The Function Of P Gene Of Newcastle Disease Virus And Its Role In Pathogenicity

Newcastle disease(ND)is one of the most serious infectious diseases of birds,and has been a major cause of economic losses in poultry industry.Its causative agent, Newcastle disease virus(NDV),is the sole member of avian paramyxovirus-1 (APMV-1),belonging to genus Avulavirus,subfamily Paramyxovirinae,family Paramyxoviridae,order Mononegaviriales.NDV possesses a negative-sense, single-stranded continuous RNA genome,which consists of 15,186 nt,15,192 nt or 15,198 nt.The genome of NDV contains six genes in the order of 3'-NP-P-M-F-HN-L-5',encoding six viral proteins(nucleoprotein,phosphoprotein,matrix protein, fusion protein,haemagglutinin-neuraminidase and large protein,respectively).Like other members of the Paramyxovirinae,NDV edits its P gene by inserting one or two G residues at the conserved editing locus(UUUUUCCC)and transcribes three P-gene-derived mRNA species.The mRNAs encode the open reading frame(ORF)of P (unedited),the V ORF(with a+1 frameshift),and the W ORF(with a+2 frameshift).P protein associates with the soluble,monomeric form of N and prevents its illegitimate self-assembly onto cellular RNA.P protein also forms complexes with assembled form of N and L during transcription and replication.V protein,which is also transcribed from P gene,proved to be associated with viral Pathogenesis and functions as an alpha interferon antagonist.In 2000,NDV strain named ZJ1 was isolated and identified from goose flocks in Zhejiang province,and subsequently the complete genomic sequence of ZJ1 strain was determined.A transcription vector containing the full length cDNA of ZJ1 genome and three helper plasmids expressing NP,P or L protein respectively were constructed to establish the reverse genetics system for NDV ZJ1 strain.After cotransfecting the transcription vector together with three helper plasmids,highly virulent infectious NDV was successfully generated.This established reverse genetics system provides a powerful tool for the research of the P gene in this study.1.Full-length genome analysis of Newcastle diseases virus strains belonging to genotypeⅢandⅨreveals the common origin of recent epizootic viruses.Ten fragments from seven NDV strain were amplified and cloned into pGEM-T vector.Based on sequencing results,the entire genome sequences of five genotypeⅨstrains and two genotypeⅢstrains were determined.Alignment of the seven full-length sequences revealed the presence of the 6 nt insert in the 5'-noncoding region(NCR)of the NP gene of five genotypeⅨNDV isolates.In other words,the genome of genotype IX NDV was 15,192 nt in length,quite different from genotypeⅢstrains.The genome size of F48E8(F48E9)indicated that NDV strains with 15,192nt genome had emerged as early as 1940s,rather than 1960s when the infections of genotypesⅤandⅥstrains were found.Phylogenetic and genetic character analysis revealed that viruses belonging to genotypeⅨshared higher homology with early genotypesⅢandⅣstrains, although they possessed 6 nt insert in the NP gene,a derived characteristic of recent four genotypes.Based on epidemiological data and phylogenetic analysis,it is inferred that the insertion of 6 nt into the genome of a genotypeⅢstrain gave rise to genotypeⅨstrains,which could be the common origin of NDV strains belonging to genotypeⅤ-Ⅷ.2.Analysis of the domains of P gene-coded viral proteins and preparation of antiserum specific for C-terminal domain ofⅤproteinFragments of P gene from NDV strains belonging to genotypeⅡ,Ⅲ,Ⅵa,Ⅶd and classⅠwere amplified with 5 pairs of specific primers.The PCR products were cloned to pGEM-T vector and sequenced to determine the exact nucleotide sequence of P gene. Based on sequencing results,the amino acid sequences of P,Ⅴand W protein were predicted,and subsequently sent to internet for second structure prediction and modeling.It was predicted that the structure ofⅤprotein of simian virus 5(SV5),a member of Rubulavirus,was adequate for modelingⅤprotein or the N moiety part of P protein of NDV,due to the homology ofⅤproteins in amino acid and second structure. Structural investigations on the P gene-encoded proteins showed that anα-helix in the phosphoprotein N-terminal domain(PNT)from 20 aa to 29 aa was identified,which was conserved in all Paramyxoviruses,acting as a chaperone for newly synthesized N, and prevent it from bingding to non-viral RNA in the infected cells.The phosphoprotein C-terminal domain(PCT)was mapped for various protein-protein interactions required for viral transcription:the putative oligomerization domain(PMD)was in the region between aa 221 and aa 290;the putative C-terminal sub-domain ofⅩdomain was found beween aa 350 and aa 392,which was proved to be a N:RNA-binding domain of Paramyxovirus;the C-terminal domain(CTD)ofⅤprotein was found beween aa 132 and aa 239,partially overlapping with PMD.According to the results of second structure analysis,the CTD of P gene from ZJ1 was amplified and cloned into pGEX-6p-l for expression.Several BALB/c mice were immunized with the expression products for more than four times to produce anti-serum specific for CTD.The indirect fluorescence assay(IFA)confirmed that anti-CTD serum reacted with NDV-infected cells,rather than other normal cells.3.Rescued NDVs with P gene derived from diverse origins showed differences in biological characteristics,especially in pathogenicity. In order to construct a plasmid for P gene substitution,a