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Journal of Virology, March 2003, p. 3647-3654, Vol. 77, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.6.3647-3654.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Triggering of Human Parainfluenza Virus 3 Fusion Protein (F) by the Hemagglutinin-Neuraminidase (HN) Protein: an HN Mutation Diminishes the Rate of F Activation and Fusion

Matteo Porotto, Matthew Murrell, Olga Greengard, and Anne Moscona*

Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029

Received 24 October 2002/ Accepted 13 December 2002

For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4°C are transferred to 22°C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HN-receptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptor-binding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22°C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HN's F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F.

* Corresponding author. Mailing address: Department of Pediatrics, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-9709. Fax: (212) 860-3316. E-mail: Anne.moscona{at}mssm.edu.

Journal of Virology, March 2003, p. 3647-3654, Vol. 77, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.6.3647-3654.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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