Dissection of Amino-Terminal Functional Domains of Murine Coronavirus Nonstructural Protein 3

Abstract

Coronaviruses, the largest RNA viruses, have a complex program of RNA synthesis that entails genome replication and transcription of subgenomic mRNAs. RNA synthesis by the prototype coronavirus mouse hepatitis virus (MHV) is carried out by a replicase-transcriptase composed of 16 nonstructural protein (nsp) subunits. Among these, nsp3 is the largest and the first to be inserted into the endoplasmic reticulum. nsp3 comprises multiple structural domains, including two papain-like proteases (PLPs) and a highly conserved ADP-ribose-1؆-phosphatase (ADRP) macrodomain. We have previously shown that the ubiquitin-like domain at the amino terminus of nsp3 is essential and participates in a critical interaction with the viral nucleocapsid protein early in infection. In the current study, we exploited atypical expression schemes to uncouple PLP1 from the processing of nsp1 and nsp2 in order to investigate the requirements of nsp3 domains for viral RNA synthesis. In the first strategy, a mutant was created in which replicase polyprotein translation initiated with nsp3, thereby establishing that complete elimination of nsp1 and nsp2 does not abolish MHV viability. In the second strategy, a picornavirus autoprocessing element was used to separate a truncated nsp1 from nsp3. This provided a platform for further dissection of amino-terminal domains of nsp3. From this, we found that catalytic mutation of PLP1 or complete deletion of PLP1 and the adjacent ADRP domain was tolerated by the virus. These results showed that neither the PLP1 domain nor the ADRP domain of nsp3 provides integral activities essential for coronavirus genomic or subgenomic RNA synthesis. IMPORTANCEThe largest component of the coronavirus replicase-transcriptase complex, nsp3, contains multiple modules, many of which do not have clearly defined functions in genome replication or transcription. These domains may play direct roles in RNA synthesis, or they may have evolved for other purposes, such as to combat host innate immunity. We initiated a dissection of MHV nsp3 aimed at identifying those activities or structures in this huge molecule that are essential to replicase activity. We found that both PLP1 and ADRP could be entirely deleted, provided that the requirement for proteolytic processing by PLP1 was offset by an alternative mechanism. This demonstrated that neither PLP1 nor ADRP plays an essential role in coronavirus RNA synthesis.

Specific recombinant proteins of porcine epidemic diarrhea virus are immunogenic, revealing their potential use as diagnostic markers

“…Of the non-structural proteins, Ac displayed strong immune reactivity ( Figs. 2A and 4 B), suggesting that it may be released into circulation or is picked up by antigen-presenting cells (Hurst-Hess et al, 2015). Therefore, the study of anti-Ac antibodies may contribute to a better understanding of the detailed function of Nsp3.…”

“…Among them, Nsp3 comprises multiple structural domains, including a highly acidic domain at the amino terminus (Ac), and a highly conserved ADP-ribose-1-phosphatase (ADRP) macrodomain. The Ac domain of Nsp3 is essential for virion assembly and plays a critical role in interaction with the viral nucleocapsid during early infection, whereas the ADRP provides activities necessary for synthesis of genomic and subgenomic RNAs (Hurst-Hess et al, 2015). As pigs of all ages are susceptible to PEDV (Alvarez et al, 2015;Annamalai et al, 2015), there is an urgent need for the development of highly sensitive and specific diagnostic assays for use in the field (Diel et al, 2016).…”

Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles

“…While the function of Ub1 has only been investigated in MHV, this domain has been found to play an essential role in initiating viral RNA synthesis, where it interacts with the viral nucleoprotein (N; (Hurst et al, 2013;Hurst-Hess et al, 2015)). This was demonstrated in experiments that attempted to delete nsp3 domains, or substitute with the corresponding domains from other coronaviruses.…”

Dimerization of Coronavirus nsp9 with Diverse Modes Enhances Its Nucleic Acid Binding Affinity

“…CoVs encode the open reading frame (ORF) 1a and ORF 1ab polyproteins, which are then processed into 15 to 16 nonstructural proteins (nsp's) by two virus-encoded proteinases, the papain-like and 3C-like proteinases (10)(11)(12)(13)(14). All of these nsp's, except for nsp1 and nsp2, are considered essential for transcription, replication, and translation of the viral RNA (15)(16)(17). nsp9 with nsp7, nsp8, and nsp10 localizes within the replication complex and is likely a member of the replication complex (18).…”