Structural Biology of the Arterivirus nsp11 Endoribonucleases

Abstract

Endoribonuclease (NendoU) is unique and conserved as a major genetic marker in nidoviruses that infect vertebrate hosts. Arterivirus nonstructural protein 11 (nsp11) was shown to have NendoU activity and play essential roles in the viral life cycle. Here, we report three crystal structures of porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) nsp11 mutants. The structures of arterivirus nsp11 contain two conserved compact domains: the N-terminal domain (NTD) and C-terminal domain (CTD). The structures of PRRSV and EAV endoribonucleases are similar and conserved in the arterivirus, but they are greatly different from that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoV), representing important human pathogens in the Nidovirales order. The catalytic center of NendoU activity is located in the CTD, where a positively charged groove is next to the key catalytic residues conserved in nidoviruses. Although the NTD is nearly identical, the catalytic region of the arterivirus nsp11 family proteins is remarkably flexible, and the oligomerization may be concentration dependent. In summary, our structures provide new insight into this key multifunctional NendoU family of proteins and lay a foundation for better understanding of the molecular mechanism and antiviral drug development.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus are two major members of the arterivirus family. PRRSV, a leading swine pathogen, causes reproductive failure in breeding stock and respiratory tract illness in young pigs. Due to the lack of a suitable vaccine or effective drug treatment and the quick spread of these viruses, infected animals either die quickly or must be culled. PRRSV costs the swine industry around $644 million annually in the United States and almost €1.5 billion in Europe every year. To find a way to combat these viruses, we focused on the essential viral nonstructural protein 11 (nsp11). nsp11 is associated with multiple functions, such as RNA processing and suppression of the infected host innate immunity system. The three structures solved in this study provide new insight into the molecular mechanisms of this crucial protein family and will benefit the development of new treatments against these deadly viruses.

Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2

“…The first two structures of Nsp15 were determined from Mouse Hepatitis Virus (MHV) (PDB id 2GTH, 2GTI (Xu et al, 2006)) and SARS coronavirus (PDB id 2H85, (Ricagno et al, 2006). Among Nsp11 endoribonucleases, there are two structures determined of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) (PDB ids 5EYI, 5DA1, (Zhang et al, 2017)), and two for EAV (Equine Arteritis Virus, PDB ids 5HC1, 5HBZ, (Zhang et al, 2017)). Among Nsp11 endoribonucleases, there are two structures determined of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) (PDB ids 5EYI, 5DA1, (Zhang et al, 2017)), and two for EAV (Equine Arteritis Virus, PDB ids 5HC1, 5HBZ, (Zhang et al, 2017)).…”

Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus

“…1C), suggesting that these proteins could form dimeric or multimeric complexes by interacting with themselves. However, we missed the previously reported self-interactions for nsp1␤ and nsp11 (40,55), which were mainly characterized by in vitro assays, suggesting that fusion domains likely have an adverse effect on the interaction. Overall, among the 29 identified interactions, 5 interaction pairs (nsp1␣-nsp1␣, nsp12-nsp12, nsp2-nsp1␣, nsp2-nsp1␤, and nsp7␣-nsp9) have been reported in PRRSV (44,45,56,57).…”

“…Identification of self-interacting nonstructural proteins. Oligomerization may be important for certain individual nsps to perform their functions in PRRSV infection, and such self-interactions have been identified for nsp1␣, nsp1␤, and nsp11 by gel filtration assay (40,55,56). Interestingly, our Y2H system missed nsp1␤ and nsp11, a failure that could be due to an adverse effect of partner fusion on the self-interaction.…”

“…Importantly, both nsp9 and nsp10 have been shown to be the key virulence determinants of the Chinese highly pathogenic PRRSV (HP-PRRSV) (38). PRRSV nsp11 is an endoribonuclease (NendoU) with the catalytic sites highly conserved in arteriviruses and coronaviruses, but its function remains poorly defined in the PRRSV life cycle (39)(40)(41). In contrast, nsp12 is an arterivirus-specific protein (42).…”

Structure and Function of PRRSV nsp11 Endoribonuclease

“…We solved the structure of PRRSV nsp11 K173A at 2.16 Å [7], the nsp11 overall structure divides into two domains: the N-terminal domain (residues 1-90, NTD) and the C-terminal domain (residues 107-222, CTD), which are connected by a long linker (residues 91-106) ( Figure 1A). The active center of NendoU locates in the CTD, which is made up of twostranded antiparallel β-sheets (βA-βB and βC-βE) and one antiparallel three-strand β-sheet (βF-βH).…”