Dependence of Coronavirus RNA Replication on an NH ₂ -Terminal Partial Nonstructural Protein 1 in cis

Abstract

Genomes of positive (؉)-strand RNA viruses use cis-acting signals to direct both translation and replication. Here we examine two 5=-proximal cis-replication signals of different character in a defective interfering (DI) RNA of the bovine coronavirus (BCoV) that map within a 322-nucleotide (nt) sequence (136 nt from the genomic 5= untranslated region and 186 nt from the nonstructural protein 1 [nsp1]-coding region) not found in the otherwise-identical nonreplicating subgenomic mRNA7 (sgmRNA7). The natural DI RNA is structurally a fusion of the two ends of the BCoV genome that results in a single open reading frame between a partial nsp1-coding region and the entire N gene. (i) In the first examination, mutation analyses of a recently discovered long-range RNA-RNA base-paired structure between the 5= untranslated region and the partial nsp1-coding region showed that it, possibly in concert with adjacent stem-loops, is a cis-acting replication signal in the (؉) strand. We postulate that the higher-order structure promotes (؉)-strand synthesis. (ii) In the second examination, analyses of multiple frame shifts, truncations, and point mutations within the partial nsp1-coding region showed that synthesis of a PEFP core amino acid sequence within a group A lineage betacoronavirus-conserved NH 2 -proximal WAPEFPWM domain is required in cis for DI RNA replication. We postulate that the nascent protein, as part of an RNA-associated translating complex, acts to direct the DI RNA to a critical site, enabling RNA replication. We suggest that these results have implications for viral genome replication and explain, in part, why coronavirus sgmRNAs fail to replicate. What constitutes the cis-acting requirements for coronavirus RNA replication has remained an intriguing question since it was discovered that the subgenomic mRNAs (sgmRNAs) of coronaviruses (used primarily to synthesize viral structural proteins) are both (i) 5= and 3= coterminal with the genome for at least ϳ70 and 1,670 nucleotides (nt), respectively, lengths greater than those of many viral RNA polymerase promoters (1-3), and (ii) are present in sgmRNA-length replication-intermediate-like doublestranded RNA structures that are involved in sgmRNA synthesis (4-6) yet fail to replicate when transfected, as synthetic transcripts, into virus-infected cells (Fig. 1) (7). If replication of the coronavirus sgmRNAs normally occurs during infection, it might be expected that they would replicate following their transfection into virus-infected cells, since all trans-acting factors required for viral RNA replication are present. In coronaviruses, the 5= twothirds of the single-stranded positive (ϩ)-strand ϳ30-kb coronavirus genome is used as mRNA for synthesis of overlapping polyproteins 1a (ϳ4,000 amino acids [aa]) and 1ab (ϳ7,000 aa), which are proteolytically processed into the 16 replicase proteins that make up the replication/transcription complex, whereas the 3= one-third of the genome is transcribed into a 3= nested set of sgmRNAs that are coterminal with ...

RNA structure analysis of alphacoronavirus terminal genome regions

“…The large number of covariant base pairs in the alphacoronavirus SL5 (Fig. 2) suggest significant constraints and a major functional role for this structure in alphacoronavirus (and, possibly, betacoronavirus) replication and there is indeed some experimental evidence to support this hypothesis (Brown et al, 2007;Su et al, 2014). Using RNA structure probing information obtained for the 5 -terminal 600 nts of HCoV-229E and HCoV-NL63, we confirmed and refined our RNA secondary structure predictions for two B-lineage alphacoronaviruses ( Figs.…”

“…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). Possible interacting partners for nsp1 remain to be identified.…”

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

“…An earlier study found that certain amino-terminal point mutations in nsp1 were severely debilitating or lethal for MHV (53), but, in retrospect, this may be attributable to disruption of cis-acting RNA structures overlapping the first half of the nsp1 ORF. A very recent analysis of the replication of a DI RNA of bovine coronavirus, which is closely related to MHV, found an absolute requirement for an intact stretch of coding sequence near the amino terminus of nsp1 (35). This nsp1 peptide was proposed to provide a means for DI RNA to be recruited to the preformed replication complexes of the helper virus.…”

“…The 5= ends of coronavirus genomes, including the 5= UTR and part of the nsp1 coding region, contain multiple cis-acting RNA structures that are critical for replication and transcription (34). In MHV and other betacoronaviruses, the genus in which these structures have been most extensively studied, the genomic 5= end contains a series of at least eight stem-loops (SL) (35)(36)(37)(38)(39), as well as a four-way junction formed by a long-range interaction between the regions upstream of SL 5 (SL5) and downstream of SL7 (40) ( Fig. 2A).…”

“…(A) Schematics of the genomic 5= ends of wild-type MHV and the N3S mutant. Numbered RNA structures, stem-loops SL1 through SL7(35,36,39) and SL8…”