Human parainfluenza virus type 3: Analysis of the cytoplasmic tail and transmembrane anchor of the hemagglutinin-neuraminidase protein in promoting cell fusion

The role of the cytoplasmic tail and transmembrane anchor of the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN) glycoprotein in promoting cell fusion was investigated. A series of amino terminal deletion mutants (d10, d20, d27, d31, d40, d44, and d73) were compared for processing, cell surface expression, and maintenance of their biological attributes by recombinant expression of mutant genes using a plasmid vector (pcDL-SR alpha-296) in CV-1 and HeLa cells. To determine the fusion promoting activity (FPA) of the various mutant proteins, a simple assay was developed which quantified the fusion of two different HeLa cell types. One of the cell types, HeLa-tat, constitutively expressed the human immunodeficiency virus type I (HIV-1) tat protein from a Moloney murine leukemia virus long terminal repeat (LTR), while the second cell type, HeLa β-gal, contained a reporter gene, beta-galactosidase, under the control of an HIV1-LTR. Fusion of mixed HeLa cell monolayers (50:50, HeLa-tat: HeLa β-gal), following transfection with appropriate plasmids, resulted in transactivation of the reporter gene which was then measured by direct staining of cells or using cell lysates with appropriate substrates. Cell fusion was observed only when both the HPIV3 F and functional HN proteins were both co-transfected into cells. Of the seven deletion mutants examined, only d10, d20, d27 and d31 were expressed to significant levels on the cell surface and only these four mutant proteins maintained FPA. Compared with the wt HN at 48 h post transfection, d10 and d20 had enhanced FPA (119% and 158%, respectively), while d27 and d31 were diminished (74% and > 4%, respectively). Analysis of protein expression suggested that the reason for the increase in FPA of the mutant proteins was that the levels of protein expressed at the cell surface was twofold or threefold higher for d10 and d20, respectively, compared to the wt HN.