JOUIRNALCOF VIROLOGY, Nov. 1993, p. 6463-6468 Vol. 67, No. 11 0022-538X/93/1 16463-06$02.00/() Copyright (© 1993, American Society for Microbiology Relative Affinity of the Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase for Sialic Acid Correlates with Virus-Induced Fusion Activity ANNE MOSCONA"2* AND RICHARD W. PELUSO3 Departments of Pediatrics,' Cell Biology/Anatomy,2 and Microbiology,3 Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, New York 10029-6574 Received 8 July 1993/Accepted 2 August 1993 The ability of enveloped viruses to cause disease depends on their ability to enter the host cell via membrane fusion events. An understanding of these early events in infection, crucial for the design of methods of blocking infection, is needed for viruses that mediate membrane fusion at neutral pH, such as paramyxoviruses and human immunodeficiency virus. Sialic acid is the receptor 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, the HN must interact with its receptor. In the present report, two variants of HPF3 with increased fusion-promoting phenotypes were selected and used to study the function of the HN glycoprotein in membrane fusion. Increased fusogenicity correlated with single amino acid changes in the HN protein that resulted in increased binding of the variant viruses to the sialic acid receptor. These results suggest that the avidity of binding of the HN protein to its receptor regulates the level of F protein-mediated fusion and begin to define one role of the receptor-binding protein of a paramyxovirus in the membrane fusion process. Enveloped viruses have evolved mechanisms of protein- membranes despite diminished receptor availability and which mediated membrane fusion that enable them to infect their have a greatly increased ability to fuse cells under normal hosts; these strategies have begun to shed light on membrane conditions are selected. These variants offer a new approach to fusion mechanisms utilized by cells (2). The envelope of the the study of the mechanism of paramyxovirus-induced cell paramyxovirus human parainfluenza virus type 3 (HPF3) con- fusion and the role of the HN protein in this process. tains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The HN glycoprotein binds to sialic acid-containing receptor molecules on the cell surface to MATERIALS AND METHODS initiate infection (24). Fusion of the viral envelope with the cell's plasma membrane is mediated by the F glycoprotein and Virus and cells. CV-1 cells were grown in monolayers in occurs at neutral pH at the cell surface, without a requirement Eagle's minimal essential medium supplemented with 10% for an acid-mediated conformational change of the fusion fetal bovine serum. Stocks of wt HPF3 were made in CV-1 cells molecule (8, 21, 25). This feature distinguishes paramyxovi- from virus that was plaque purified four times. Virus titer was ruses from viruses, such as influenza virus, that require a low determined by a plaque assay with CV-1 cells. Cells were pH to mediate fusion (reviewed in reference 13). Infection infected by aspirating the medium, washing the monolayers results in fusion between adjacent cells, which involves the with medium lacking serum, and then adding medium lacking interaction of F protein, expressed on the surface of an serum with various amounts of wt or C variant HPF3 to the infected cell, with the membrane of an adjacent uninfected cells, as described in the text. After incubation at 37°C for 60 cell. min, the medium was aspirated; either medium lacking serum We have reported that the HN glycoprotein is essential for or agar overlay medium was added. Cells were photographed cell fusion mediated by HPF3 and that interaction of HN with through a phase-contrast microscope. its receptor is required in order for F to promote membrane Neuraminidase treatment. Monolayer cultures of infected fusion during viral infection (18). We now report that the cells were treated with neuraminidase (from Clostridium per- avidity of binding of virus to the sialic acid receptor correlates fringens; Sigma Chemical Co., St. Louis, Mo.) by washing the with the level of F protein-mediated fusion. cells with medium lacking serum and then adding serum-free medium neuraminidase to the cells Syncytium formation by wild-type (wt) HPF3 can be pre- containing (19). Isolation of HPF3 variants. cultures of CV-1 vented in a low-multiplicity infection by the addition of neura- Monolayer cells in 60-mm dishes were infected with HPF3 and treated minidase to the cells; by removing some cell surface sialic acid receptors, the neuraminidase treatment prevents the requisite with 0.1 U of C. perfringens neuraminidase as described interaction of the wt viral HN glycoprotein with its receptor previously (19). Supernatant fluid from these infected cultures (19). While the neuraminidase-treated infected cells do not was collected and used in plaque assays. Large plaques were fuse, the viral infection spreads throughout the culture, and all picked and plaque purified twice, and a single plaque was used the cells become persistently infected (19, 20). Under these to infect each CV-1 cell monolayer for preparation of stocks of variant viruses. conditions, variant viruses which are able to fuse with cell Protein analysis. 