This research proposal was conceived as a three-year project, with this report encompassing Year 1. PRRSV is recognized as one of the major virus threats facing the swine industry in the USA and worldwide. Whilst the introduction of vaccines has been moderately successful in the control of the disease, new strains are constantly emerging. Live virus vaccines have been shown to be the only type of vaccine capable of establishing protective immunity against PRRSV. Indeed, currently the most efficient way to produce live virus vaccines is to create stable recombinant live virus vaccines with altered growth phenotypes, yet are still capable of generating a protective immune response. Identifying and characterizing unique and important stages in the virus life cycle can achieve this, by attenuating the efficiency of virus protein function through targeted mutagenesis coupled to reverse genetic strategies. The objective of this research project to industry is to exploit knowledge of the virus nucleocapsid (N) protein to subvert virus biology and to deliver growth attenuated recombinant viruses for use as live virus vaccines. Year 1 (this report) was focused on basic research of N protein biology and its role in binding to viral RNA. This provided detailed information for Year 2 (currently ongoing) to create and characterize growth attenuated recombinant viruses in cell culture based on mutated N proteins deficient in viral RNA binding. This leads into Year 3 (funding applied for) where the recombinant viruses will be characterized and tested in vivo focusing on vaccine suitability and the generation of protective immunity when challenged with wild-type virus.
Year 1 of the project focused on determining the features of N protein that were involved in binding of the protein to the viral RNA genome. This is a multi-partite process involving the N protein binding to itself through self-association (called oligomerization), post-translational modifications and specific amino acids in the protein. All of these modifications and sites were thoroughly investigated and mapped using both in silico, in vitro and in cell analysis. Such techniques included mass spectrometry to map post-translational modifications, circular dichroism (CD) to determine protein structure, sequential mutagenesis to map RNA binding amino acids and self-association motifs.
Thus in Year 1 of the project we have assembled a complete picture of how N protein is involved in binding viral RNA, which then allows us to target specific features and processes of the N protein for generating recombinant viruses for potential growth attenuated vaccine candidates.
