Bovine Coronavirus Nonstructural Protein 1 (p28) Is an RNA Binding Protein That Binds Terminal Genomic cis -Replication Elements

Abstract

Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-crosslinking analyses that cellular proteins of ϳ60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5 untranslated region (UTR)-and one 3 UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5 UTR with ϳ2.5 M affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.Coronaviruses (CoVs) (59) cause primarily respiratory and gastroenteric diseases in birds and mammals (35,71). In humans, they most commonly cause mild upper respiratory disease, but the recently discovered human CoVs (HCoVs), HCoV-NL63 (65), HCoV-HKU1 (73), and severe acute respiratory syndrome (SARS)-CoV (40) cause serious diseases in the upper and lower respiratory tracts. The SARS-CoV causes pneumonia with an accompanying high (ϳ10%) mortality rate (69). The ϳ30-kb positive-strand CoV genome, the largest known among RNA viruses, is 5Ј capped and 3Ј polyadenylated and replicates in the cytoplasm (41). As with other characterized cytoplasmically replicating positive-strand RNA viruses (3), translation of the CoV genome is an early step in replication, and terminally located cis-acting RNA signals regulate translation and direct genome replication (41). How these happen mechanistically in CoVs is only beginning to be understood.In the highly studied group 2 mouse hepatitis coronavirus model (MHV A59 strain) and its close relative the bovine CoV (BCoV Mebus strain), five higher-order cis-replication signals have been identified in the 5Ј and 3Ј untranslated regions (UTRs). These include two in the 5Ј UTR required for BCoV defective interfering (DI) RNA replication (Fig. 1A) described as stem-loop III (SLIII) (50) and SLIV (51). Recently, the SLI regi...

Coronavirus cis-Acting RNA Elements

“…Furthermore, using MHV/BCoV chimera, a region downstream of SL5 was revealed to be engaged in long-range interactions with the nsp1-coding region, possibly forming an extensive higher-order RNA structure (Guan et al, 2012). Furthermore, a mutagenesis study using BCoV DI RNA indicated that this multipartite RNA structure may involve several SL substructures identified in earlier studies (Gustin et al, 2009;Raman and Brian, 2005) but require refolding of other RNA structures suggested earlier to be essential for DI RNA replication (Brown et al, 2007). A recent study provided evidence that a short oligopeptide from the N-terminal domain of nsp1 may be an essential cisacting protein factor involved in betacoronavirus replication, thus adding to the multiple other functions of this protein (Brockway and Denison, 2005;Huang et al, 2011a,b;Kamitani et al, 2006Kamitani et al, , 2009Lei et al, 2013;Lokugamage et al, 2012;Narayanan et al, 2008a;Tanaka et al, 2012;Tohya et al, 2009;Wathelet et al, 2007;Z€ ust et al, 2007).…”

“…Elements The majority of the 5 0 -proximal RNA structures and sequences essential for coronavirus genome replication have first been characterized for BCoV using DI RNA-based systems (Brown et al, 2007;Chang et al, 1994Chang et al, , 1996Gustin et al, 2009;Raman and Brian, 2005;Raman et al, 2003). The 5 0 -proximal 215 nts of the BCoV genome were predicted to harbor four stem-loops (SLs) that, in the older literature, were termed SL I (comprised of Ia and Ib), II, III, and IV.…”

RNA structure analysis of alphacoronavirus terminal genome regions

“…Also, in a study using MHV/BCoV chimera, a region downstream of SL4 was revealed to be engaged in long-range interactions with the nsp1-coding region, thus possibly forming an extensive higher-order RNA structure (Guan et al, 2012). A subsequent BCoV DI RNA mutagenesis study further suggested that this multipartite RNA structure may involve several stem-loop (sub)structures identified in earlier studies (Gustin et al, 2009;Raman and Brian, 2005) but require refolding of other RNA structures suggested earlier to be essential for DI RNA replication (Brown et al, 2007). The study by Su et al (2014) also identified an intriguing requirement in cis of an oligopeptide sequence in the Nproximal part of nsp1, suggesting that nsp1 may be an essential cis-acting protein factor in betacoronavirus replication, in addition to its multiple other functions (Brockway and Denison, 2005;Huang et al, 2011a,b;Kamitani et al, 2006Kamitani et al, , 2009Lei et al, 2013;Lokugamage et al, 2012;Narayanan et al, 2008a;Tanaka et al, 2012;Tohya et al, 2009;Wathelet et al, 2007;Züst et al, 2007).…”

“…Cis-acting RNA structures in the 5 -terminal region of the coronavirus genome have first been studied for BCoV using DI RNAbased systems (Brown et al, 2007;Chang et al, 1994Chang et al, , 1996Gustin et al, 2009;Raman et al, 2003;Raman and Brian, 2005). In the 5terminal 215 nts of the BCoV genome, four stem-loops (designated I [comprised of Ia and Ib], II, III, and IV) were defined.…”

Cis-acting RNA elements in human and animal plus-strand RNA viruses

“…The 5′-genomic region of BCoV contains at least 6 stem loop structures, numbered SL-I to SL-VI in the 5′ to 3′ direction, whose structures were confirmed by enzymatic probing (Fig. 9) [195][196][197][198][199]. Stem loops I and II were found to be essential for RNA replication.…”