35S-radiolabeled virus proteins were pre- pared as previously described (17). At 3, 6, or 9 h after infection with either wt or C variant HPF3, CV-1 cell mono- * Corresponding author. layers were cultured for 15 min in methionine-free minimal 6463 6464 MOSCONA AND PELUSO J. VIROL. wt C-O C-22 plaque assay 24 hrs * X v~~~~~~~~~/1S , ,g a @.; ? se: ¼B plaque " 'A:#b; assay i22t ''::¾?1?Jh^}? 24 hrs * *..,?sS# ......;.s... ,., 3 X'iLS..i....'S infected monolayer (moi=0.1) 18 hrs p.i. FIG. 1. Fusion properties of HPF3 variants. CV-1 cell cultures and HPF3 were grown as described in Materials and Methods. Confluent cell monolayers were infected with either wt HPF3 or C variant HPF3 at an MOI of 0.1 PFU/cell. For observation of plaques, overlay medium was prepared and added after a 90-min adsorption period. The photographs show the plaque size and morphology and the extent of CPE in a monolayer at 1 day postinfection (p.i.). essential medium and then for 1 h in methionine-free minimal was then added to each well. The plates were photographed 90 essential medium containing [35S]methionine (50 ,xCi/ml). min after the addition of erythrocytes. Mock-infected cell proteins were prepared in parallel with the Sequence analysis. The F and HN genes of C variant viruses infected cell proteins at each time point. were sequenced after reverse transcription-polymerase chain Assays of viral neuraminidase. Neuraminidase activities, reaction (PCR) amplification of each gene, either by direct substrate specificities, and pH optima were determined for wt sequencing of the PCR product or by cloning into pUC19 and HPF3 and C variants by the thiobarbituric acid assay (14). 2,3- subsequent dideoxy sequencing of plasmid DNA. Total RNA and 2,6-neuraminyllactose substrates were purchased from from infected cells was prepared as described previously (17). Sigma Chemical Co. The primers used for the reverse transcriptase reaction were Hemagglutination inhibition assays. Hemagglutination in- complementary to the 3'-terminal 19 nucleotides of the F gene hibition assays were performed at 4°C (22) with fetal calf mRNA sequence (5) and the 3' 32 nucleotides of the HN gene serum (FCS) or bovine fetuin as the competitor. Four hemag- mRNA sequence (27). The upstream primers used for PCR glutination units of virus in 50 il was added to each dilution of amplification corresponded to the 5'-terminal 15 nucleotides FCS (in 50 pul of phosphate-buffered saline [PBS]) or to of the F gene mRNA sequence and the 5'-terminal 16 nucle- dilutions of fetuin in the wells of a microtiter plate. Fifty otides of the HN mRNA sequence. Primers for sequencing microliters of a 0.5% suspension of human erythrocytes in PBS were synthesized to be complementary to the F or HN gene VOL. 67, 1993 HUMAN PARAINFLUENZA VIRUS TYPE 3 MEMBRANE FUSION 6465 wt C-O C-22 HPF3 alone + neuraminidase HPF3 alone + neuraminidase HPF3 alone + neuraminidase low moi ( 01) FIG. 2. Effect of MOI and of neuraminidase addition on CPE produced by HPF3 variants. Monolayer cultures of CV-1 cells were infected with wt or C variant HPF3 at either a low MOI (0.01 PFU/cell) or a high MOI (5 PFU/cell). For each virus, the cells were either infected with virus alone or received 0.1 U of C. perfringens neuraminidase after viral infection, as described in Materials and Methods. The photographs show CPE 1 day after infection. In cells infected with C variant HPF3, infection at a high MOI did not block fusion; the CPE in these cells is equivalent to that in those infected at a low MOI. In addition, in cells infected with variant HPF3, exogenous neuraminidase addition did not prevent cell fusion. sequence at 200-bp intervals. Sequence alterations were con- effect of neuraminidase on cell fusion and are able to fuse firmed by repeating at least twice the RNA isolation from efficiently under conditions of decreased receptor availability. infected cells, reverse transcription-PCR amplification, and We determined whether the variant fusion phenotype could sequencing of the altered region. be explained by altered growth characteristics of the virus. Figure 3 shows that the growth characteristics of the variant RESULTS viruses do not differ from those of the wt.