Respiratory syncytial virus (RSV) is the leading cause of serious respiratory infections in children as

Human respiratory syncytial virus (RSV) is a serious respiratory pathogen in infants and

Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host's cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly.

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Membrane lipid raft domains are thought to be sites of assembly for many enveloped viruses. The roles of both classical lipid rafts and lipid rafts associated with the membrane cytoskeleton in the assembly of Newcastle disease virus (NDV) were investigated. The lipid raft-associated proteins caveolin-1, flotillin-2, and actin were incorporated into virions, while the non-lipid raft-associated transferrin receptor was excluded. Kinetic analyses of the distribution of viral proteins in lipid rafts, as defined by detergent-resistant membranes (DRMs), in non-lipid raft membranes, and in virions showed an accumulation of HN, F, and NP viral proteins in lipid rafts early after synthesis. Subsequently, these proteins exited the DRMs and were recovered quantitatively in purified virions, while levels of these proteins in detergent-soluble cell fractions remained relatively constant. Cholesterol depletion of infected cells drastically altered the association of viral proteins with DRMs and resulted in an enhanced release of virus particles with reduced infectivity. Decreased infectivity was not due to effects on subsequent virus entry, since the extraction of cholesterol from intact virus did not significantly reduce infectivity. Particles released from cholesterol-depleted cells had very heterogeneous densities and altered ratios of NP and glycoproteins, demonstrating structural abnormalities which potentially contributed to their lowered infectivity. Taken together, these results indicate that lipid rafts, including cytoskeletonassociated lipid rafts, are sites of NDV assembly and that these domains are important for ordered assembly and release of infectious Newcastle disease virus particles.

Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought to undergo dramatic conformational changes upon activation. How the F protein accomplishes extensive conformational rearrangements is unclear. Since several viral fusion proteins undergo disulfide bond rearrangement during entry, we asked if similar rearrangements occur in NDV proteins during entry. We found that inhibitors of cell surface thiol/disulfide isomerase activity-55-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin, and anti-protein disulfide isomerase antibody-inhibited cell-cell fusion and virus entry but had no effect on cell viability, glycoprotein surface expression, or HN protein attachment or neuraminidase activities. These inhibitors altered the conformation of surface-expressed F protein, as detected by conformation-sensitive antibodies. Using biotin maleimide (MPB), a reagent that binds to free thiols, free thiols were detected on surface-expressed F protein, but not HN protein. The inhibitors DTNB and bacitracin blocked the detection of these free thiols. Furthermore, MPB binding inhibited cell-cell fusion. Taken together, our results suggest that one or several disulfide bonds in cell surface F protein are reduced by the protein disulfide isomerase family of isomerases and that F protein exists as a mixture of oxidized and reduced forms. In the presence of HN protein, only the reduced form may proceed to refold into additional intermediates, leading to the fusion of membranes.Cell entry by enveloped viruses requires fusion of the viral envelope with host cell membranes, a step in infection that is mediated by viral fusion proteins. Viral fusion proteins have been categorized into two and possibly three groups based on their structures and mechanisms for mediating fusion (22,58,70). Class 1 fusion proteins, which fold as trimers, include paramyxovirus F proteins, influenza virus hemagglutinin (HA) proteins, and retrovirus envelope (Env) proteins. These proteins, synthesized as inactive precursors, are cleaved into two subunits, F 1 and F 2 in the case of paramyxoviruses. The sequence at the new amino terminus generated by this cleavage is the fusion peptide (FP), which inserts into the target membrane upon fusion activation (reviewed in references 12, 23, 49, and 70). These proteins also contain two important heptad repeat (HR) domains. The F protein HR domains are located just carboxyl terminal to the fusion peptide (HR1) and adjacent to the transmembrane (TM) domain (HR2). The HR1 and HR2 peptides have a strong affinity and form a very stable six-stranded coiled coil, with HR1 forming an interior trimer and HR2 binding in the grooves of the trimer in an antiparallel orientation (3). Inhibition of fusion with either the HR1 or HR2 peptide su...

To explore the role of N-linked carbohydrate in the activities of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV), the six glycosylation addition sites (G1-G6) in the HN sequence of the AV strain of NDV were mutated. Migration of mutant protein on polyacrylamide gels as well as endoglycosidase H digestion of mutant protein showed that four of the addition sites (G1, G2, G3, and G4 at amino acids 119, 341, 433, and 481, respectively) are used while two (G5 and G6 at amino acids 508 and 538, respectively) are not used. Proteins expressed from single and all possible combinations of double and triple mutant DNAs as well as the unglycosylated molecule were characterized for the presence of specific antigenic sites, formation of disulfide-linked dimers, stability, transport to the cell surface, and biological activity. Results showed that glycosylation at positions G1 and G2 play little detectable role in the folding, stability, or transport of the molecule either singly or in combination with other mutations. Mutation of these sequons, however, significantly increased the cell attachment and fusion promotion activities of the protein, particularly in combination. Mutation of the glycosylation site at G4 either singly or in combination with other site-eliminating mutants inhibited the formation of the mature protein, while a mutation eliminating the addition site at G3 had a slight effect on the efficiency of folding, particularly in combination with mutation of the site G4. When normalized to surface expression, elimination of carbohydrate addition sites at G3 and G4 singly or in combination with other mutations depressed in particular the neuraminidase activity of the protein but not the fusion promotion activity. Thus two oligosaccharides do not have a detectable role in maturation but do modulate the biological activities of the protein. The other two oligosaccharides influence both folding and activity of the protein.