A zinc finger-containing papain-like protease couples subgenomic mRNA synthesis to genome translation in a positive-stranded RNA virus

Abstract

The genome expression of positive-stranded RNA viruses starts with translation rather than transcription. For some viruses, the genome is the only viral mRNA and expression is regulated primarily at the translational level and by limited proteolysis of polyproteins. Other virus groups also generate subgenomic mRNAs later in the reproductive cycle. For nidoviruses, subgenomic mRNA synthesis (transcription) is discontinuous and yields a 5′ and 3′ coterminal nested set of mRNAs. Nidovirus transcription is not essential for genome replication, which relies on the autoprocessing products of two replicase polyproteins that are translated from the genome. We now show that the N-terminal replicase subunit, nonstructural protein 1 (nsp1), of the nidovirus equine arteritis virus is in fact dispensable for replication but crucial for transcription, thereby coupling replicase expression and subgenomic mRNA synthesis in an unprecedented manner. Nsp1 is composed of two papain-like protease domains and a predicted N-terminal zinc finger, which was implicated in transcription by site-directed mutagenesis. The structural integrity of nsp1 is essential, suggesting that the protease domains form a platform for the zinc finger to operate in transcription.

The Autocatalytic Release of a Putative RNA Virus Transcription Factor from Its Polyprotein Precursor Involves Two Paralogous Papain-like Proteases That Cleave the Same Peptide Bond

“…Although the function of the N-terminal region of pp1a/pp1ab is not known, both the transcription-negative phenotype of an alphavirus X domain mutant (31) and the conservation of a transcription factor-like zinc finger in coronavirus PLpros (32) indicated that p195/p210 might be involved in coronavirus RNA synthesis. This hypothesis is strongly supported by a recent report in which the equine arteritis virus nonstructural protein 1, which, most probably, is a distant homolog of the coronavirus PLpros, is shown to be a transcriptional factor that is indispensable for sg mRNA synthesis (33).…”

In Vitro Characterization of a Purified NS2/3 Protease Variant of Hepatitis C Virus

“…Accessory proteases fall predominantly within the papain family, are found mostly in the N-terminal region of positive-stranded RNA virus polyproteins, and are wide spread among positive-stranded RNA viruses. Although not directly involved in genome replication, accessory proteases appear to be indispensable for virus reproduction (20,32,33). Remarkably, functional HCV subgenomic RNAs replicate in the absence of the structural proteins and NS2 in cell culture and suggest that the NS2/3 protease activity is not essential for RNA replication (34).…”

Equine arteritis virus

“…These two precursor proteins are extensively processed after translation into at least 13 nsps (nsp1-12, including nsp7 a/b; Figs. 4 and 5, Table 1) by three viral proteases (nsp1, nsp2, and nsp4) (Barrette-Ng et al, 2002;den Boon et al, 1995;Snijder et al, 1995a;Snijder and Meulenberg, 1998;Snijder et al, 1994Snijder et al, , 1995bSnijder et al, , 2001Tijms et al, 2001;van Aken et al, 2006a,b,c;van den Born et al, 2007;van Dinten et al, 1996van Dinten et al, , 1999Wassenaar et al, 1997;Ziebuhr et al, 2000). The greatest variation in the EAV replicase gene occurs in the portion of ORF1a encoding the nsp2 protein, with considerable variation at amino acids 388-488 (Balasuriya et al, 2004b;Miszczak et al, 2012;Zhang et al, 2008a,b,c).…”

“…However, PCPa may possess RNA-binding and/or protein-binding activities (along with PCPb), serving as a platform for the ZF during transcription. Therefore, the structural integrity of nsp1 is essential since all three domains are required for trans-activation of sg RNA synthesis (Tijms et al, 2001). It has been reported that nsp1 is essential for sg RNA transcription but dispensable for genome replication (Tijms et al, 2007).…”

“…Snijder and colleagues did pioneering work to characterize the molecular biology of EAV and other nidoviruses using the pEAV030 infectious cDNA clone Snijder et al, 1995aSnijder et al, , 2005Snijder, 1998). Specifically, they used this infectious cDNA clone to characterize the mechanism of EAV replication along with processing of viral non-structural proteins (den Boon et al, 1995;Snijder, 1998Snijder, , 2001van Aken et al, 2006a,b;van Dinten et al, 1999), transcription and subgenomic mRNA synthesis (Tijms et al, 2001(Tijms et al, , 2007Tijms and Snijder, 2003;van den Born et al, 2005a;van Dinten et al, 2000;van Marle et al, 1999a,b), and the viral replication complex . This clone also apparently contributed to the publications of other groups Glaser et al, 1998).…”