Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G

Parainfluenza virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-Å resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the "rule of six" that governs the genome packaging of the Paramyxovirinae subfamily of viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.nucleoprotein | nucleocapsid ring | atomic structure | paramyxovirus | ribonucleoprotein T he Paramyxoviridae family of nonsegmented negative-strand RNA viruses includes many serious pathogens of humans and animals including mumps virus, measles virus, parainfluenza viruses 1-5, Nipah virus, Hendra virus, and Newcastle disease virus, all of which are in the Paramyxovirinae subfamily, and respiratory syncytial virus (RSV) and human metapneumovirus, which are in the subfamily Pneumoviridae. The Paramyxoviridae belong in the order Mononegavirales of membrane enveloped negative-strand RNA viruses (NSV), an order that includes the Rhabdoviridae (rabies virus and vesicular stomatitis virus; VSV), Orthomyxoviridae (influenza virus) and Filoviridae (Ebola virus) (1). Parainfluenza virus 5 (PIV5) is a paramyxovirus that was initially isolated from rhesus monkey-kidney cell cultures (2) and although PIV5 is not known to cause human disease (3), it is used as a prototype in the study of paramyxoviruses.PIV5 has a nonsegmented single-stranded RNA genome of negative polarity of 15,246 nucleotides that encodes eight proteins (1): Three integral membrane proteins, fusion protein F and attachment protein hemagglutinin-neuraminidase (HN) and a small hydrophobic protein (SH). Inside the virion envelope lies a helical nucleocapsid core containing the R...

Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.