Loss of the N-linked glycan at residue 173 of human parainfluenza virus type 1 hemagglutinin-neuraminidase exposes a second receptor-binding site

Abstract

BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.

FIG. 1.

Structure of the lead compound and BCX 2798. (A) Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetylneuraminic acid); (B) BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid).

FIG. 2.

Elution of the parent and the N173S mutant virus from RBCs. In HA assays, both viruses were diluted to provide eight HAU with 0.5% chicken (upper rows) and turkey (lower rows) RBCs after 1 h at 4°C. Then, the plate was shifted to 37°C, and elution of viruses from RBCs was recorded after 0.5, 3, and 18 h of incubation.

FIG. 3.

Electron micrographs of LLC-MK₂ cells infected with the parent (A) and the N173S mutant virus (B). Cells in 80-mm-diameter dishes were infected with viruses at a high MOI of 50 and processed for subsequent transmission electron microscopy 18 h after infection. Note the few virions (arrows) associated with the cell membrane, which occurs during normal release (A), and a large number of membrane-associated virions in mutant-infected cells (B). Bar = 1 μm.

FIG. 4.

The relative locations of the catalytic and second receptor-binding sites (A) and possible conservation of a second receptor-binding site in hPIV-1 HN (B). (A) Shown are the superimposed dimers of NDV HN (green; PDB code 1usr) and hPIV-3 HN (cyan; PDB code 1v2i) with the thiosialoside (yellow bonds) that identified the second receptor-binding site in the NDV HN structure and the relative locations of the catalytic and second receptor-binding sites. (B) Focus on the second receptor-binding site at the dimer interface. The residue numbering is for hPIV-1/NDV HN. Where hPIV-1 HN has a different amino acid relative to hPIV-3 HN, the structure of hPIV-3 HN has been mutated to show the possible location of the hPIV-1 HN residue. This was the case for Asn173 (Lys in hPIV-3), Arg520 (Ala in hPIV-3), and Leu521 (Ile in hPIV-3).

FIG. 5.

Electrophoretic mobility of HN for the parent and the N173S mutant virus. Concentrated purified [³⁵S]methionine-cysteine-labeled viruses (lanes: 1, parent; 2, N173S), glycoproteins (lanes: 3, parent; 4, N173S), and PNGase F-treated glycoproteins (lanes: 5, parent; 6, N173S) were analyzed under reducing conditions with 10% SDS-PAGE. Separation of viral glycoproteins is described in Materials and Methods. PNGase F-treated HN and F are indicated by asterisks. Molecular mass standards in kilodaltons are indicated at right. Below is shown an enlargement of lanes 3 to 6 to show more clearly the mobilities of HN for the parent and the N173S mutant viruses.

FIG. 6.

Growth kinetics of the parent and the N173S mutant virus. (A) LLC-MK₂ cells were infected with the parent (•) and the N173S mutant virus (▴) at an MOI of 0.001. Virus titers were determined for culture supernatant fluids by plaque assays at the times indicated on the x axis. Values presented are the means of results from three independent experiments, plotted with error bars indicating the standard errors of the means (SEM). (B) The 129x1/SvJ mice were infected with the parent (•) and the N173S mutant virus (▴) at a dose of 10³ PFU per mouse. Lungs were collected at 1, 3, 5, and 7 days after infection, and the lung virus titers were determined by plaque assays. Values are the mean titers of virus from three animals, plotted with error bars indicating the SEM. An asterisk indicates a significant difference in titers for the N173S mutant virus at that time point compared to the parent virus (P < 0.05).

FIG. 7.

Effect of pretreatment with BCX 2798 on survival (A) and weight loss (B) of mice infected with a lethal dose of the N173S mutant virus. BCX 2798 (10 mg/kg per day; ▴) was intranasally administered to mice (10 per group) for 5 days; administration began 4 h before infection with one MLD₉₀ of the N173S mutant virus. Infected control mice were treated only with PBS (•). Mean values for weight loss are plotted with error bars indicating the standard errors of the means (SEM). An asterisk indicates a significant difference in survival or weight change for the drug-treated group compared to the untreated control group (P < 0.05).