The 24-Angstrom Structure of Respiratory Syncytial Virus Nucleocapsid Protein-RNA Decameric Rings

Abstract

Respiratory syncytial virus (RSV), a nonsegmented, negative-sense RNA-containing virus, is a common cause of lower respiratory tract disease. Expression of RSV nucleocapsid protein (N) in insect cells using the baculovirus expression system leads to the formation of N-RNA complexes that are morphologically indistinguishable from viral nucleocapsids. When imaged in an electron microscope, three distinct types of structures were observed: tightly wound short-pitch helices, highly extended helices, and rings. Negative stain images of N-RNA rings were used to calculate a three-dimensional reconstruction at 24 Å resolution, revealing features similar to those observed in nucleocapsids from other viruses of the order Mononegavirales. The reconstructed N-RNA rings comprise 10 N monomers and have an external radius of 83 Å and an internal radius of 40 Å. Comparison of this structure with crystallographic data from rabies virus and vesicular stomatitis virus N-RNA rings reveals striking morphological similarities.Respiratory syncytial virus (RSV) is a clinically important virus that is the leading viral cause of lower respiratory tract infection in infants. According to the World Health Organization, RSV infects approximately 60 million people and is responsible for an estimated 160,000 deaths annually worldwide. Classified within the Pneumovirinae subfamily of the Paramyxoviridae family, order Mononegavirales, the virus has a nonsegmented negative-sense RNA genome that is encapsidated by multiple copies of the nucleocapsid (N) protein, forming a helical ribonucleoprotein complex termed the nucleocapsid. In addition to protecting the RNA from damage, N mediates the interaction between the genomic RNA and the virally encoded RNA-dependent RNA polymerase, which is composed of the phosphoprotein (P), large (L) polymerase, and M2-1 proteins. The matrix (M) protein forms a layer between the nucleocapsid and the virion envelope, which is derived from the host-cell plasma membrane and studded with the fusion (F), attachment (G), and small hydrophobic (SH) proteins. The remaining three proteins encoded by RSV, M2-2 and nonstructural proteins 1 (NS1) and NS2, are found in negligible amounts within the virion and are thought to regulate RNA synthesis or the host's innate immunity (8).Domains of N responsible for interactions with associated cofactors have been mapped for several members of Mononegavirales, including the rhabdovirus rabies virus (RV) (17), Sendai virus (SeV) (23), measles virus (MeV), (both members of the Paramyxovirinae [3]), and RSV (13, 16, 22). Although differences exist among the viruses, there is an overall homology in modular components of the N proteins: the amino terminus of N is largely responsible for the assembly of the nucleocapsid, while the carboxyl terminus contains elements that are necessary for the interaction with the polymerase and M protein (22).Nucleocapsid formation proceeds concurrently with genome or antigenome synthesis, and N is thought to be delivered to the nascent RNA strand in comple...

Structure of Crimean-Congo Hemorrhagic Fever Virus Nucleoprotein: Superhelical Homo-Oligomers and the Role of Caspase-3 Cleavage

“…Here, we have determined the crystal structure of CCHFV N in a double superhelical oligomeric form and a monomeric form. The double superhelical structure of CCHFV N presented in this report is in a relaxed helical state and could represent the physiological organization of CCHFV RNP, as previous studies have revealed that, unlike nucleocapsids of nonsegmented viruses, which are tightly packed rigid helical structures (24), nucleocapsids of bunyaviruses and arenaviruses are more relaxed and form irregularly shaped RNPs (24,29,33,35,37). The bending of the stalk domain in CCHFV N appears to be crucial for the oligomerization of CCHFV N, as it mediates the interaction with the adjacent molecule in the superhelical structure and allows dimerization of the superhelices to form an antiparallel double super helix.…”

Crystal structure of the Lassa virus nucleoprotein–RNA complex reveals a gating mechanism for RNA binding

“…The Ns of influenza virus and Rift Valley fever virus and the N-terminal domain of LASV NP are compact, rather than more extended like the Ns of the nonsegmented RABV, VSV, RSV, and Borna disease virus. Furthermore, electron microscopy of RNPs of the segmented Pichinde arenavirus, influenza, and Rift Valley fever viruses demonstrates the RNP complexes of segmented viruses are less helical in nature than those of the nonsegmented viruses (26)(27)(28). Furthermore, LASV NP could have some partial sequence specificity or preferences, but no specificity was observed in N-RNA complexes of the nonsegmented viruses RSV, RABV, and VSV.…”

Mutagenesis of the nucleocapsid protein of Nipah virus involved in capsid assembly

“…Transmission electron microscopic studies revealed that all the paramyxovirus nucleocapsids are helical in structure, but there are significant differences among the virus genera (Bhella et al, 2002) such as Sendai virus (SeV; Myers et al, 1999), measles virus (MeV;Bhella et al, 2004) and respiratory syncytial virus (RSV; MacLellan et al, 2007). This is most likely due to the differences in the primary structure of the N proteins of these viruses which determine the tertiary and quaternary structures of the nucleocapsids.…”