This study provides direct evidence that SARS human coronavirus is capable of infecting the central nervous system, and that Mig might be involved in the brain immunopathology of SARS.

Hepatitis E virus (HEV), a small, non-enveloped RNA virus in the familyHepeviridae, is associated with endemic and epidemic acute viral hepatitis in developing countries. Our 3.5-Å structure of a HEV-like particle (VLP) shows that each capsid protein contains 3 linear domains that form distinct structural elements: S, the continuous capsid; P1, 3-fold protrusions; and P2, 2-fold spikes. The S domain adopts a jelly-roll fold commonly observed in small RNA viruses. The P1 and P2 domains both adopt ␤-barrel folds. Each domain possesses a potential polysaccharide-binding site that may function in cell-receptor binding. Sugar binding to P1 at the capsid protein interface may lead to capsid disassembly and cell entry. Structural modeling indicates that native T ‫؍‬ 3 capsid contains flat dimers, with less curvature than those of T ‫؍‬ 1 VLP. Our findings significantly advance the understanding of HEV molecular biology and have application to the development of vaccines and antiviral medications.capsid ͉ HEV V iral hepatitis is principally caused by 5 distinct viruses named hepatitis A-E. Despite their similar names, the 5 viruses are unrelated, and they have totally different genome structures with distinct replication mechanisms. Hepatitis E virus (HEV) is responsible for endemic hepatitis as well as sporadic epidemics of acute, enterically transmitted hepatitis in the developing world, including parts of Asia, the Middle East, Africa, and Mexico (1, 2). HEV accounts for more than 50% of acute viral hepatitides in young adults in these regions, with a case fatality of 1-2% in regular patients and up to 20% in pregnant women.Given the lack of a robust cell culture system, and because HEV is not closely related to any other well-characterized virus, little is known about the molecular biology of HEV or its strategy for replication (1). HEV is a small, non-enveloped virus with a 7.2 kb, positive-sense RNA genome. Its genomic RNA is polyadenylated and contains 3 ORFs. Located near the 5Ј-end, ORF1 encodes a non-structural polyprotein with multiple functional domains, including those for methyltransferase, protease, helicase, and polymerase. The viral capsid protein (CP) is encoded by ORF2 near the 3Ј-end. ORF3, which partially overlaps with the other 2 ORFs, codes for an immunogenic protein of unknown function. HEV was originally classified in the Caliciviridae family because of its structural similarity to other caliciviruses; however, it is now the sole member of the Hepeviridae family. The genomic RNA of HEV exhibits several distinct features compared to the genomic RNA of caliciviruses, including a methylated cap at the 5Ј-end and an ORF1 with functional domains arranged in a different order (1, 3).Previous studies of HEV assembly have primarily focused on the overexpression of viral proteins. The ORF2 capsid protein, HEV-CP, contains a total of 660 amino acid residues. At the HEV-CP N terminus is a signal peptide followed by an arginine-rich domain that potentially play a role in viral RNA encapsidation during assem...

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The nucleocapsid (N) protein plays an essential role in SARS-CoV genome packaging and virion assembly. We have previously shown that SARS-CoV N protein forms a dimer in solution through its C-terminal domain. In this study, the crystal structure of the dimerization domain, consisting of residues 270 -370, is determined to 1.75 Å resolution. The structure shows a dimer with extensive interactions between the two subunits, suggesting that the dimeric form of the N protein is the functional unit in vivo. Although lacking significant sequence similarity, the dimerization domain of SARS-CoV N protein has a fold similar to that of the nucleocapsid protein of the porcine reproductive and respiratory syndrome virus. This finding provides structural evidence of the evolutionary link between Coronaviridae and Arteriviridae, suggesting that the N proteins of both viruses have a common origin.Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses that infect a variety of mammals and birds. Although previously identified human coronaviruses cause only mild respiratory infections, in the 2003 outbreak of severe acute respiratory syndrome (SARS), 3 a disease caused by a new type of coronavirus (SARS-CoV), there were more than 8,000 cases resulting in 774 deaths (ϳ10% mortality). Phylogenetic analysis suggests that SARSCoV diverged early from group 2 coronaviruses and has evolved independently for a long period of time (1).The coronavirus genome, containing ϳ30,000 bases, is the largest among positive-sense RNA viruses (2, 3). It encodes non-structural proteins including the RNA polymerase and helicase, as well as the spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins. The coronavirus virion is about 120 nm in diameter and consists of a lipid envelope containing three or four anchored glycoproteins and a helical ribonucleoprotein core (4). The surface projections forming the crown-like structure observed via electron microscopy are made up of the S protein, which is responsible for receptor recognition and membrane fusion (5-9). The integral membrane proteins M and E are essential for virus budding. When co-expressed in animal cells, the M and E proteins are sufficient to form virus-like particles (10). The N protein interacts with the viral genome to form the ribonucleoprotein core and has been shown to be involved in viral RNA synthesis, transcriptional regulation of genomic RNA, translation of viral proteins, and budding (2,11,12).Coronaviruses are related to arteriviruses by their similar genome organizations and viral replication mechanisms (3, 13). Recently, Coronaviridae and Arteriviridae were united to form the new order Nidovirales. The name of the order comes from a property common to both viruses, a nested set of subgenomic mRNAs for structural protein expression (Latin nidus, meaning nest). The replicase genes of arteriviruses and coronaviruses are thought to ha...

