The Network of Interactions Among Porcine Reproductive and Respiratory Syndrome Virus Non-structural Proteins

Abstract

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.

Nucleotide-binding oligomerization domain-like receptor X1 restricts porcine reproductive and respiratory syndrome virus-2 replication by interacting with viral Nsp9

“…Noteworthy, multiple novel antiviral treatments, including Nsp9 specific Camel single-domain antibodies, and siRNA targeting Nsp9, as well as vaccine strategy of de-optimization of codon pair bias in Nsp9, significantly decreased PRRSV replication in vitro and in vivo Liu et al, 2015;Xie et al, 2014;Zheng et al, 2015;Zhu et al, 2018). Finally, Nsp9 was found to associate with at least 9 PRRSV encoded proteins, including Nsp1α,Nsp1β,Nsp3,Nsp7α,Nsp7β,Nsp8,Nsp11,Nsp12,and nucleocapsid (N) protein, validating the notion of this protein as a core component of the viral replication/transcription complex (RTC) Liu et al, 2016;Nan et al, 2018). Further exploring the interaction of Nsp9 with host cellular proteins and analyzing the biological significance are necessary for fully understanding the replication mechanisms and pathogenesis of PRRSV .…”

The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis

“…Nonetheless, arterivirus sgmRNA synthesis depends on the Nsp1 autoprotease [10,40,41]. PRRSV Nsp1α reportedly binds directly to Nsp9 [17], and EAV Nsp1 controls the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis [42]. We speculate that the component necessary for PRRSV gRNA synthesis differs from that required for sgmRNA synthesis, which may be the reason that gRNA is not influenced by Nsp12.…”

“…It has been proposed that Nsp2, Nsp3 and Nsp5 of arteriviruses participate in modulating membrane curvature and DMV formation [12,35]. PRRSV is closely related to EAV, and Nsp2, Nsp3, and Nsp5 of PRRSV may also have the same roles in DMV formation [17]. As the arterivirus RNA polymerase, Nsp9 has been recognized as a core component of RTCs, and PRRSV Nsp9 plays a central role in the RTC machinery and interacts with the N protein [36], Nsp7α [37], Nsp1α, Nsp1β, Nsp3, Nsp7β, Nsp8, Nsp11, and Nsp12 [17].…”

“…PRRSV is closely related to EAV, and Nsp2, Nsp3, and Nsp5 of PRRSV may also have the same roles in DMV formation [17]. As the arterivirus RNA polymerase, Nsp9 has been recognized as a core component of RTCs, and PRRSV Nsp9 plays a central role in the RTC machinery and interacts with the N protein [36], Nsp7α [37], Nsp1α, Nsp1β, Nsp3, Nsp7β, Nsp8, Nsp11, and Nsp12 [17]. Furthermore, RNA synthesis efficiency correlates closely with Nsp9 and consistently with PRRSV virulence [13,38,39].…”

“…However, all of these studies focused on the interaction of host proteins with Nsp12, but no information describes how Nsp12 participates in the replication phase of the PRRSV lifecycle. A recent study attempting to explore the interaction network involving most PRRSV Nsps demonstrated that Nsp12 may serve as a hub of the Nsp interactome along with Nsp9 [17]; this study also indicated that Nsp12 may be a major component of the replication and transcription complexes (RTCs) [17].…”