Packaging of porcine reproductive and respiratory syndrome virus replicon RNA by a stable cell line expressing its nucleocapsid protein

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, is one of the most common and economically important swine pathogens. Although both live-attenuated and killed-inactivated vaccines against the virus have been available for a decade, PRRSV is still a major problem in the swine industry worldwide. To explore the possibility of producing single-round infectious PRRSV replicon particles as a potential vaccine strategy, we have now generated two necessary components: 1) a stable cell line (BHK/Sinrepl9/PRRSV-N) that constitutively expresses the viral nucleocapsid (N) protein localized to the cytoplasm and the nucleolus and 2) a PRRSV replicon vector (pBAC/PRRSV/Replicon-AN) with a 177-nucleotide deletion, removing the 3'-half portion of ORF7 in the viral genome, from which the self-replicating propagation-defective replicon RNAs were synthesized in vitro by SP6 polymerase run-off transcription. Transfection of this replicon RNA into N protein-expressing BHK-21 cells led to the secretion of infectious particles that packaged the replicon RNA, albeit with a low production efficiency of 0.4 × 10(2) to 1.1 × 10(2) infectious units/ml; the produced particles had only single-round infectivity with no cell-to-cell spread. This trans-complementation system for PRRSV provides a useful platform for studies to define the packaging signals and motifs present within the viral genome and N protein, respectively, and to develop viral replicon-based antiviral vaccines that will stop the infection and spread of this pathogen.

Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PARP-1 as a cellular target whose interaction is critical for virus biology

“…This protein has important roles in the virus life cycle and may have critical interactions with the host cell ( Rowland et al, 1999; Sun et al, 2012 ). At a basic level N protein complexes with virus genomic RNA to form the ribonucleocapsid core and as such is an integral component of the enveloped virus particle ( Song et al, 2011 ). Because the N protein forms a close association with genomic RNA and other virus proteins such as those involved in replication and components of the viral envelope, the protein has been postulate to play an important role in virus RNA synthesis and virion assembly, perhaps in the modulation of these events ( Lee et al, 2006 ).…”

Research progress on the N protein of porcine reproductive and respiratory syndrome virus

“…This approach is considered a promising vaccination strategy to enhance the control of PRRS. Song et al ( 2011 ) explored the application of replicon particles in vaccine development. They synthesized a self-replicating, non-propagating replicon RNA in vitro and transfected it into cells expressing the N protein.…”

Porcine Reproductive and Respiratory Syndrome Virus Reverse Genetics and the Major Applications

“…Deletion of the gene encoding N protein completely abolishes viral infectivity. However, single-round infectious PRRSV replicon particles can be rescued by using a trans-complementation system with a recombinant cell line stably expressing N protein [98]. N protein is a serine phosphoprotein that exists as a homodimer [28].…”