Establishment Of Reverse Genetics System For Goose Parvovirus And Its Application In The Mutation Of The Nuclear Localization Of Viral Capsid

Gosling plague is an infectious disease,named as goose parvovirus?GPV?,which can infect goslings showing viremia and intestinal embolism.The GPV infectious clones are important technical platforms for studying the pathogenic and replication mechanisms of GPV.The classical nuclear localization signal?NLS?is a special amino acid sequence consisting of arginine and lysine.The proteins containing the classical NLS sequence can be recognized by the karyopherins and targeted into nucleus through the nuclear pore complex?NPC?.The NLS is critical to the life cycle of parvovirus,but the GPV NLS sequence remains unknown.In this study,a GPV infectious clone was constructed based on the GPV RC16 strain to study the key amino acid within NLS in the localization process of viral capsid protein,which contribute to constrction of vaccine skeleton.In order to construct a GPV infectious clone,the complete sequence of GPV RC16 was amplified through polymerase chain reaction?PCR?,the PCR products were sequenced and assembly of subcloned fragments.A molecular phylogenetic tree was constructed based on the obtained complete sequence and it futher indicated that the GPV RC16 is GPV.A GPV infectious clone containing the full-length genome of GPV RC16 strain was constructed through ligation of the subcloned fragment by homologous recombination method,which was named as pIGPV RC16.The pIGPV RC16 was transfected into the goose embryo fibroblasts to rescue the virus.The date of indirect immunofluorescence,identification of genetic markers,and sequencing of PCR products indicated that the virus was successfully rescued.The growth curve of the second generation recombinant virus was determined and the rusults indicate that the second generation recombinant virus can proliferate in GEF.The GPV VP1 sequence was analyzed by PSORT II program to predict the putative NLS sequence.The predicted results showed that there was a lysine-rich basic region(BR,¹⁶⁰YPVVKKPKLTEE¹⁷¹)between the 160 and 171 amino acid sequences of GPV VP1,which may be a putative NLS.Based on this prediction,we constructed pEGFP-GPV NLS and transfected it into cells to directly observe the localization of GFP protein,which confirmed that the BR(¹⁶⁰YPVVKKPKLTEE¹⁷¹)can target small molecular protein?GFP,27 kDa?into the nucleus;meanwhile,the pEGFP-GPV NLS-?-Gal was constructed and transfected into cells to directly observe the localization of GFP protein,the SV40 strong nuclear localization signal?PKKKRKV?as a positive control,which confirmed that the BR(¹⁶⁰YPVVKKPKLTEE¹⁷¹)can't translocate large molecular weight protein??-Gal,100kDa?into the nucleus.In summary,the BR(¹⁶⁰YPVVKKPKLTEE¹⁷¹)has NLS activity,but it is not a strong nuclear localization signal.To further identify the key amio acid of this BR,we performed alanine mutation scanning on the 164 K,165 K,and 167 K amino acid positions in VP1 and the data indicated that the 164 K,165 K,and 167 K play a key role in import of GPV VP1 into the nucleus.In order to investigate the role of 164 K,165 K,and 167 K in the life cycle of GPV,we constructed the infectious clone with NLS mutant by overlapping PCR,which are named as pIGPV RC16K164A,pIGPV RC16K165A and pIGPV RC16K167A respectively,and transfected them into GEF to try to rescue virus.The results showed that the K164,K165 and K167 were vital for progeny virus proliferation.In this paper,the infectious clone of GPV RC16 strain was constructed firstly,and the recombinant virus was successfully rescued in GEF;the ¹⁶⁰YPVVKKPKLTEE¹⁷¹ was identified as GPV NLS,and the 164 K,165 K and 167 K were key amino acid sites of GPV NLS;the pIGPV RC16K164A,pIGPV RC16K165A and pIGPV RC16K167A were constructed based on pIGPV RC16 to investigate the role of K164,K165 and K167 in the GPV lifecycle.The dates indicated that the K164,K165 and K167 are required for progeny virus proliferation.This study laid the foundation for further construction of attenuated vaccine.