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 ...

Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicasetranscriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3 35 direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2 -O-methylated RNA substrates and required divalent metal ions for activity. A range of 5 -labeled ssRNA substrates were processed to final products of Ϸ8 -12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5 -labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.replication ͉ ribonuclease ͉ severe acute respiratory syndrome

Coronaviruses are important pathogens that cause acute respiratory diseases in humans. Replication of the Ϸ30-kb positive-strand RNA genome of coronaviruses and discontinuous synthesis of an extensive set of subgenome-length RNAs (transcription) are mediated by the replicase-transcriptase, a barely characterized protein complex that comprises several cellular proteins and up to 16 viral subunits. The coronavirus replicase-transcriptase was recently predicted to contain RNA-processing enzymes that are extremely rare or absent in other RNA viruses. Here, we established and characterized the activity of one of these enzymes, replicative nidoviral uridylate-specific endoribonuclease (NendoU). It is considered a major genetic marker that discriminates nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) from all other RNA virus families. Bacterially expressed forms of NendoU of severe acute respiratory syndrome coronavirus and human coronavirus 229E were revealed to cleave single-stranded and double-stranded RNA in a Mn 2؉ -dependent manner. Single-stranded RNA was cleaved less specifically and effectively, suggesting that doublestranded RNA is the biologically relevant NendoU substrate. Double-stranded RNA substrates were cleaved upstream and downstream of uridylates at GUU or GU sequences to produce molecules with 2-3 cyclic phosphate ends. 2-O-ribose-methylated RNA substrates proved to be resistant to cleavage by NendoU, indicating a functional link with the 2-O-ribose methyltransferase located adjacent to NendoU in the coronavirus replicative polyprotein. A mutagenesis study verified potential active-site residues and allowed us to inactivate NendoU in the full-length human coronavirus 229E clone. Substitution of D6408 by Ala was shown to abolish viral RNA synthesis, demonstrating that NendoU has critical functions in viral replication and transcription. H uman coronavirus 229E (HCoV-229E), a group 1 coronavirus, is one of the major viral pathogens causing upper respiratory tract illness in humans (1), whereas severe acute respiratory syndrome coronavirus (SARS-CoV), a group 2 coronavirus, has been identified as the causative agent of SARS, a life-threatening form of pneumonia that caused Ͼ8,000 fatalities in a worldwide epidemic in 2003 (2, 3). The extremely large positive-strand RNA genomes of HCoV-229E (27.3 kb) and SARS-CoV (29.7 kb) contain 8 and 14 ORFs, respectively (4, 5). The two most 5Ј-terminal ORFs, 1a and 1b, encode the major subunits of the replicative machinery, whereas the more downstream ORFs encode structural and virus-specific accessory proteins.Coronavirus gene expression involves a series of complex transcriptional, translational, and posttranslational regulatory mechanisms (6, 7). After receptor-mediated entry, two large replicative polyproteins, pp1a (Ͼ450 kDa) and pp1ab (Ͼ750 kDa), are translated from the genome RNA. The polyproteins are encoded by the replicase gene (Ͼ20,000 bases) that comprise ORFs 1a and 1b (Fig. 1A) (8). Expression of pp1ab involves ribosomal frameshifting into...

A previously unknown coronavirus (CoV) is the aetiological agent causing severe acute respiratory syndrome (SARS), for which an effective antiviral treatment is urgently needed. To enable the rapid and biosafe identification of coronavirus replicase inhibitors, we have generated a non-cytopathic, selectable replicon RNA (based on human CoV 229E) that can be stably maintained in eukaryotic cells. Most importantly, the replicon RNA mediates reporter gene expression as a marker for coronavirus replication. We have used a replicon RNA-containing cell line to test the inhibitory effect of several compounds that are currently being assessed for SARS treatment. Amongst those, interferon-a displayed the strongest inhibitory activity. Our results demonstrate that coronavirus replicon cell lines provide a versatile and safe assay for the identification of coronavirus replicase inhibitors. Once this technology is adapted to SARS-CoV replicon RNAs, it will allow high throughput screening for SARS-CoV replicase inhibitors without the need to grow infectious SARS-CoV.

Listeria monocytogenes causes meningitis and encephalitis in humans and crosses the blood-brain barrier by yet unknown mechanisms. The interaction of the bacteria with different types of endothelial cells was recently analyzed, and it was shown that invasion into, but not adhesion to, human brain microvascular endothelial cells (HBMEC) depends on the product of the inlB gene, the surface molecule InlB, which is a member of the internalin multigene family. In the present study we analyzed the role of the medium composition in the interaction of L. monocytogenes with HBMEC, and we show that invasion of HBMEC is strongly inhibited in the presence of adult human serum. The strong inhibitory activity, which is not present in fetal calf serum, does not inhibit uptake by macrophage-like J774 cells but does also inhibit invasion of Caco-2 epithelial cells. The inhibitory component of human serum was identified as being associated with L. monocytogenes-specific antibodies present in the human serum. Human newborn serum (cord serum) shows only a weak inhibitory activity on the invasion of HBMEC by L. monocytogenes.Listeria monocytogenes, a gram-positive, facultatively intracellular bacterium, is known to cause meningitis, encephalitis, and brain abscesses, mainly in immunocompromised individuals (21). Central nervous system (CNS) penetration by L. monocytogenes suggests that invasion of brain microvascular endothelial cells may be an important way of crossing the blood-brain barrier. During the last couple of years, several groups have reported on the capacity of L. monocytogenes to invade different types of human endothelial cells. However, the absolute values of invasion, as well as the dependency of invasion on the inlB gene product, differed markedly among the studies (5,11,12,17,22). It has previously been shown that invasion of, but not adhesion to, human brain microvascular endothelial cells (HBMEC) by L. monocytogenes is strictly dependent on the presence of the product of the inlB gene (2, 10, 11). InlB is a 630-amino-acid protein of the internalin family of leucine-rich repeat proteins which is found at the cell surface but is also secreted into the supernatant (3, 7, 9). Parida et al. (17) have reported a similar inlB-dependent invasion of human umbilical vein endothelial cells (HUVEC), which we could not detect in an earlier study (12). Furthermore, Drevets et al. (5) first reported an InlA-dependent invasion of HUVEC and later an inlA-and inlB-independent invasion of human microvascular endothelial cells (22). The differences in InlB dependency of endothelial cell invasion might be due, at least partially, to the different types of inlB mutants used in the studies as well as to differences in the target cells. On the other hand, differences in experimental conditions might also have influenced the outcomes of the experiments.In the present study we analyzed the roles of normal human serum (HS) and fetal calf serum (FCS) in adhesion to and invasion of HBMEC by L. monocytogenes. We show that antibodies present ...