Neuraminidase-deficient Sendai virus HN mutants provide protection from homologous superinfection

Abstract

Binding of hemagglutinin-neuraminidase proteins (HN) to sialylated receptors initiates the infection process of several paramyxoviruses, whereas later in the viral life cycle, the neuramindase (NA) activity of newly synthesized HN destroys all receptors. Prior to NA action, expressed HN has to bind the receptor. To evaluate this HN-receptor complex with respect to receptor inactivation, three temperature-sensitive Sendai virus HN mutants carrying amino acid exchanges at positions 262, 264 and/or 461 were created that uncoupled NA activity from receptor binding at 39 degrees Celsius. Interestingly, at elevated temperature, when there is no detectable neuramindase activity, all infected cells are protected against homologous superinfection. Mutated HN protein on the cell surface is mainly bound to sialylated cell-surface components but can be released by treatment with NA. Thus, continuous binding to HN already inactivates the receptors quantitatively. Furthermore, mutant HN bound to receptors is prevented from being incorporated into virus particles in the absence of NA. It is shown here for the first time that during paramyxoviral infection, quantitative receptor inactivation already occurs due to binding of receptors to expressed HN protein without involvement of NA and is independent of NA activity of viral progeny. NA subsequently functions in the release of HN from the complex, coupled with desialysation of receptors. These findings could have implications for further antiviral drug development.

Fig. 1

Characterization of mutant HN proteins. LLC-MK₂ cells were infected with wt SeV or HN mutants at an m.o.i. of ≥4. a For detection of HN protein in virus particles, cells were cultured at 33°C for 47 h. Virus particles were concentrated by ultracentrifugation of cell supernatants. Virus pellets were solubilized in RIPA buffer, and the protein concentration was determined. Virus proteins (1 μg per lane) were separated by SDS–PAGE. Immunodetection was performed with an HN-specific (VII3c-4F) monoclonal antibody. For detection of cytoplasmically expressed viral proteins, cells were cultured at 33 or 39°C for 46 h. Cell extracts were prepared, and proteins (5 μg per lane) were separated by SDS–PAGE. Immunodetection was performed with an HN-specific (VII3c-4F) monoclonal antibody. b Neuraminidase assays were carried out 24 h after infection with X-Neu5Ac as substrate [7]. c For detection of mutant HN on the cell surface by immunofluorescence, binding of anti-HN S2 antibodies [30] was performed 24 h after infection, followed by fixation with methanol. Anti-mouse Alexa Fluor 568-conjugated antibody was used as a secondary antibody

Fig. 2

Optimization of superinfection with homologous SeV. Cells were infected with wt SeV at 33°C (m.o.i. > 4) and challenged after 5 h a or 24 h b with SeV P-eGFP (m.o.i. > 4), and eGFP-specific fluorescence was detected after 48 h. c Cells infected with wt SeV at 33°C (m.o.i. > 4) were tested for infection by the FLAT method

Fig. 3

Protection of SeV HN-mutant-infected cells against superinfection. a LLC-MK₂ cells were infected with one of the three HN mutants (m.o.i. ≥ 6) and subjected to a hemadsorption test 24 h p.i. (HAd). Parallel cultures were challenged 24 h p.i. with SeV P-eGFP (m.o.i. ≥ 4) and examined for eGFP fluorescence 47 h after challenge (SeV-eGFP superinfection). HN 262+264-infected cells (39°C) were treated additionally for 30 min with NA prior to challenge (262+264+NA); LLC-MK₂ cells infected only with SeV P-eGFP (SeV-eGFP infection); b LLC-MK₂ cells were infected with mutant 262+461 and challenged 24 h later with MeV P-DsRed (m.o.i. ≥ 2) Ds-Red fluorescence was determined 70 h after challenge (MeV-DsRed superinfection). Distinct cytopathic effect (CPE) of cells by heterologous superinfection is shown. Cells were infected with MeV P-DsRed only (MeV-DsRed infection)

Fig. 4

Characterization of receptor binding to mutant HN. a Cells infected with HN 262+264+461, HN 262+461 or HN 262+264 and incubated at 39°C for 24 h were treated with V.C. NA or not treated and tested for erythrocyte binding at 4°C (HAd). b, c Cells infected with wt SeV or HN mutants at an m.o.i. of ≤40, leading to 100% HAd activity at 33°C. b After incubation for 24 h at 33 and 39°C, a 0.5% suspension of human erythrocytes was added for 1 h at 4°C. Unbound erythrocytes were removed, and the percentage of cells covered with erythrocytes was determined. c Infected cells were treated prior to the HAd test with 10 mU V.C. NA for 1 h, and HAd activity was determined after removing the enzyme by extensive cell washing

Fig. 5

HN protein content in mutant virus progeny generated at 39°C. Virus production was performed essentially as described in Fig. 1a. Protein separation by SDS–PAGE was performed with quantities corresponding to 1/60 of the respective total virus protein. Immunodetection was performed with HN-specific (VII3c-4F) mAB and monospecific anti-N serum. Signals of diluted and undiluted N protein served as an internal control and for quantification. X-ray films were exposed to antibody-treated membranes for about 4 s