Human parainfluenza virus 3 fusion protein cleavage: a key determinant of infection and spread

ABSTRACT Human parainfluenza virus 3 (HPIV3) entry into target cells depends on a viral fusion complex composed of hemagglutinin-neuraminidase (HN) and fusion protein (F). In addition to attachment, HN activates F to mediate membrane fusion, while its neuraminidase activity promotes viral release. This creates a biological paradox: virions must repeatedly engage sialic acid-rich substrates in the airway without prematurely triggering F until they reach the target cell sialic acid receptor. Unlike the F proteins of laboratory strains that are cleaved intracellularly by furin to process F from its pro-protein to the active form that is essential for fusion, the F proteins of HPIV3 field strains lack a furin cleavage site and undergo partial cleavage mediated by extracellular serine proteases. We show that virions isolated directly from infected humans contain both cleaved and uncleaved F. In viral migration assays, reducing the proportion of cleaved F on virions increases the distance virions travel before infecting a cell. Even virions bearing only uncleaved F upon egress from infected cells can establish infection in vivo . These findings support a model in which uncleaved F on viral surfaces permits virions to engage and disengage sialylated substrates without undergoing premature fusion protein triggering, allowing virions to traverse the airway. Cleavage of F at the target cell then licenses membrane fusion, ensuring that virions initiate infection at the correct site. HPIV3 exploits host protease environments to produce virions with heterogeneity in F cleavage, equipped to either establish local infection or spread to distant target cells. IMPORTANCE Parainfluenza viruses employ their surface glycoproteins, the receptor-binding (hemagglutinin-neuraminidase) and fusion (F) proteins, to fuse with target cell membranes and infect the airway. F is made as an inactive precursor that is activated when host cell proteases cleave it to produce the fusion-competent form. Previous studies, utilizing laboratory-adapted viruses, assumed that F proteins are pre-cleaved on virus produced by infected people. We show that virions isolated directly from infected humans contain both cleaved and uncleaved F proteins. In viral migration assays, reducing the proportion of cleaved F on virions increases the distance virions travel before infecting a cell. The uncleaved F proteins enable virions to travel from their cell of origin without undergoing premature fusion protein activation, allowing virions to traverse the airway and infect the proper target cells. HPIV3 exploits host protease environments to produce a spectrum of virions suited to either local infection or intra-host spread.