The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.

The SARS coronavirus protein nsp1 can suppress host gene expression at a post-transcriptional level, with previous work showing a reduction in mRNA abundance. Now a direct effect on protein synthesis is revealed, as nsp1 modifies transcripts and also inactivates the 40S ribosomal subunit. Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression, including type I interferon production, by promoting host mRNA degradation and inhibiting host translation, in infected cells. We present evidence that nsp1 uses a novel, two-pronged strategy to inhibit host translation and gene expression. Nsp1 bound to the 40S ribosomal subunit and inactivated the translational activity of the 40S subunits. Furthermore, the nsp1–40S ribosome complex induced the modification of the 5′ region of capped mRNA template and rendered the template RNA translationally incompetent. Nsp1 also induced RNA cleavage in templates carrying the internal ribosome entry site (IRES) from encephalomyocarditis virus, but not in those carrying IRES elements from hepatitis C or cricket paralysis viruses, demonstrating that the nsp1-induced RNA modification was template-dependent. We speculate that the mRNAs that underwent the nsp1-mediated modification are marked for rapid turnover by the host RNA degradation machinery.

SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5′-end of a reporter mRNA having a short 5′ untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5′ untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5′ cap structure and 3′ poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5′-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection. Severe acute respiratory syndrome (SARS) coronavirus (SCoV) is the causative agent of SARS. The nsp1 protein of SCoV blocks host protein synthesis, including type I interferon, a general inhibitor of virus replication, in infected cells. This finding suggests that SCoV nsp1 protein plays a key role in the severe symptoms that accompany SARS infection. Nsp1 binds to the 40S ribosome subunit, which is an essential component for protein synthesis, and inactivates the translation activity of the ribosome. Furthermore, nsp1 binding to the 40S ribosome induces the modification of host mRNAs, leading to the accelerated decay of these RNAs in SCoV-infected cells. We found that the nature of nsp1-induced RNA modification was RNA cleavage and that nsp1 did not recognize specific nucleotides in host mRNAs to induce this cleavage. Interestingly, nsp1 did not induce RNA cleavage in SCoV mRNAs. These data indicate that nsp1 induces RNA cleavage of host mRNAs to suppress the expression of host genes, including those having antiviral functions; yet viral mRNAs are spared from such cleavage events, which, most likely, facilitate efficient SCoV protein synthesis and virus replication in infected cells.

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.

Bumping the priming sites for RT-LAMP based on thermodynamic predictions can dramatically reduce non-specific background amplification.