virulence of this recombinant virus are the same as those of the parental PRV.Using r PRV-Anchor3-m Cherry as a reporter virus and employing single-virus live-cell imaging and three-dimensional reconstruction technology,this study performed real-time visualization and quantitative analysis of PRV’s complete replication cycle for the first time.The study found that PRV can invade mouse neuroblastoma cells（N2a）through endocytosis and membrane fusion.The viral genome enters the cell nucleus 1-1.5 hours after infection,and the late viral protein g M is assembled onto progeny virus particles 8±0.25 hours after infection.The complete replication cycle of the virus is 12.5±0.25 hours.Additionally,a standard curve for obtaining virus titers in real-time through fluorescence measurement was established.It was determined that viral genome replication can trigger nuclear exhaustion,and precise localization and quantification of the PRV genome during latency were performed,confirming that the latent viral genome is randomly distributed within the cell nucleus.This study found that PRV’s UL54 protein plays a key role in the latent infection process.UL54 is an immediate-early protein homologous to the ICP27 protein of herpes simplex virus type 1（HSV-1）.ICP27 can sense environmental changes and regulate the virus’s latent infection and reactivation.Using an established in vitro temperature-controlled latent infection model of PRV,this study found that deletion of the early gene UL54 does not affect PRV latent infection but significantly reduces the efficiency of virus reactivation.Subsequently,six differentially expressed host molecules were identified using the PRV latent infection model.Among them,the host meiotic regulator protein（XMR）co-localizes with the PRV genome within the cell nucleus,and overexpression of XMR protein can significantly inhibit PRV replication.Further research found that UL54 protein inhibits XMR protein expression by inhibiting XMR m RNA elongation,thereby promoting PRV latent infection reactivation.In summary,this study aimed to analyze the latent infection mechanism of PRV and established models of PRV latent infection and reactivation in mouse neuroblastoma N2a cells and mice.Based on a novel ANCHOR DNA labeling system,a recombinant virus with dual fluorescence labeling of the genome and envelope was constructed.Using this recombinant virus,the replication cycle of PRV in N2a cells was traced.It was found that PRV can invade N2a cells through endocytosis and membrane fusion,viral genome replication can trigger nuclear exhaustion,and the viral genome during latency was localized and quantified.The UL54 gene of PRV was found to promote the reactivation efficiency of PRV.The host molecule XMR related to PRV latency can significantly inhibit PRV replication.UL54protein promotes PRV latent infection reactivation by inhibiting XMR protein expression through inhibition of XMR m RNA elongation.This study provides a reference for research on the molecular mechanism of PRV latent infection and offers new target molecules for developing vaccines and drugs to block PRV latent infection reactivation.