Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. IMPORTANCE It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.

Prions are proteins that can fold into multiple conformations some of which are self-propagating. Such prion-forming proteins have been found in animal, plant, fungal and bacterial species, but have not yet been identified in viruses. Here we report that LEF-10, a baculovirus-encoded protein, behaves as a prion. Full-length LEF-10 or its candidate prion-forming domain (cPrD) can functionally replace the PrD of Sup35, a widely studied prion-forming protein from yeast, displaying a [PSI⁺]-like phenotype. Furthermore, we observe that high multiplicity of infection can induce the conversion of LEF-10 into an aggregated state in virus-infected cells, resulting in the inhibition of viral late gene expression. Our findings extend the knowledge of current prion proteins from cellular organisms to non-cellular life forms and provide evidence to support the hypothesis that prion-forming proteins are a widespread phenomenon in nature.

The RNA synthesis of porcine reproductive and respiratory syndrome virus (PRRSV), a positive-strand RNA virus, is compartmentalized in virus-induced double-membrane vesicles where viral proteins and some cellular proteins assemble into replication and transcription complexes (RTCs). The viral replicase proteins are the major components of the RTCs but the physical associations among these non-structural proteins (nsps) remain elusive. In this study, we investigated the potential interactions between PRRSV nsps by yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and pull-down assays. Our analyses revealed a complex network of interactions involving most of PRRSV nsps. Among them, nsp9 and nsp12 were identified as the hubs of the nsp interactome; transmembrane proteins nsp2 and nsp5 both interacted with nsp3, indicating that the three membrane-bound proteins might bind together to form the scaffold to support the association of RTCs with the intracellular membrane. The PRRSV nsp interactions identified in this study may provide valuable clues for future researches on the RTC formation and function.