Severe Acute Respiratory Syndrome Coronavirus Protein nsp1 Is a Novel Eukaryotic Translation Inhibitor That Represses Multiple Steps of Translation Initiation

Abstract

Severe acute respiratory syndrome (SARS) coronavirus nonstructural protein 1 (nsp1) binds to the 40S ribosomal subunit and inhibits translation, and it also induces a template-dependent endonucleolytic cleavage of host mRNAs. nsp1 inhibits the translation of cap-dependent and internal ribosome entry site (IRES)-driven mRNAs, including SARS coronavirus mRNAs, hepatitis C virus (HCV), and cricket paralysis virus (CrPV) IRES-driven mRNAs that are resistant to nsp1-induced RNA cleavage. We used an nsp1 mutant, nsp1-CD, lacking the RNA cleavage function, to delineate the mechanism of nsp1-mediated translation inhibition and identify the translation step(s) targeted by nsp1. nsp1 and nsp1-CD had identical inhibitory effects on mRNA templates that are resistant to nsp1-induced RNA cleavage, implying the validity of using nsp1-CD to dissect the translation inhibition function of nsp1. We provide evidence for a novel mode of action of nsp1. nsp1 inhibited the translation initiation step by targeting at least two separate stages: 48S initiation complex formation and the steps involved in the formation of the 80S initiation complex from the 48S complex. nsp1 had a differential, mRNA template-dependent, inhibitory effect on 48S and 80S initiation complex formation. nsp1 inhibited different steps of translation initiation on CrPV and HCV IRES, both of which initiate translation via an IRES-40S binary complex intermediate; nsp1 inhibited binary complex formation on CrPV IRES and 48S complex formation on HCV IRES. Collectively, the data revealed that nsp1 inhibited translation by exerting its effect on multiple stages of translation initiation, depending on the mechanism of initiation operating on the mRNA template. Severe acute respiratory syndrome (SARS) coronavirus (SCoV) is the causative agent of the respiratory disease SARS, which emerged in southern China in 2002 and spread to different countries of the world during a 2002-2003 epidemic (6, 18-20, 26, 30). SCoV carries a large, single-stranded, positive-sense RNA genome. The 5=-most two-thirds of the genome contains two large overlapping open reading frames that encode two polyproteins, which are cleaved by virus-encoded proteinases into 16 nonstructural proteins (nsp1 to nsp16), most of which are involved in viral RNA synthesis.Coronavirus (CoV) nsp1 proteins share a biological function to inhibit host gene expression; nsp1 of different CoVs employ different strategies to inhibit host gene expression (11,15,16,36). Several lines of evidence indicate a strong possibility that CoV nsp1 is a major CoV virulence factor (22,36,37,39). SCoV nsp1 suppresses the host innate immune functions by inhibiting type I interferon expression (22) and host antiviral signaling pathways (37) in infected cells. Mouse hepatitis virus nsp1 inhibits the type I interferon system, and a mutant virus lacking the nsp1 gene is severely attenuated in infected mice (39). Hence, studies using CoV nsp1 proteins have begun to tease out the common as well as the divergent mechanisms involved in the CoV-i...

RNA structure analysis of alphacoronavirus terminal genome regions

“…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.…”

Coronavirus cis-Acting RNA Elements

“…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).…”

Identification of the hyper-variable genomic hotspot for the novel coronavirus SARS-CoV-2

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