A Cell Fusion-Inhibiting Monoclonal Antibody Binds to the Presumed Stalk Domain of the Human Parainfluenza Type 2 Virus Hemagglutinin-Neuraminidase Protein

Abstract

Previously, we obtained a neutralizing monoclonal antibody directed against the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (PIV2), which was able to prevent cell fusion without affecting the hemagglutinating and neuraminidase activities. In this study, four escape mutants of PIV2 have been obtained under pressure of the monoclonal antibody. Intriguingly, the HN protein of each mutant proved to have two amino acid substitutions, one of which is at 83Asn or 91Lys, and another one is at 150Leu, 160Ala, or 186Met. One mutant designated F13, which has substitutions at 83Asn and 186Met in the HN protein, could not cause cell fusion in HeLa cells despite its multiple replication, while the other mutants formed typical syncytial cells. The deduced amino acid sequence of F13 fusion (F) protein proved to be identical to that of wild-type F protein, and furthermore, protein expression analyses have revealed that the low-fusion phenotype of F13 was due to its mutated HN protein, whose antigenicity to the monoclonal antibody was abolished by the single mutation at 83Asn. These observations have suggested that the principal epitope for the monoclonal antibody resides in the presumed stalk domain of the HN protein, which may play an important role in promoting cell fusion.

Identification of regions on the fusion protein of human parainfluenza virus type 2 which are required for haemagglutinin-neuraminidase proteins to promote cell fusion.

“…Previous studies using a number of chimaeric HN proteins indicated that the presumed stalk domain on the HN protein of PIV-2 and Sendai virus might be involved in the type-specific interaction with homologous F proteins (Tanabayashi & Compans, 1996 ;Tsurudome et al, 1995). Besides the stalk domain, the transmembrane (TM) domain was also required for HN proteins of PIV-3 and NDV (Deng et al, 1995) to display their type-specificity.…”

“…A cDNA fragment encoding the HN or F protein of PIV-2 or SV-41 was inserted into the plasmid expression vector pcDL-SRα296 (SRα) as described previously (Takebe et al, 1988 ;Tsurudome et al, 1995). Similarly, a cDNA fragment encoding the HN protein of NDV (D26 strain) (Sato et al, 1987) or that encoding the F protein of NDV (Miyadera strain) (Toyoda et al, 1987) was inserted into plasmid SRα.…”

“…Introduction of a mutation-generating synthetic oligonucleotide into the target recombinant plasmid was performed as described previously (Tsurudome et al, 1995) with a Transformer Site-Directed Mutagenesis kit (Clontech). Since a ScaI site was present in plasmid SRα (in the ampicillin-resistance gene) but not in cDNAs for SV-41 F and PIV-2 F genes, elimination of this site was a useful marker for selection of mutant plasmids in our present study.…”

“…The amount of F proteins expressed on the cell surface was measured by flow cytometric analysis as described previously (Tabata et al, 1994 ;Yuasa et al, 1995) with some modifications. We have shown previously that an anti-PIV-2 F MAb (117-1A) cross-reacted with and was able to immunoprecipitate the SV-41 F protein (Tsurudome et al, 1989(Tsurudome et al, , 1990.…”

“…Cell fusion was quantified as described previously (Tsurudome et al, 1995). Briefly, a monochrome photograph was taken, which corresponded to an area containing approximately 2n5i10% single cells.…”