fragment of ZJ1 cDNA from 3641 nt(BglⅡ)to 4748 nt(BstZ17Ⅰ)was amplied and ligated with BglⅡ-digested plasmid pNDV/Z J1,containing the full-length cDNA of NDV ZJ1.The newly constructed plasmid was identified and called pTX37.The fragments of P gene were amplified from three NDV strains as follows:lentogenic strain LaSota,belonging to genotypeⅡ;mesogenic strain JS/05/9/Go,belonging to genotypeⅢ;velogenic strain JS/05/5/Go,belonging to genotypeⅦ.The PCR products of three P gene were digested with Smal and Apal,and ligated into the digested pTX37 to replace the P gene of pTX37.Subsequently,the recombinant plasmid and pNDV/ZJl were digested with XbaI and BstZ17I,and the products of digestion were ligated together to construct recombinant full-length cDNA of ZJ1 with heterologous P gene.The transcription vectors containing recombinant full-length cDNA together with three helper plasmids expressing viral protein NP,P and L were cotransfected into BSR-T7/5 cells,expressing T7 RNA polymerase.Three recombinant NDV strains were successfully rescued and named RZJ-LP,RZJ-4P and RZJ-IP respectively.To compare the biological characteristics of rescued viruses and wild-type NDV strain ZJ1,the mean death time (MDT),intracerebral pathogenicity index(ICPI),intravenous pathogenicity index(IVPI) and viral growth kinetics test were carried out.The results showed that recombinant NDV strains with P from LaSota or JS/05/9/Go were less virulent than wild-type strain ZJ1.In Vero,CEF and GEF cells,RZJ-LP and RZJ-4P grew more slowly and produced 10-fold fewer infectious progeny compared to ZJ1.The IVPI of RZJ-LP was 1.51,much lower than 2.54 of ZJ1.The pathogenicity of the three recombinant viruses indicated that P gene played an important role in virulence.4.Rescued NDV ZJ1 strain with partial P gene replacement showed differences in biological characteristics.The N-terminal moiety and C-terminal moiety of P gene of LaSota was respectively subcloned into plasmid pTX37 to replace the counterpart of P gene of ZJ1.As described above,the recombinant plasmids were ligated into pNDV/ZJ1,and subsequently two recombinant ZJ1 strains with chimeric P gene were rescued,called RZJ-LaN and RZJ-LaC.Two point mutations were introduced after the RNA edition motif in the P gene of plasmid pTX37 to inhibit the expression of the C-terminal domain(CTD)of V protein,while the expression of phosphoprotein was not changed.The resultant plasmid was ligated into full-length cDNA of ZJ1 and subsequently two CTD-deficient NDVs, named RZJ-VS and RZJ-VS/GFP,were rescued.To compare the biological characteristics of rescued viruses and wild-type NDV strain ZJ1,pathogenicity tests of MDT,ICPI,IVPI and viral growth kinetics test were carried out.The IVPI of RZJ-LaN and RZJ-LaC was 2.26 and 2.46,a bit lower than that of ZJ1,indicating that all the domains rather than single domain of P gene contributed to the low-virulence of RZJ-LP. It is noted that RZJ-LP,RZJ-LaN and RZJ-LaC grew equally in Vero cells,a cell lineage lacking interferon system,while the growth of RZJ-LaN in CEF and GEF was faster than those of others and that of RZJ-LaC was the lowest.RZJ-LaN produced 100-fold fewer infectious progeny than RZJ-LaC in vitro,indicating that the antagonism of the CTD of ZJ1 was better than LaSota.Although the rescued strain RZJ-VS encoded a V protein without CTD,it grew well in cells and eggs,even the same as wild-type strain.The biological characteristics of RZJ-VS were contrary to the results of similar research on other strains.In indirect fluorescent assy(IFA),many fluorescent cells were observed in RZJ-VS/GFP or RZJ/GFP-infected cells 12 h post-infection,indicating that ZJ1 predominated in propagation,which could also be an immune evasion mechanism.In addition,the cytopathic effect(CPE)of RZJ-VS and RZJ-VS/GFP was obviously different from that of ZJ1,indicating that certain function of CTD might not be discovered.Conclusion:(1)The entire genome sequences of five genotypeⅨstrains and two genotypeⅢstrains were determined and analyzed.Alignment of the seven full-length sequences revealed that the genome size of genotypeⅨNDV isolates was 15,192 nt,quite different from genotypeⅢstrains.Based on epidemiological data and phylogenetic analysis,it is inferred that the insertion of 6 nt into the genome of a genotypeⅢstrain gave rise to genotypeⅨstrains,which could be the common origin of NDV strains belonging to genotypeⅤ-Ⅷ.(2)The amino acid sequences of P,V and W protein were predicted and sent to internet for second structure prediction and modeling.Finally,all the functional domains were roughly located in P gene.(3)By using reverse genetics technology,five recombinant ZJ1 strains were rescued, three of which contained heterologous P gene,the others containing chimeric P gene.The biological characteristics of recombinant strains showed that P gene play an important role in virulence.(4)By using reverse genetics technology,two recombinant ZJ1 strains encoding CTD-deficientⅤprotein were rescued.The biological characteristics of recombinant strains showed that CTD of ZJ1 was more powerful than that of LaSota.The CTD-lacking strains grew as well as parental strain,contrary to the results of similar research on other strains,indicating that ZJ1 predominated in propagation,which could also be an immune evasion mechanism.(5)The cytopathic effect(CPE)of RZJ-VS and RZJ-VS/GFP was obviously altered, indicating that certain function of CTD might not be discovered.