The Molecular Mechanism Of Nucleolin Inhibiting The Replication Of Peste Des Petits Ruminants Virus

Peste des Petits Ruminants(PPR)is an acute,highly contagious,and fatal disease caused by Peste des Petits Ruminants Virus(PPRV),which mainly infects cloven-hoofed small ruminants.The disease is characterized by pyrexia,stomatitis,pneumonia,conjunctivitis,mucopurulent ocular and nasal discharges,severe diarrhea,and high morbidity and mortality.PPRV belongs to the genus Morbillivirus in the family of Paramyxoviridae and its genome encodes six structural proteins and two nonstructural proteins.Although there have been advances in the molecular biology of PPRV,such as its structure and function,the molecular pathogenesis and immune mechanism of PPRV are still lack of relevant information.However,the interaction between pathogen and host is the best way to study its molecular pathogenic mechanism,and there are few studies in this field at present.In this paper,the interaction of virus and host protein was used as a starting point to study the role of NCL in the replication of PPRV and its mechanism.PPRV N protein is highly conserved and the most immunogenic protein,which plays an important role in virus replication and cell tropism.In addition,the PPRV N protein interacts with many host regulators.Therefore,in this study,we aim to screen host proteins interacting with PPRV N protein by immunoprecipitation and mass spectrometry.We successfully screened the host molecule nucleolin(NCL)that interacts with N protein,and then further confirmed the direct interaction between NCL and N protein through Co-IP and GST-pull down experiments.Confocal microscopy assays showed that NCL and PPRV N protein co-located in the nucleus.By constructing truncated eukaryotic expression plasmids of PPRV N protein and NCL and conducting Co-IP experiments,we successfully located the functional domains of interaction between N protein and NCL.The results showed that CTD domain of nucleolin mediated its interaction with N protein,while NTD domain of N protein mediated its interaction with NCL.Secondly,we successfully constructed an over-expressed NCL cell line using lentivirus packaging system,which laid a foundation for studying the role of NCL in the growth of PPRV.When NCL was overexpressed in Vero-SLAM cells,the expression of PPRV N protein decreased,the number of genomic copies of PPRV decreased and the viral titer decreased.When endogenous NCL was down-regulated by siRNA,the expression of PPRV N protein increased,the number of genomic copies of PPRV increased and the viral titer increased.Therefore,NCL plays a negative regulatory role in PPRV replication.In addition,we preliminarily found that NCL promotes the production of IFN-? by luciferase assay.In summary,this study is the first to discover the interaction between NCL and PPRV N protein.Moreover,NCL is a negative regulator of PPRV replication.Our results suggested that NCL can affect the pathogenesis of PPRV by regulating PPRV replication,which would be helpful for us to understand the mechanism of PPRV infection.