Structure And Function Of Porcine Reproductive And Respiratory Syndrome Virus Nonstructural Protein1β

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of theArteriviridae family of Nidovirales, is the etiological agent responsible for a disease inpigs called porcine reproductive and respiratory syndrome (PRRS). PRRS ischaracterized by late-term abortion, early farrowing, stillbirth, and the birth of weakpiglets, which is initially referred to as "blue-ear pig disease" by its clinical symptoms.Since its first report in1987, it has spread to the majority of swine-producing countriesaround the world and has caused enormous economic losses in the swine industry. APRRS epidemic occurred in China in late1995, quickly transmitted to most provincesand is considered as one of the major problems in the swine industry. Due to the highmutation rate of its genome, various strains possess great differences, which curbs thedevelopment of vaccines, and more effective medications are needed.As the second nonstructural protein coded in the genome of PRRSV, nsp1βreleases itself from the replicase polyprotein through self-cleavage by itsC-terminal papain-like cysteine protease (PCP) domain, which is named PCPβ.It is known that nsp1β is able to function as protease, and is of vital importanceto the replication of PRRSV and the pathogenesis of PRRS, yet the mechanismof such processes is lack of sufficient illustration.We report here the three-dimensional structure of PRRSV nsp1β, revealing thecommon known PCPβ domain and some other regions. N-terminal sequencing ofauto-cleaved nsp2fragment from an nsp1β-nsp2N fusion protein shows the exactcleavage site between nsp1β and nsp2. By analyzing the interaction between thePCPβ domain and the C-terminal extension (CTE) as a putative substrate, wecan explain the mechanism in the interaction between PCPβ and its substrate.We also discovered and explained the phenomenon that mature nsp1β lost itsproteolytic activity. The results of gel-filtration and cross-linking assay showsthat nsp1β should exist as a homodimer in solution, which suggests a cisself-processing mode of nsp1β by the crystal structure of the homodimer.Besides, we expect a possible nuclease activity of N-terminal domain (NTD) bycomparing protein structures in3D, and demonstrate our anticipation by in vitro nuclease assay with λ-DNA. Further examination on the nuclease activity ofnsp1β reveals its metal dependence on Mn2+and Mg2+and substrate selectivityinclined to dsDNA and ssRNA. The combination of the fact that nsp1βpossesses the nuclease activity on total RNA extracted from porcine liver andthe result of the sub-cellular localization assay of nsp1β hints some possibleinfluence on the host cell of PRRSV. In sum, these results shed light on thebiological function of nsp1β and offer a multi-target template for future drugdiscovery.