The severe acute respiratory syndrome coronavirus (SARS-CoV) devotes a significant portion of its genome to producing nonstructural proteins required for viral replication. SARS-CoV nonstructural protein 9 (nsp9) was identified as an essential protein with RNA/DNA-binding activity, and yet its biological function within the replication complex remains unknown. Nsp9 forms a dimer through the interaction of parallel ␣-helices containing the protein-protein interaction motif GXXXG. In order to study the role of the nsp9 dimer in viral reproduction, residues G100 and G104 at the helix interface were targeted for mutation. Multi-angle light scattering measurements indicated that G100E, G104E, and G104V mutants are monomeric in solution, thereby disrupting the dimer. However, electrophoretic mobility assays revealed that the mutants bound RNA with similar affinity. Further experiments using fluorescence anisotropy showed a 10-fold reduction in RNA binding in the G100E and G104E mutants, whereas the G104V mutant had only a 4-fold reduction. The structure of G104E nsp9 was determined to 2.6-Å resolution, revealing significant changes at the dimer interface. The nsp9 mutations were introduced into SARS-CoV using a reverse genetics approach, and the G100E and G104E mutations were found to be lethal to the virus. The G104V mutant produced highly debilitated virus and eventually reverted back to the wild-type protein sequence through a codon transversion. Together, these data indicate that dimerization of SARS-CoV nsp9 at the GXXXG motif is not critical for RNA binding but is necessary for viral replication.

Interferon-λ 1 (IFN-λ1, also known as IL-29) belongs to the recently discovered group of type III IFNs. All type III IFNs initiate signaling processes through formation of specific hetero-dimeric receptor complexes consisting of IFN-λR1 and IL-10R2. We have determined the structure of human IFN-λ1 complexed with human IFN-λR1, a receptor unique to type III IFNs. The overall structure of IFN-λ1 is topologically similar to the structure of IL-10 and other members of the IL-10 family of cytokines. IFN-λR1 consists of two distinct domains having fibronectin type III topology. The ligand/receptor interface includes helix A, loop AB and helix F on the IFN site, and loops primarily from the N-terminal domain and inter-domain hinge region of the IFN-λR1. Composition and architecture of the interface that includes only a few direct hydrogen bonds supports an idea that long-range ionic interactions between ligand and receptor govern the process of initial recognition of the molecules while hydrophobic interactions finalize it.

The coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS), SARS-CoV, encodes two large polyproteins (pp1a and pp1ab) that are processed by two viral proteases to yield mature non-structural proteins (nsps). Many of these nsps have essential roles in viral replication, but several have no assigned function and possess amino acid sequences that are unique to the CoV family. One such protein is SARS-CoV nsp1, which is processed from the N-terminus of both pp1a and pp1ab. The mature SARS-CoV protein is present in cells several hours post-infection and colocalizes to the viral replication complex, but its function in the viral life cycle remains unknown. Furthermore, nsp1 sequences are highly divergent across the CoV family, and it has been suggested that this is due to nsp1 possessing a function specific to viral interactions with its host cell or acting as a host specific virulence factor. In order to initiate structural and biophysical studies of SARSCoV nsp1, a recombinant expression system and a purification protocol have been developed, yielding milligram quantities of highly purified SARS-CoV nsp1. The purified protein was characterized using circular dichroism, size exclusion chromatography, and multi-angle light scattering.The Severe Acute Respiratory Syndrome (SARS) outbreak of 2002-2003, followed by a much smaller outbreak in 2004, caused over 8000 illnesses and nearly 800 deaths (World Health Organization; http://www.who.int/csr/sars/country/table2004_04_21/en/index.html). The infectious agent responsible for this disease was quickly identified as a new member of the coronavirus (CoV) family, SARS-coronavirus (SARS-CoV) [1][2][3], most closely related to the group 2 CoVs [4]. This newly emerged virus prompted a renewed interest in CoV research. Prior to the SARS outbreak, only two CoVs (HCoV-229E and HCoV-OC43) were known to infect humans [5]. These two CoVs have been estimated to cause up to 30% of common colds and mild respiratory illnesses [6]. Other CoVs are widespread in both domestic and wild animals, with several posing significant economic impact on livestock and poultry industries.Following the emergence of SARS, two additional human CoVs associated with upper and lower respiratory tract diseases were identified. Three groups independently identified in young children what is likely a single CoV species, and this new CoV has been variously designated NL63,. The second new CoV was discovered in an elderly patient * Corresponding author: Mailing Address: Timothy C. Umland, Ph.D., Hauptman-Woodward Medical Research Institute, 700 Ellicott St., Buffalo, NY 14203-1102, E-mail: umland@hwi.buffalo.edu, Phone: 716-898-8632, Fax: 716-898-8660. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please ...

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