The transmembrane domain sequence affects the structure and function of the Newcastle disease virus fusion protein.

The role of specific sequences in the transmembrane (TM) domain of Newcastle disease virus (NDV) fusion (F) protein in the structure and function of this protein was assessed by replacing this domain with the F protein TM domains from two other paramyxoviruses, Sendai virus (SV) and measles virus (MV), or the TM domain of the unrelated glycoprotein (G) of vesicular stomatitis virus (VSV). Mutant proteins with the SV or MV F protein TM domains were expressed, transported to cell surfaces, and proteolytically cleaved at levels comparable to that of the wild-type protein, while mutant proteins with the VSV G protein TM domain were less efficiently expressed on cell surfaces and proteolytically cleaved. All mutant proteins were defective in all steps of membrane fusion, including hemifusion. In contrast to the wild-type protein, the mutant proteins did not form detectable complexes with the NDV hemagglutinin-neuraminidase (HN) protein. As determined by binding of conformation-sensitive antibodies, the conformations of the ectodomains of the mutant proteins were altered. These results show that the specific sequence of the TM domain of the NDV F protein is important for the conformation of the preactivation form of the ectodomain, the interactions of the protein with HN protein, and fusion activity.

Assembly and immunological properties of Newcastle disease virus-like particles containing the respiratory syncytial virus F and G proteins.

Human respiratory syncytial virus (RSV) is a serious respiratory pathogen in infants and young children as well as elderly and immunocompromised populations. However, no RSV vaccines are available. We have explored the potential of virus-like particles (VLPs) as an RSV vaccine candidate. VLPs composed entirely of RSV proteins were produced at levels inadequate for their preparation as immunogens. However, VLPs composed of the Newcastle disease virus (NDV) nucleocapsid and membrane proteins and chimera proteins containing the ectodomains of RSV F and G proteins fused to the transmembrane and cytoplasmic domains of NDV F and HN proteins, respectively, were quantitatively prepared from avian cells. Immunization of mice with these VLPs, without adjuvant, stimulated robust, anti-RSV F and G protein antibody responses. IgG2a/IgG1 ratios were very high, suggesting predominantly T(H)1 responses. In contrast to infectious RSV immunization, neutralization antibody titers were robust and stable for 4 months. Immunization with a single dose of VLPs resulted in the complete protection of mice from RSV replication in lungs. Upon RSV intranasal challenge of VLP-immunized mice, no enhanced lung pathology was observed, in contrast to the pathology observed in mice immunized with formalin-inactivated RSV. These results suggest that these VLPs are effective RSV vaccines in mice, in contrast to other nonreplicating RSV vaccine candidates.

Assembly and biological and immunological properties of Newcastle disease virus-like particles.

Virus-like particles (VLPs) released from avian cells expressing the Newcastle disease virus (NDV) strain AV proteins NP, M, HN (hemagglutinin-neuraminidase), and F were characterized. The VLP-associated HN and F glycoproteins directed the attachment of VLPs to cell surfaces and fusion of VLP membranes with red blood cell membranes, indicating that they were assembled into VLPs in an authentic conformation. These particles were quantitatively prepared and used as an immunogen, without adjuvant, in BALB/c mice. The resulting immune responses, detected by enzyme-linked immunosorbent assay (ELISA), virus neutralization, and intracellular cytokine staining, were comparable to the responses to equivalent amounts of inactivated NDV vaccine virus. HN and F proteins from another strain of NDV, strain B1, could be incorporated into these VLPs. Foreign peptides were incorporated into these VLPs when fused to the NP or HN protein. The ectodomain of a foreign glycoprotein, the Nipah virus G protein, fused to the NDV HN protein cytoplasmic and transmembrane domains was incorporated into ND VLPs. Thus, ND VLPs are a potential NDV vaccine candidate. They may also serve as a platform to construct vaccines for other pathogens.

Interacting domains of the HN and F proteins of newcastle disease virus.

The activation of most paramyxovirus fusion proteins (F proteins) requires not only cleavage of F(0) to F(1) and F(2) but also coexpression of the homologous attachment protein, hemagglutinin-neuraminidase (HN) or hemagglutinin (H). The type specificity requirement for HN or H protein coexpression strongly suggests that an interaction between HN and F proteins is required for fusion, and studies of chimeric HN proteins have implicated the membrane-proximal ectodomain in this interaction. Using biotin-labeled peptides with sequences of the Newcastle disease virus (NDV) F protein heptad repeat 2 (HR2) domain, we detected a specific interaction with amino acids 124 to 152 from the NDV HN protein. Biotin-labeled HR2 peptides bound to glutathione S-transferase (GST) fusion proteins containing these HN protein sequences but not to GST or to GST containing HN protein sequences corresponding to amino acids 49 to 118. To verify the functional significance of the interaction, two point mutations in the HN protein gene, I133L and L140A, were made individually by site-specific mutagenesis to produce two mutant proteins. These mutations inhibited the fusion promotion activities of the proteins without significantly affecting their surface expression, attachment activities, or neuraminidase activities. Furthermore, these changes in the sequence of amino acids 124 to 152 in the GST-HN fusion protein that bound HR2 peptides affected the binding of the peptides. These results are consistent with the hypothesis that HN protein binds to the F protein HR2 domain, an interaction important for the fusion promotion activity of the HN protein.