The aim of the study was to establish the life cycle of severe acute respiratory syndrome-associated coronavirus (SARS CoV) in host cells and determine the pathogenesis of SARS. Vero E6 cells (African green monkey kidney cells) were inoculated with SARS coronavirus for 3, 7, 24, 48, and 72 hr, respectively, and were observed under electron microscope. It was found that the SARS coronavirus entered the cells through membrane fusion instead of endocytosis, and then the nucleocapsids assembled in the RER and matured by budding into the smooth vesicles, which were derived from the Golgi apparatus. The smooth vesicles fused with the cell membrane, and the mature particles were released. A special phenomenon was that some virus-like particles appeared in the nucleus. We propose a scheme of the life cycle of SARS coronavirus and discuss the mechanism of its replication in Vero E6 cells.

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The viral nucleocapsid (N) protein plays an essential role in viral RNA packaging. In this study, recombinant SARS-CoV N protein was shown to be dimeric by analytical ultracentrifugation, size exclusion chromatography coupled with light scattering, and chemical cross-linking. Dimeric N proteins selfassociate into tetramers and higher molecular weight oligomers at high concentrations. The dimerization domain of N was mapped through studies of the oligomeric states of several truncated mutants. Although mutants consisting of residues 1-210 and 1-284 fold as monomers, constructs consisting of residues 211-422 and 285-422 efficiently form dimers. When in excess, the truncated construct 285-422 inhibits the homodimerization of full-length N protein by forming a heterodimer with the full-length N protein. These results suggest that the N protein oligomerization involves the C-terminal residues 285-422, and this region is a good target for mutagenic studies to disrupt N protein self-association and virion assembly.Coronaviruses are responsible for ϳ30% of human upper respiratory infections each year. In November 2002, a new coronavirus, known as the severe acute respiratory syndrome (SARS) 1 -associated coronavirus, SARS-CoV, emerged in China and caused more than 8000 cases of SARS worldwide. Approximately 10% of these cases were fatal. Similar to other coronaviruses, SARS-CoV is an enveloped, single-stranded (ss) RNA virus. The SARS-CoV genome contains ϳ29,700 nucleotides (1, 2), encoding the RNA-dependent RNA polymerase and four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). In addition, intergenic regions encode several open reading frames for nonstructural proteins of unknown function (1, 2). The S protein is a surface glycoprotein that mediates viral entry by binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) (3) and inducing membrane fusion. The receptorbinding domain has been mapped to amino acids 318 -510 (4, 5), and structures of the heptad repeat region of S protein (6, 7) indicate that it is a class I membrane fusion protein. The M protein of coronavirus is the most abundant protein component of the envelope. This protein plays a predominant role in the formation and release of the virion envelope. When co-expressed with the E protein, virus-like particles with sizes and shapes similar to those of virions are assembled (8, 9). Recently, virus-like particles of SARS-CoV were obtained by recombinant expression of S, E, and M proteins in insect cells (10). Inside the envelope, the N protein associates with the genomic RNA to form a long, flexible, helical ribonucleoprotein. The N protein is typically 350 -450 amino acids in length, highly basic, and serine-phosphorylated, but the extent and physiological relevance of phosphorylation is unclear (11, 12). In addition to its structural role, several additional functions are postulated for the N protei...