Molecular Mechanism Underlying Host Protein BAG3 Degradation Induced By PRV PUL56 And Its Role During Viral Lytic Infection

Pseudorabies disease is an acute infectious disease which is caused by pseudorabies virus(PRV)infection.PRV belongs to the Herpesviridae family,Alphaherpesviridae subfamily members.The infection of PRV in piglets leads to clinical symptoms from mild subclinical symptom to death.PRV can invade the host peripheral nervous system through epithelial cell infection.Then,the viruses could establish a long-term latent infection in the peripheral sensory ganglia.The latent viruses are reactivated by appropriate environment or stimuli,and then assembling into infectious virions.The function of PRV protein UL56(pUL56)was first reported in 2016.The study showed that the PRV UL56 gene was involved in viral transmission in nervous system.To investigate the function and host interaction of pUL56,this project first identified the molecular mechanism underlying pUL56 subcellular localization.Our data indicated pUL56 predominantly localized at the Golgi and trans-Golgi network(TGN)through its predicted C-terminal transmembrane helix in transfected cells.Further,we found the 174 proline(P),181leucine(L),185L and 191L were essential for maintaining perinuclear accumulation,suggesting an important role of leucine.Finally,the smallest pUL56 mutant 174P/A-177L/A-181L/A-185L/A-191L/A-194L/A-195I/A-196-197L/A-200L/A fully lost Golgi-TGN localization.Bcl2-associated athanogene 3(BAG3)plays a pivotal role in the lytic cycle of several viruses.However,the effects of BAG3 on PRV remain unknown.To investigate whether BAG3 could modulate PRV life cycle during a lytic infection,we first identified pUL56 as a novel BAG3 interactor by co-immunoprecipitation and co-localization analyses.The study confirmed that pUL56 interacted with BAG3 through four PPxY motifs.Further study showed that overexpression of pUL56 could significantly downregulate endogenous BAG3 and the overexpressed BAG3 in the co-transfected HEK293T cells.First,we tried to identify if PPxY played a role in the downregulation of BAG3 induced by pUL56.The results showed the PPxY motifs did not function in the BAG3 degradation induced by pUL56 using PPxY/AAxY mutants and PPxY containing domain truncations.Further study identified the pUL56 C-terminus was critical on the BAG3 degradation.Based on subcellular localization study,thirteen pUL56 C-terminal transmembrane domain mutants were employed and the 181 and 185L were identified to be the critical amino acids that contributed to BAG3 degradation.The pUL56 mutants 181L/A-185L/A no longer mediated BAG3 degradation.The occurrence of this degradation strongly depends on the Golgi localization of pUL56 and lysosomal pathway.Then,the ?UL56 PRV was constructed using CRISPR/Cas9 technique.The regulation of BAG3 by pUL56 during viral lytic infection in different cell lines was investigated using WT and?UL56 PRV.BAG3 was not observed to be degraded in either WT or ?UL56 PRV infected cells as compared to mock infected ones,whereas additional two adjacent BAG3 cleaved products were found in the infected cells in a species-specific manner.The overexpression and knockdown assays were employed to study the effects of BAG3 during PRV lytic infection.The results showed overexpression of BAG3 could significantly suppress the protein level of gB,and reduce viral titers in infected cells.In contrast,knockdown of BAG3 could significantly elevate the protein level of gB,and increase viral titers in infected cells.Thus,BAG3 plays a negative regulation role during a lytic infection of PRV in cells.Collectively,this study revealed a novel molecular mechanism on host protein degradation induced by pUL56,and clarified the molecular mechanism underlying subcellular localization of pUL56.Additionally,this study also clarified the regulation role of BAG3 during PRV lytic infection.These data would be a molecular basis for clarifying the molecular mechanism underlying the neurovirulence of pUL56 in PRV.