The Molecular Mechanism Of VP1Protein Contributes To The Virulence Of Infectious Bursal Disease Virus

Infectious bursal disease (IBD) caused by infectious bursal disease virus (IBDV) is an acute and highlycontagious disease. IBD is one of the most economically significant diseases of chickens worldwide. Besidesthe classical virulent strains, the pathogenic serotype1viruses also include attenuated strains, antigenicvariant strains and very virulent IBDV (vvIBDV) strains. The emergence of vvIBDV causing severe andacute IBD with more than sixty percent mortality since1987brings new challenges for effective preventionand control of IBD. The exploration of the molecular determinants of vvIBDV virulence is an area of activeresearch. Some reports showed that segment A was not the sole factor that determines the virulence of IBDV.The segment A was not the major contributing factor in the emergence and expansion of vvIBDV. But thevvVP1played an important role in the initiation of vvIBDV expansion. This study further confirmed the VP1protein contributed to IBDV pathogenicity.The complete genome sequences of HuB-1, HeB10XS02and SD10LY01were cloned and the furtheranalysis of the nucleotide and amino acid sequences showed that VP1of IBDVs were divided into threedistinct subgroups, subgroup I (non-vvIBDV), subgroup II (China vvIBDV) and subgroup III (typicalvvIBDV). The VP1proteins of HuB-1and SD10LY01belong to subgroup III and HeB10XS02belongs tosubgroup II. The alignment of VP1deduced amino acid sequences showed that a total of17conservativeamino acid changes were found among I, II and III subgroups. Gt, HLJ0504and HuB-1with different VP1sfrom different subsets were screened by analysis of the genome sequences.Three recombinant cell-adapted viruses and three recombinant non-cell-adapted viruses were rescuedbased on the sequences of the attenuated IBDV Gt and vvIBDV HuB-1. The results showed that VP1ofIBDV contributed to its replication and virulence. The VP1proteins from subgroups II and III enhanced thevirulence of IBDV due to their positive effects on viral replication in vitro and in vivo. However, VP1fromsubgroup I reduced the virulence of IBDV due to its negative effects on viral replication in vitro and in vivo.According to the three-dimensional structure of VP1, VP1is divided into N-terminal, the center of theactive activity and the C-terminal functional domains. Three recombinant cell-adapted viruses were rescuedby replacing N-terminal, central active domain and C-terminal of the B-segment of HuB-1(HuBB) with thecorresponding region of the B-segment of Gt (GtB). Meanwhile, a recombinant non-cell-adapted virus wasrescued by replacing N-terminal of GtB with the corresponding region of HuBB. The virulence of the viruswas enhanced by vvVP1N-domain due to its negative effects on viral replication in vitro but positive effectson viral replication in vivo. The results of VP1N-terminal (1-167aa) amino acid sequences alignment showedthat, the145-147amino acids formed three unique "triplet" motifs, and different subgroup had different motif.Subgroup I was "NEG", subgroup II was "TEG" and subgroup III was "TDN". On this basis, two site-mutantcell-adapted viruses (rGtTEG, rGtTDN) and three site-mutant non-cell-adapted viruses (rHuBNDN,rHuBTEG, and rHuBNEG) were rescued. The results showed that the replacement of "NEG" with "TEG" or"TDN" enhanced the virulence of Gt due to its increased ability to replicate in vivo and reduced ability toreplicate in vitro. On the other hand, the replacement of "TDN" with "TEG" or "NEG" reduced viralvirulence of HuB-1due to its reduced ability to replicate in vivo and enhanced ability to replicate in vitro. The results of the detection of polymerase activity indicated that the polymerase activity was significantlyreduced by replacing "NEG" with "TEG" or "TDN". Otherwise, the polymerase activity was significantlyincreased by replacing "TDN" with "NDN","TEG" or "NEG". One of molecular mechanism of145-147amino acids attributing to viral virulence was the polymerase activity changed that further hanged the viralreplication.To further explore the molecular mechanism of VP1polymerase activity, the study was carried out fromvirus and host aspects. The results showed that VP3of IBDV was necessary for VP1polymerase activity.However, there was no positive relationship between the impact of VP3on the viral polymerase activity andthe different polymerase activity induced by VP1145-147amino acids mutation. On the other hand, the hostprotein eIF4AII was proved to interact with VP1by yeast co-transformation, CO-IP and confocal. Thepolymerase activity of VP1was inhibited by eIF4AII protein, inhibiting the viral replication of IBDV. Thehost protein eIF4AII inhibited the polymerase activity of wild-type VP1and its mutants by polymeraseactivity assay. However, there was no positive relationship between the inhibition of polymerase activityinduced by eIF4AII and the different polymerase activity induced by VP1145-147amino acids mutation.In summary, VP1is an important factor of IBDV virulence. The amino acids145-147located at VP1Nterminus is the molecular basis of the enhance virulence of vvIBDV and the molecular mechanism is themutation of145-147amino acids changed the viral polymerase activity and further changed the viralreplication. The viral proteins (VP3) and host factors (eIF4AII) are involved in the regulation of VP1polymerase activity. The eIF4AII interacts with VP1, which inhibits its viral polymerase activity and reducedthe replication of IBDV. The eIF4AII was shown to be a natural host defense factor against viral infection.