Mammalian sialidase Neu3 overexpression in Cos-7 cells : effects on Newcastle disease virus-cell interaction

Newcastle Disease Virus (NDV) is an avian single-stranded RNA enveloped virus that belongs to the Paramyxoviridae family. The interaction of enveloped viruses with cell surface receptors is the first step in the viral cycle and an important determinant of viral host range. Although it is estabilished that NDV, such as the most of Paramyxovirus, infects cells binding sialic acid-containing glycoconjugates, the exact nature of these receptors is not well known. It is thought that both sialoglycoproteins and gangliosides may serve as viral receptors. Infection by NDV requires the combined action of its two glycoproteins: the hemagglutinin-neuraminidase (HN) protein and the fusion (F) protein. HN protein has three main biological activities: hemagglutinin, sialidase and fusion promotion. The hemagglutinin activity allows the binding of the virus to the host cell surface by recognizing specific cellular receptors. The sialidase activity facilitates the viral spread of newly born virions, while the fusion promotion activity induces F protein to begin the fusion between the virus and host membranes. After fusion, viral nucleocapsid is delivered into the cytoplasm and here the replication cycle takes place. After synthesis, once correctly folded and assembled, HN and F are transported to the host plasma membrane. F protein is synthetized as a precursor, Fo, and it becomes fusogenic only after cleavage into disulfide-linked F1 and F2 polypeptide before catching up the host membrane (Iwata et al., 1994). The NDV clone used in our experiments is “Clone 30” that belongs to the lentogenic group. The lentogenic viruses’ F protein can be cleaved only by tripsin-like enzyme. In this work we investigated the effects caused by mammalian sialidase Neu3 overexpression in COS7 cells in terms of NDV infection, NDV-cell interaction and its entry. First, we studied if the overexpression of Neu3 sialidase could interfere with the virus entry. To this end, we performed immunofluorescence assays in collaboration with the Department of Biochemistry and Molecular Biology of the University of Salamanca, and we observed that NDV infection was reduced (19%) in Neu3 overexpressing COS7 cells compared to controls. Because Neu3 sialidase is mainly located in regions of the membrane named “lipid rafts” (Simons et al., 1997), we studied the possible involvement of these domains in the infection process by Western Blot analysis and HN activity assays. Mock and Neu3 overexpressing COS7 infected with 1 moi of NDV at 4°C were collected after infection. Cell lysates were separated on an optiprep gradient. The 8 collected fractions were analyzed for sialidase activity toward Mu-NeuAc substrate and by western blot with antibodies a-caveolin 1 and a-NDV. We observed that NDV’s neuraminidase is concentrated in the fraction corresponding to that of lipid rafts. Gangliosides have been identified as key receptors for some of caveolae-dependent viruses, such as for the polyomaviruses SV40 and Py (Tsai et al., 2003). Since it has been reported that NDV interacts with several gangliosides bearing both the Neu5 Ac and N-glycolylneuraminic sialic acid forms (Suzuky et al., 1985) and in vitro with different monosialogangliosides, disialogangliosides and trisialogangliosides, carrying sialic acid residues attached to a terminal or internal galactose (Ferreira et al.,2004), we tried to delineate if one or more gangliosides may act as receptors. We made a metabolic labelling with [1-3H]Sphingosine with a 2h pulse followed by a 48h chase; at 24h of chase cells were infected with NDV at 1 or 10 moi at 37°C and collected after infection. Total lipids from lyophilized cells were extracted. [3H]Sphingolipids from aqueous and organic phases were separated by HPTLC. No differences could be detected between infected cells and controls in both aqueous and organic phases. It seems that NDV probably only hydrolize a very small number of sialo-glycosphingolipid to infect COS7 cells. Subsequently, we defined the glycosphingolipid pattern of COS7 cells at 24h post-infection, and we could observe a 50% decrease of GD1a accompained by a 46% increase of GM1 in infected cells compared to controls. This suggests that the newly syntetized HN specifically hydrolizes GD1a present on the host’s surface to allow the virus spread by budding. To be sure that newly syntetized HN hydrolized only GD1a present on the host cell surface, we labelled COS7 with [1-3H]Sphingosine with a 2h pulse followed by a 48h chase: at 24h of chase, we made a co-colture for 24h of the labeled cells with COS7 cells infected by NDV. Analyzing the lipids from aqueous and organic phases, we could observe that GD1a of neighboring cells wasn’t hydrolized by HN present on the infected cell’s surface. The present study demonstrates that:  The overexpression of Neu3 sialidase leads to a decrease in the ability of NDV to infect host cells.  NDV’s neuraminidase (HN) co-localizes with lipid rafts.  NDV’s neuraminidase acts preferentially on ganglioside GD1a of host cells. GD1a of neighboring cells is not hydrolized by HN present on the infected cell’s surface. Iwata et al.,(1994), J Virol. 68, 3200-3206 Simons et al., (1997) Nature 387, 569-572 Suzuky et al., (1985) JB 97,1189-1199 Ferreira et al., (2004) ICBCB 36, 2344-2356