Identification of cellular proteins that interact with Newcastle Disease Virus and human Respiratory Syncytial Virus by a two-dimensional virus overlay protein binding assay (VOPBA).

Although it is well documented that the initial attachment receptors for Newcastle Disease Virus (NDV) and Respiratory Syncytial Virus (RSV) are sialic acid-containing molecules and glycosaminoglycans respectively, the exact nature of the receptors for both viruses remains to be deciphered. Moreover, additional molecules at the host cell surface might be involved in the entry mechanism. With the aim of identifying the cellular proteins that interact with NDV and RSV at the cell surface, we performed a virus overlay protein binding assay (VOPBA). Cell membrane lysates were separated by two dimensional (2D) gel electrophoresis and electrotransferred to PVDF membranes, after which they were probed with high viral concentrations. NDV interacted with a Protein Disulfide Isomerase from chicken fibroblasts. In the case of RSV, we detected 15 reactive spots, which were identified as six different proteins, of which nucleolin was outstanding. We discuss the possible role of PDI and nucleolin in NDV and RSV entry, respectively.

Cholesterol dependence of Newcastle Disease Virus entry.

Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane in Newcastle Disease Virus (NDV) entry. We used methyl-ß-cyclodextrin (MßCD) to deplete cellular cholesterol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral binding, fusion and infectivity. MßCD had no effect on the expression of sialic acid containing molecule expression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cholesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-resistant membrane microdomains (DRMs) at 4°C and then shifted to detergent-soluble fractions at 37°C. These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells.

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.

Newcastle disease virus may enter cells by caveolae-mediated endocytosis.

The entry into cells of Newcastle disease virus (NDV), a prototype member of the paramyxoviruses, is believed to occur by direct fusion at the plasma membrane through a pH-independent mechanism. In addition, NDV may enter host cells by an endocytic pathway. Treatment of cells with drugs that block caveolae-dependent endocytosis reduced NDV fusion and infectivity, the degree of inhibition being dependent on virus concentration. The inhibitory effect was reduced greatly when drugs were added after virus adsorption. Cells treated with methyl beta-cyclodextrin, a drug that sequesters cholesterol from membranes, reduced the extent of fusion, infectivity and virus-cell binding; this indicates that cholesterol plays a role in NDV entry. Double-labelling immunofluorescence assays performed with anti-NDV monoclonal antibodies and antibodies against the early endosome marker EEA1 revealed the localization of the virus in these intracellular structures. Using fluorescence microscopy, it was found that cell-cell fusion was enhanced at low pH. It is concluded that NDV may infect cells through a caveolae-dependent endocytic pathway, suggesting that this pathway could be an alternative route for virus entry into cells.