Dissecting The Role Of Porcine Reproductive And Respiratory Syndrome Virus Nucleocapsid Protein In Virus Replication

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is enveloped single strand positive sense RNA virus. Two genotypes were identified, type I (European type) and type II (North American type). The genome of PRRSV includes at least 9 open reading frames (ORFs), ORF1 encoded non-structural proteins (nsps) in charge of viral genome replication and suggenomic mRNA (sgmRNA) transcription. Structural proteins were expressed by ORF2 to ORF7. Nucleocapsid protein (N protein) expressed by ORF7 is a basic multifunctional protein with molecular weight of about 15kD. Generally, type I and type II PRRSV N proteins consist of 128 and 123 amino acids (aa), but the length of type I N protein exhibited size polymorphism. It is intrested to dissect the translational control mechanism of ORF7 because of the 319nt leader sequence before the start codon of ORF7. N was documented to play crucial roles in virion assembly, but the minimal requirements of N for virus functionality have not been charactered.This study was based on the platform of full-length PRRSV infectious cDNA clones. The first part focuses on the translational control of N protein. Three mutants were constructed based on the infectious cDNA clone pORF673 which had separated the repeated sequences between ORF6 and ORF7 and inserted three restriction enzyme sites. It proved that the translation of N protein prones to utilize the first AUG before N coding region, and the first 11 aa of N protein could be fully changed without affecting virus infectivity. Also,N terminal could tolerate 12 aa insertion without blocking virus viability.The second part focuses on the role of terminal and internal regions of N protein for virus replication. We targeted the terminal N protein and constructed a series of N- and C-terminal truncations. The transfection and virus rescue assay indicated that residues 5-13 at the N terminus and the last four residues at the C terminus were non-essential for virus viability. Unexpectedly, the recovered viruses were accompanied by various patterns of second-site mutations, including substitution, in-frame deletion, in- or out-of-frame insertion within N protein. Immunofluorescence assay and RT-PCR analysis for non-viable mutants indicated that the conserved 14-20 aa region may contained the essential elements for virus replication.To further study whether the N protein can tolerate internal deletion per se, or if the latter has to be accompanied by the terminal deletion, we introduced two internal deletions found in C?3/121-123 ppvs back into the wild-type pAPRRS, named as pN?39-42 and pN?48-52. Meanwhile, two double mutants containing the originally engineered C-terminal 3-aa deletion, together with second-site mutation Y61C and the residue 48-52 deletion were reconstructed. The resultant plasmids were designated as pC?3/N?48-52 and pC?3/Y61C, respectively. These mutants developed visible CPE after transfection, and the growth property exhibited indistinguishable growth kinetics from the wild type APRRS. It was worth noting that the recovered viruses N?48-52 and C?3/N?48-52 still provoked additional mutations, including substitution and aa insertion. The results indicated that the internal regions of N protein could tolerate deletion. In conclusion, we found that N protein of PRRSV had several properties: (1) its expression proned to utilize the first AUG followed with leader sequences; (2) the sequence specificity of the first 11 aa was non-essential for virus viability; (3) N-terminal residues 5 to13 were non-essential for type II PRRSV viability; (4) the deletion of 14-20 aa at N-terminal rendered virus nonviable and blocked genome replication and sgmRNA transcription; (5) the last 4 residues at C terminus could be deleted without affecting virus infectivity; (6) the internal region (aa39-52) could tolerate deletion. Almost all of the recovered viruses were accompanied by second-site mutations in N protein. This is belived to be the first report that second-site mutations arose in N protein of arterivirus. This study lays a foundation for further molecular dissection of structural and function relationship of N protein, and development of genetically tagged vaccines against PRRS.