[PubMed] [Google Scholar]. viral membrane (lipid) contains two glycoproteins, an attachment protein and a F protein, and a nonglycosylated M protein. The attachment protein of Newcastle disease virus, an avian paramyxovirus, is the HN protein. The core of the virus contains the genomic RNA and associated nucleocapsid protein in a helical RNP complex, which also contains the L viral polymerase and a P protein. (B) Generation of ND VLPs, which are very efficiently released from cells expressing the viral NP, M, HN and F proteins after a transient transfection of cells with pCAGGS vectors [58,59] containing cDNAs encoding each of these viral proteins. These cDNAs were derived from the AustraliaCVictoria strain of Newcastle disease virus. The cleavage site GNE 2861 of the F protein was mutated to mimic the site in the F protein from the vaccine strain of Newcastle PMCH disease virus, resulting in an uncleaved F protein. Cells may be mammalian (COS7 or 293T) or avian cells (ELL-0), although avian cells release Newcastle disease VLPs more efficiently. In avian cells, 84% of total expressed M protein is released as particles [45]. F: Fusion; HN: HemagglutininCneuraminidase; L: Large; M: Membrane; ND: Newcastle disease; NP: Nucleocapsid protein; P: Phosphoprotein; VLP: Virus-like particle. It has been reported that many different paramyxovirus VLPs can be produced upon expression of the M protein or M protein with various combinations of the glycoproteins and NP [32,38C43]. Indeed, as depicted in Figure 1B, cells expressing the Newcastle disease virus HN, F, NP and M proteins release particles that structurally and functionally resemble virus particles [44,45]. What distinguishes Newcastle disease VLPs from other paramyxovirus VLPs and, indeed, from many other types of VLPs, is their striking efficiency of release [45]. The reported efficiency of release of other paramyxovirus VLPs, as measured by the efficiency of M protein released, ranged from 10 to GNE 2861 50% [32,38C43], while Newcastle disease VLP release is 84% [44,45]. As a result, quantitative amounts of Newcastle disease VLPs are relatively easy to prepare even from transiently transfected cells [46]. They can be purified using protocols utilized for virus purification and the purified VLPs have minimal cell protein contamination (Figure 2) [44,46]. Furthermore, the ratios of viral proteins were similar to those in virus particles (Figure 2) [44]. Open in a separate window Figure 2 Protein content of purified Newcastle disease virus-like particles compared with virusVLPs harvested from the supernatant of cells expressing the Newcastle disease NP, M, HN and F proteins are purified by sequential sedimentation and flotation in protocols similar to those used for virus purification, as described in Pantua [45] and McGinnes [44]. Proteins in the purified VLPs, separated by electrophoresis on polyacrylamide gels GNE 2861 and detected by silver stain, are compared with proteins in equivalent amounts of purified, avirulent vaccine virus grown in embryonated chicken eggs. Proteins shown in lanes 1 and 2 were separated on polyacrylamide gels in the absence of reducing agent (?ME) in order to resolve the disulfide linked F1?F2 heterodimer of the cleaved form of F protein present in egg-grown virus and the disulfide-linked HN homodimer of the correctly folded HN protein of the AV strain of Newcastle disease virus. The HN protein of the vaccine strain is not disulfide linked. ME: -mercaptoethanol; F: Fusion; F1: Cleaved form of the F protein; Fnr: Nonreduced form of F protein (F1+F2); HN: HemagglutininCneuraminidase; HN dimer: Disulfide-linked HN protein; M: Membrane protein; NP: Nucleocapsid protein; VLP: Virus-like particle. Adapted with permission from [44] ? American Society for Microbiology. Glycoproteins assembled into Newcastle disease VLPs are likely in their authentic conformation. The most stringent test of the conformation of a glycoprotein is the preservation of the biological activities of virion-associated glycoproteins. Indeed, HN protein associated with Newcastle disease VLPs mediates cell binding and possesses neuraminidase activity [44]. The HN protein associated with these particles also directs hemagglutination with titers comparable to equivalent amounts of virus [44]. F protein in these particles can direct the fusion of the VLP membrane with red blood cell membranes [44]. The effectiveness of Newcastle disease VLPs as an immunogen was demonstrated in a murine model and compared with responses stimulated by immunization with comparable amounts of a UV-inactivated, vaccine strain of Newcastle disease virus [44]. Levels of soluble antibodies, characterized by ELISA and by neutralizing.