Mechanisms and enzymes involved in SARS coronavirus genome expression

Abstract

A novel coronavirus is the causative agent of the current epidemic of severe acute respiratory syndrome (SARS). Coronaviruses are exceptionally large RNA viruses and employ complex regulatory mechanisms to express their genomes. Here, we determined the sequence of SARS coronavirus (SARS-CoV), isolate Frankfurt 1, and characterized key RNA elements and protein functions involved in viral genome expression. Important regulatory mechanisms, such as the (discontinuous) synthesis of eight subgenomic mRNAs, ribosomal frameshifting and posttranslational proteolytic processing, were addressed. Activities of three SARS coronavirus enzymes, the helicase and two cysteine proteinases, which are known to be critically involved in replication, transcription and/or post-translational polyprotein processing, were characterized. The availability of recombinant forms of key replicative enzymes of SARS coronavirus should pave the way for high-throughput screening approaches to identify candidate inhibitors in compound libraries. INTRODUCTIONSevere acute respiratory syndrome (SARS) is a lifethreatening form of pneumonia (Peiris et al., 2003a). In the course of a few months in 2003, an epidemic emerged that has spread from its likely origin in Guangdong Province, China, to 32 countries. By 11 June 2003 more than 8400 cases and 789 deaths had been recorded by the World Health Organization. The rapid transmission by aerosols (and probably also the faecal-oral route) and the high mortality rate make SARS a global threat for which no efficacious therapy is available. There is now clear evidence that SARS is caused by a previously unknown coronavirus, provisionally termed SARS coronavirus (SARS-CoV) (Peiris et al., 2003b;Drosten et al., 2003;Ksiazek et al., 2003;Fouchier et al., 2003). Genome sequences of SARS-CoV isolates obtained from a number of index patients have been published recently and provide important information on the organization, phylogeny and variability of the 29?7 kb positive-strand RNA genome of SARS-CoV (Rota et al., 2003;Marra et al., 2003;Ruan et al., 2003). By analogy with other coronaviruses (Lai & Holmes, 2001;Gorbalenya, 2001), SARS-CoV gene expression is expected to involve complex transcriptional, translational and post-translational regulatory mechanisms, whose molecular details remain to be determined. SARS-CoV genome expression starts with the translation of two large replicative polyproteins, pp1a (486 kDa) and pp1ab (790 kDa), which are encoded by the viral replicase gene (21 221 nt) that comprises ORFs 1a and 1b (Fig. 1). Expression of the ORF1b-encoded region of pp1ab is predicted to involve ribosomal frameshifting into the 21 frame just upstream of the ORF1a translation termination codon (Brierley et al., 1989). The pp1a and pp1ab polyproteins are processed by viral proteinases to yield the functional components of the membrane-bound replicase complex (Ziebuhr et al., 2000). In contrast to most other coronaviruses, which use three proteinase activities for replicase polyprotein processing (Ziebuhr et ...

Mechanisms of Host Defense following Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) Pulmonary Infection of Mice

“…In addition to the specific neutralizing Ab made in response to SARS-CoV infection by BALB/c mice that was mentioned previously, a Th1 response occurs in response to vaccination of BALB/c mice with SARS-CoV spike protein (15,33,34 We were able to detect viral genome in the lungs of infected B6 mice by RT-PCR beyond day 9 postinfection, yet at these same time points the virus could not be cultured or demonstrated by in situ hybridization. This could simply reflect the presence of defective or latent virions, neutralized virus or, alternatively, persistent very low level viral infection (38,39). The latter possibility is interesting in light of our discovery of SARS-CoV in the brain of B6 mice after the lung infection appeared to have cleared.…”

Understanding Helicases as a Means of Virus Control

“…The SARS-CoV polyproteins are then processed by two viral proteases into mature viral peptides, which include both structural and nonstructural proteins. The helicase is part of nonstructural protein 13 (nsp13), which is processed from the C-terminal portion of pp1ab [51]. SARS-CoV helicase is localized on the endoplasmic reticulum of SARS-CoV infected cells, where RNA replication is likely to take place [52].…”

Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

“…SARS-CoV is a member of group 2 of the Coronaviridae family within the order Nidovirales (13), which is composed of enveloped, singlestranded, positive-sense RNA viruses relevant in animal and human health (5,9). Two-thirds of the 29.7-kb SARS-CoV genome carries the replicase gene, which comprises two overlapping open reading frames, ORF 1a and ORF 1b, the latter being translated by a ribosomal frameshift mechanism (29). Translation of both ORFs results in the synthesis of two polyproteins that are processed by viral proteinases to release the components of the replication-transcription complex (36,37).…”