The Use of a Quantitative Fusion Assay to Evaluate HN–Receptor Interaction for Human Parainfluenza Virus Type 3

Abstract

Sialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity. The putative active sites are in the extracellular domain of this type II integral membrane protein. However, HN is not available in crystalline form; the exact locations of these sites, and the structural requirements for binding to the cellular receptor, which has not yet been isolated, are unknown. Nor have small molecular synthetic inhibitors of attachment or fusion that would provide insight into these processes been identified. The strategy in the present study was to develop an assay system that would provide a measure of a specific step in the viral cycle-functional interaction between viral glycoproteins and the cell during attachment and fusion-and serve to screen a variety of substances for inhibitory potential. The assay is based on our previous finding that CV-1 cells persistently infected (p.i.) with HPF3 do not fuse with one another but that the addition of uninfected CV-1 cells, supplying the critical sialic acid containing receptor molecules that bind HN, results in rapid fusion. In the present assay two HeLa cell types were used: we persistently infected HeLa-LTR-betagal cells, assessed their fusion with uninfected HeLa-tat cells, and then quantitated the beta-galactosidase (betagal) produced as a result of this fusion. The analog alpha-2-S-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2SMe) interfered with fusion, decreasing betagal production by 84% at 50 mM and by 24% at 25 mM. In beginning to extend our studies to different types of molecules, we tested an unsaturated derivative of sialic acid, 2,3-dehydro-2-deoxy-n-acetyl neuraminic acid (DANA), which is known to inhibit influenza neuraminidase by virtue of being a transition-state analog. We found that 10 mM DANA inhibited neuraminidase activity in HPF3 viral preparations. More significantly, this compound was active in our assay of HN-receptor interaction; 10 mM DANA completely blocked fusion and betagal production, and hemadsorption inhibition by DANA suggested that DANA blocks attachment. In plaque reduction assays performed with the compounds, the active analog alpha-Neu5thioAc2SMe reduced plaque formation by 50% at a 50 mM concentration; DANA caused a 90% inhibition in the plaque reduction assay at a concentration of 25 mM. Our results indicate that specific sialic acid analogs that mimic the cellular receptor determinant of HPF3 can block virus cell interaction and that an unsaturated n-acetyl-neuraminic acid derivative with affinity to the HN site responsible for neuraminidase activity also interferes with HN-receptor binding. Strategies suggested by these findings are now being pursu...

Mutations in Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Causing Increased Receptor Binding Activity and Resistance to the Transition State Sialic Acid Analog 4-GU-DANA (Zanamivir)

“…293T (human kidney epithelial) cells were grown in Dulbecco's modification of Eagle's medium (DMEM; Mediatech Cellgro) supplemented with 10% fetal bovine serum and antibiotics. WT virus stock was prepared by infecting CV-1 cell monolayers at a multiplicity of infection of 0.1 as described previously (15). Variant virus stocks were made in CV-1 cell monolayers from virus that was plaque purified three times.…”

“…Greengard et al and Levin Perlman et al showed that 4-GU-DANA and related sialic acid analogs inhibit not only the NA activity but also the receptor binding activity of HN and thus its ability to promote attachment and fusion (6,15). The demonstration that a single compound can block both HPF3 HNЈs receptor binding and receptor cleaving activities was consistent with subsequently presented crystallographic studies on the HN of Newcastle disease virus (NDV), which suggested that a single active site provides both the receptor binding and hydrolytic functions (4).…”

Premature Activation of the Paramyxovirus Fusion Protein before Target Cell Attachment with Corruption of the Viral Fusion Machinery

“…Plaque Reduction Assay-The effect of the compounds on plaque number was assessed as described (34,35). Briefly, CV-1 cell monolayers were infected with 100 plaque-forming units of WT HPIV3 in the presence of serial dilutions of the indicated compounds.…”

Kinetic Dependence of Paramyxovirus Entry Inhibition

“…For this experiment, in addition to wildtype (wt) HPIV3 (16) and wt HeV and NiV (1), we used an HPIV3 variant bearing an HN with the N551D mutation (designated HN-N551D), known to be more efficient at triggering HPIV3 F (19,22). The effects of peptides on plaque numbers were assessed by a plaque reduction test (12) for HPIV3 and by a viral detection assay (1) for HeV/NiV. For the experiment with results presented in Fig.…”