N-linked glycan at residue 523 of human parainfluenza virus type 3 hemagglutinin-neuraminidase masks a second receptor-binding site

Abstract

The hemagglutinin-neuraminidase (HN) glycoprotein plays a critical role in parainfluenza virus replication. We recently found that in addition to the catalytic binding site, HN of human parainfluenza virus type 1 (hPIV-1) may have a second receptor-binding site covered by an N-linked glycan at residue 173, which is near the region of the second receptor-binding site identified in Newcastle disease virus (NDV) HN (I. A. Alymova, G. Taylor, V. P. Mishin, M. Watanabe, K. G. Murti, K. Boyd, P. Chand, Y. S. Babu, and A. Portner, J. Virol. 82:8400-8410, 2008). Sequence analysis and superposition of the NDV and hPIV-3 HN dimer structures revealed that, similar to what was seen in hPIV-1, the N-linked glycan at residue 523 on hPIV-3 HN may cover a second receptor-binding site. Removal of this N-linked glycosylation site by an Asn-to-Asp substitution at residue 523 (N523D) changed the spectrum of the mutant virus's receptor specificity, delayed its elution from both turkey and chicken red blood cells, reduced mutant sensitivity (by about half) to the selective HN inhibitor BCX 2855 in hemagglutination inhibition tests, and slowed its growth in LLC-MK(2) cells. The neuraminidase activity of the mutant and its sensitivity to BCX 2855 in neuraminidase inhibition assays did not change, indicating that the mutation did not affect the virus's catalytic-binding site and that all observed effects were caused by the exposure of the purported second receptor-binding site. Our data are consistent with the idea that, similar to the case for hPIV-1, the N-linked glycan shields a second receptor-binding site on hPIV-3 HN.

FIG. 1.

Superposition of the HN dimer structures of NDV (green) and hPIV-3 (magenta). (A) The relative locations of the catalytic and second sites. The area highlighted in gray is shown in panels B and C. (B) Location of the second site with thiosialoside (shown in yellow) bound in the NDV HN crystal structure. The NAG carbohydrate N-linked to Asn523 is shown; residues that form the conserved hydrophobic pocket accommodating the methyl of the SA acetamido group are drawn without labeling. (C) The same region as in panel B, rotated 90° around a horizontal axis. The hydrogen bonds made between Arg516 and the carboxylate group of SA in NDV HN are shown as black dotted lines.

FIG. 2.

SDS-PAGE analysis of the purified parent and mutant viruses' proteins. (A) Viruses were grown in LLC-MK₂ cells, concentrated, and purified through a 35% sucrose cushion. Two micrograms of purified parent (lane 1), N523D (lane 2), or H552Q (lane 3) virus was loaded on a 10% Bis-Tris gel. Gel was stained with Page Blue protein stain (Fermentas, Inc., Glen Burnie, MD) to visualize proteins. (B) LLC-MK₂ cell monolayers were infected with viruses. At 20 h after infection, the cells were metabolically radiolabeled with [³⁵S]methionine-cysteine (500 μCi per 10⁷ cells) (Perkin-Elmer, Boston, MA). Cells were then lysed and subjected to immunoprecipitation with anti-hPIV-3 HN monoclonal antibody. Half of the immunoprecipitate was treated with PNGase F (New England Biolabs, Beverly, MA) to remove N-glycosylated moieties. The untreated HNs of the parent (lane 1) or N523D (lane 2) and PNGase F-treated HNs (indicated by asterisks) of the parent (lane 3) or N523D (lane 4) are shown. Molecular mass standards (in kilodaltons) are shown to the left of the gel.

FIG. 3.

Neuraminidase activities of the parent and mutant viruses at different pH levels. Purified concentrated parent (•), N523D (▪) mutant, and H552Q (▴) mutant viruses were analyzed for optimum pH for NA activity by using a modified fluorometric assay (1) with 200 μM MUNANA in citrate-phosphate buffer (McIlvaine's buffer system) at a pH range of 3.0 to 8.0. One microgram of total viral proteins was used for the reaction. HN protein loading was determined by parallel SDS-PAGE analysis of viral proteins. Specific signal is expressed as nanomoles of product formed per milligram of HN after 1 h of incubation at 37°C.

FIG. 4.

Growth kinetics of the parent and mutant viruses. LLC-MK₂ cells in 24-well plates were infected with the parent (•), N523D (▪), and H552Q (▴) viruses at an MOI of 0.0001. After 1 h of incubation at room temperature, the cells were washed with PBS and DMEM supplemented with 0.3% BSA, and 1 μg/ml acetylated trypsin was added to the wells. 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 at least three independent experiments, plotted with error bars indicating the standard errors of the means (SEM). *, P < 0.05 compared with results for the parent virus by Student's t test.