Partial Biological And Immunological Characteristics Of PRV Ribonucleotide Reductase

Pseudorabies virus(PRV)has a wide variety of hosts,and swine is the only natural host of PRV.The infection of PRV in piglets is commonly lethal,thus bringing enormous economic losses to pig industry.During the replication of PRV,the interaction of virus-host and viral proteins plays pivotal roles in viral life cycle.Alphaherpesviral ribonucleotide reductase(RNR)is a heterotetramer composed of large(pUL39,RR1)and small(pUL40,RR2)subunits.This enzyme can catalyze the conversion of ribonucleotides to deoxynucleoside diphosphate(d NDP),which is further phosphorylated to deoxynucleoside triphosphate(d NTP).d NTP is a substrate for viral DNA synthesis in infected cells.The enzymatic activity of RNR depends on the interaction between RR1 and RR2.However,the molecular mechanism underlying alphaherpesviral RNR complex formation is still largely unknown.Co-immunoprecipitation(Co-IP)and confocal microscopy were employed to investigate pUL39-pUL40 interaction in this study.The results showed that the interaction between pUL39 and pUL40 was essential for the formation of PRV RNR.In order to determine the interaction domains in pUL39 and pUL40,the interaction regions in pUL39 and pUL40 were mapped using a series of truncated proteins.Consequently,the 90-210 aa in pUL39 were identified to be responsible for the interaction with pUL40.In turn,the 66-152,218-258 and 280-303 aa in pUL40 could interact with pUL39,respectively.Deletion of 90-210 aa in pUL39 completely abrogated the interaction with pUL40.Deletion of 66-152,218-258and 280-303 aa in pUL40 remarkably weakened the interaction with pUL39,whereas a weak interaction could still be observed.Compared to the RNR subunits of VZV,BHV1,EHV1 and DEV,amino acid sequence alignments showed that the interaction domains identified in PRV pUL39 and pUL40 were relatively non-conserved.However,they were relatively conserved among PRV,HSV-1 and HSV-2.Further,the CRISPR/Cas9 gene editing technology was successfully used to construct PRV UL39-deleted(?UL39)or UL40-deleted(?UL40)recombinant viruses.Through plaque assay,growth kinetic curve and experimental infections of mice,we found that the replication of both mutant strains in the cultured cell line were weakly reduced as compared to the wild-type(WT)virus.Their virulence in mice was weakened,and did not cause death of mice,thus indicating fine safety.The?UL39,but not?UL40,can induce a full protection immunity in mice against 10^4.0 TCID₅₀ WT challenge.Collectively,this study reveals the molecular mechanism underlying PRV RNR formation and its pathogenicity,and provides some molecular targets for inhibition of pUL39-pUL40 interaction to antagonize viral replication in PRV infected hosts and a novel strategy for vaccine development of PRV variant strains.