The nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its N-terminal domain and multimerization properties.

The coronavirus nucleocapsid (N) protein packages viral genomic RNA into a ribonucleoprotein complex. Interactions between N proteins and RNA are thus crucial for the assembly of infectious virus particles. The 45 kDa recombinant nucleocapsid N protein of coronavirus infectious bronchitis virus (IBV) is highly sensitive to proteolysis. We obtained a stable fragment of 14.7 kDa spanning its N-terminal residues 29-160 (IBV-N29-160). Like the N-terminal RNA binding domain (SARS-N45-181) of the severe acute respiratory syndrome virus (SARS-CoV) N protein, the crystal structure of the IBV-N29-160 fragment at 1.85 A resolution reveals a protein core composed of a five-stranded antiparallel beta sheet with a positively charged beta hairpin extension and a hydrophobic platform that are probably involved in RNA binding. Crosslinking studies demonstrate the formation of dimers, tetramers, and higher multimers of IBV-N. A model for coronavirus shell formation is proposed in which dimerization of the C-terminal domain of IBV-N leads to oligomerization of the IBV-nucleocapsid protein and viral RNA condensation.

Structure of the SARS coronavirus main proteinase as an active C2 crystallographic dimer.

The 34 kDa main proteinase (Mpro) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV Mpro is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pH 6.5 in the orthorhombic space group P2(1)2(1)2 that diffract to a resolution of 1.9 A. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.

Structure of internalin C from Listeria monocytogenes.

The crystal structure of internalin C (InlC) from Listeria monocytogenes has been determined at 2.0 A resolution. Several observations implicate InlC in infection: inlC has the same transcriptional activator as other virulence genes, it is only present in pathogenic Listeria strains and an inlC deletion mutant is significantly less virulent. While the extended concave receptor-binding surfaces of the leucine-rich repeat (LRR) domains of internalins A and B have aromatic clusters involved in receptor binding, the corresponding surface of InlC is smaller, flatter and more hydrophilic, suggesting that InlC may be involved in weak or transient associations with receptors; this may help explain why no receptor has yet been discovered for InlC. In contrast, the Ig-like domain, to which the LRR domain is fused, has surface aromatics that may be of functional importance, possibly being involved in binding to the surface of the bacteria or in receptor binding.

X-ray structures of the N- and C-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation.

Coronaviruses cause a variety of respiratory and enteric diseases in animals and humans including severe acute respiratory syndrome. In these enveloped viruses, the filamentous nucleocapsid is formed by the association of nucleocapsid (N) protein with single-stranded viral RNA. The N protein is a highly immunogenic phosphoprotein also implicated in viral genome replication and in modulating cell signaling pathways. We describe the structure of the two proteolytically resistant domains of the N protein from infectious bronchitis virus (IBV), a prototype coronavirus. These domains are located at its N- and C-terminal ends (NTD and CTD, respectively). The NTD of the IBV Gray strain at 1.3-A resolution exhibits a U-shaped structure, with two arms rich in basic residues, providing a module for specific interaction with RNA. The CTD forms a tightly intertwined dimer with an intermolecular four-stranded central beta-sheet platform flanked by alpha helices, indicating that the basic building block for coronavirus nucleocapsid formation is a dimeric assembly of N protein. The variety of quaternary arrangements of the NTD and CTD revealed by the analysis of the different crystal forms delineates possible interfaces that could be used for the formation of a flexible filamentous ribonucleocapsid. The striking similarity between the dimeric structure of CTD and the nucleocapsid-forming domain of a distantly related arterivirus indicates a conserved mechanism of nucleocapsid formation for these two viral families.