Structural Analysis And Functional Study Of Porcine Reproductive And Respiratory Syndrome Virus Helicase

Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)is one of the most serious pathogens in the pig industry,causing huge economic losses to the global pig industry every year,without effective drugs.In 2006,the highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV)broke out in China,which virulence enhancement is related to the variation of RNA dependent RNA polymerase(RdRp,nsp9)and Helicase(Helicase,nsp10),but the specific molecular mechanism remains unclear.In addition,nsp9 and nsp10 are the most important components of the PRRSV replication transcription complex(RTC),and the molecular mechanism involved in replication is unknown.In this study,the first full-length structure of the PRRSV Helicase of the arteritis virus family was analyzed by crystallography,revealing a novel conformation of the 1B domain and showing the C-terminal domain for the first time.The biochemical experiments confirmed that the 1B domain has an important influence on the binding of Helicase to the nucleic acid substrate,and the C-terminal domain plays a crucial role of in ATPase activity.This study provides a good structural basis for drug design and explaining the molecular mechanism of nsp10 involved in viral replication.The specific work content is as follows:1.PRRSV nsp10 structural analysis.The PRRSV nsp10 soluble protein was purified in vitro by E.coli prokaryotic expression system.High resolution crystals were obtained by preliminary screening and optimization of crystals,and the three-dimensional crystal structure with resolution of 3.0 ? was analyzed by X-Ray diffraction.PRRSV nsp10 is a monomeric structure consisting of three domains: an N-terminal zinc-binding domain(ZBD),a non-characteristic 1B domain,and a C-terminal Helicase core domain.We found that the PRRSV nsp10 1B domain is different from other Helicases and compared it with the existing SF1 family Helicase.It is found that 1B is a very flexible domain,which is not a domain in which the Helicase must exist and its position in the sequence and structure also has a large change.We structurally revealed that the PRRSV nsp10 1B domain has different orientations because the 126 th leucine(L)and the 128 th threonine(T)on 1B forms a hydrogen bonding force with and the 19 th alanine(A)and20th cysteine(C)on ZBD,and the 129 th cysteine of 1A,respectively.In addition,thispaper demonstrates the C-terminal domain of the Arteritis Viridae Helicase is composed of three ?-sheets and two mutually perpendicular ?-helix for the first time.This regular structure suggests that the region may play a role in the process.2.Functional study of the PRRSV nsp10 1B domain and the C-terminal domain.In order to study the function of 1B domain and C-terminal domain,we constructed the nsp10 ?1B,nsp10 1-389 recombinant plasmid and purified the corresponding soluble protein.First,the binding of nsp10 ?1B protein to dsDNA and dsRNA was detected by EMSA method.The results showed that nsp10 ?1B reduced the binding ability to dsDNA by about 50% compared with wild type protein;ATPase activity is weakened but not completely lost,the results of Helicase activity assays showed that there was no significant difference between 1B deletion protein and wild type protein.Similarly,in order to study the function of nsp10 C-terminal domain,we examined the activity of ATPase and Helicase of nsp10 1-389 protein,and the results showed that the deletion of the C-terminal domain completely lost the activity of ATPase,but the activity of Helicase activity is not affected.This indicates that the C-terminal structure of PRRSV nsp10 is an important component of ATPase activity.