Dissection Of Structure And Function Of PRRSV Nsp10

Porcine Reproductive and Respiratory Syndrome (PRRS) is a severe infectious disease. It is characterized by mild to severe respiratory failure in sows and gilts, and respiratory problems in piglets. The causative agnt is identified as PRRS virus (PRRSV), which was first isolated almost simultaneously in European and North American; the strains are designated Lelystad and VR-2332. There are still lots of mysteries about PRRSV genome replication, transcription and protein translation. Particularlly after a nationwide outbreak of "Porcine High Fever Syndrome" in2006, the highly pathogenic (HP) PRRSV, as a major cause pathogen, has become hotspot. PRRSV Nsp10, which is comprised of a C-terminal superfamily1helicase domain and an N-terminal putative zinc-binding domain(ZBD), is improtant for both genomic and sg RNA synthesis. Based on our established PRRSV infectious clone of European and North American strain, we intended to dissect structure and function of PRRSV Nsp10, which establish a foundation for further dissection of structure and function of PRRSV genetic components and its pathogenesis. The detailed experimentations are discussed as follows.1. Dissection of structure and function of PRRSV Nsp10ZBD by RGSN-Terminal ZBD of PRRSV Nsp10is comprised of a set of comserved Cys and His residues, which may bind Zn2+to form Zinc finger. In order to dissect structure and function of PRRSV Nsp10ZBD, firstly we made a prediction of the Zinc finger of PRRSV Nsp10by SMART, and then a total of19mutants, all of them carrying specific point mutations, were tested in the ZBD of PRRSV full length infectious cDNA clone, Upon DNA transfection into Marc-145cells, four mutants (pMHZM5, pMHH28C, pMHS55P, pMHS55A) rescued viruses. The mutant viruses had genetic stability, and the growth curve of mutant viruses were phenotypically indistinguishable from the parental virus, but the plaque morphology of mutant viruses were smaller than that of the parental virus except vMHS55P. But for other15mutants, no viable virus was rescued without the expression of N-protein and sgmRNA7synthesis except pMHH43C(detected the expression of N-protein). The results indicate that conserved Cys and His residues are essential for viral genome replication and subgenome transcription, and a set of13Cys and His residues may be involved in the coordination of Zn2+, and His-28can be replaced by Cys; the region containing residue Ser-55may play the role of" hinge spacer", which connects the ZBD to the rest of Nsp10; the mutant pMHH43C was transcription competent but did not produce infectious progeny virus, indicating that this mutant may interfere with the packaging of virion. Moreover, these nonviable mutants could not be trans-complemented, suggesting that ZBD may play cis-acting role in PRRSV Nsp10, it is impossible to establish Nsp10function when this subunit is not produced as an integral part of the PRRSV replicase polyprotein. In general, our results also reveal that Nsp10ZBD is significant for viral genome replication, subgenome transcription and virion biogenesis.2. Dissection of structure and function of PRRSV Nsp10helicase by RGSPRRSV Nsp10helicase contains seven concerved motifs, which may be essential for the helicase function. A set of mutants, all of them carrying specific point mutations, were tested in helicase motif â…  and â…¡ of European PRRSV full length infectious cDNA clone, in order to dissect structure and function of PRRSV Nsp10helicase. Upon DNA transfection into Marc-145cells, two mutants (pMHY223F and pMHD225E) rescued viruses. The mutant viruses had genetic stability, the growth curve of the mutant viruses were phenotypically indistinguishable from the parental virus, but the plaque morphology of vMHD225E was smaller than that of vMHY223F and the parental virus. But for other mutants (pMHS153A, pMHK155R, pMHE226D), no viable virus was rescued without the expression of N-protein and sgmRNA7synthesis except pMHS153A(detected the expression of N-protein). The results indicate that conserved Lys-155and Glu-226are improtant for motif â…  and â…¡; mutations of conserved residuses of motif â…  and â…¡ proved to be more deleterious than mutations of nonconserved residuses; the mutant pMHS153A may interfere with the packaging of virion. Moreover, these nonviable mutants could not be trans-complemented, suggesting that Nsp10helicase may play cis-acting role in viral genome replication, subgenome transcription, and an as yet unidentified step of virion biogenesis.3. Construction of Chimeric Clones of two PRRSV genotype Nsp10Two PRRSV genotype Nsp10have similar ZBD and helicase with homology of53.9%in nucleotide, may have similar function. In order to know whether two genotype Nsp10with similar structure have similar function, we constucted a chimeric clone (pCHN10) that replaced North American PRRSV infectious clone (pAPRRS) Nsp10with that of European PRRSV infectious cDNA clone (pMSHE). Upon DNA transfection into Marc-145cells, the chimeric clone did not produce infectious progeny virus without sgmRNA7synthesis and the expression of N-protein and Nsp2. The results proved that European PRRSV Nsp10could not substitute for the role of North American PRRSV Nsp10, the two may differ in function. We speculate that it may be attributed to the low homology in nucleotide, European PRRSV Nsp10can not become an integral part of North American PRRSV replicase polyprotein. Futhermore, our results also suggest that PRRSV Nsp10plays an essential role in viral genome replication, subgenome transcription.