Requirements for syncytium formation mediated by the fusion glycoprotein of human parainfluenza virus type 3.

Abstract

Recombinant vaccinia viruses VF and VHN were shown to express glycoproteins with molecular weights similar to authentic HPIV3 F and HN proteins that were transported to the cell surface and recognized by monoclonal antibodies specific for HPIV3 F and HN. The HN glycoprotein in VHN-infected cells exhibited both hemagglutinin and neuraminidase activities. Both cleaved and uncleaved forms of the F glycoprotein were immunizers from VF-infected cell lysates. Fusogenic activity of the F protein was demonstrated only upon coinfection of HeLa T4$\sp+$ cells with VF + VHN and resulted in syncytium formation and hemolysis. Recombinant adenoviruses AdF and AdHN, expressed the HPIV3 F and HN proteins on the surface of infected HeLa T4$\sp+$ cells, respectively. AdHN-infected cells produced HN protein that was glycosylated and exhibited both hemagglutinin and neuraminidase activities. AdF infection led to the synthesis of glycosylated F$\sb0$ that was proteolytically cleaved. As was observed for vaccinia virus recombinants, syncytium formation was only observed upon coinfection of HeLa T4$\sp+$ cells with AdF + AdHN The F and HN proteins expressed by recombinant adenoviruses were recognized by monoclonal antibodies specific for the HPIV3 F or HN protein and were immunogenic in mice. Sera from AdF-or AdHN-inoculated mice immunoprecipitated appropriate glycoproteins from lysates of HPIV3-infected cells and neutralized HPIV3. Amino acid substitutions were introduced into three regions of the HPIV3 F protein to identify sequences involved in fusogenic activity. Substitution of uncharged amino acids for positively charged residues within the cytoplasmic tail, or introduction of a charged residue within the transmembrane domain, did not appear to affect fusogenic activity. Several different mutations designed to disrupt the leucine zipper or the heptad repeat motifs resulted in mutant F proteins that not only exhibited decreased fusogenic activity but concomitantly lost the ability to react with monoclonal antibody c128, which recognized an epitope in antigenic site B of the HPIV3 F glycoprotein. An association between the heptad repeat and leucine zipper domains, therefore, appears to be required for the HPIV3 F glycoprotein to adopt a conformation that is recognized by antibody c128 and that appears to be important for fusogenic activity.