2025 Theses Doctoral

Human parainfluenza virus 3 fusion protein cleavage regulation is a key determinant of viral fitness

Stearns, Kyle

Entry by human parainfluenza virus 3 (HPIV3) into a target cell depends on viral fusion machinery that has been honed by evolution to have a balance of receptor binding affinity, membrane fusion activation, and neuraminidase-driven release of virus, that favor spread within and between hosts. A critical, yet unaddressed, regulator of the viral fusogenicity is the proportion of cleaved and thereby functional fusion proteins on virions.

HPIV3 infection is driven by the coordinated action of a fusion complex consisting of the viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Upon receptor engagement, HN triggers F to insert into the target cell membrane and drive virion-cell membrane fusion. For cell entry to occur, the fusion protein precursor (F0) must first be cleaved by host proteases into its active form. In this body of work, starting with our discovery that field strains and laboratory strains of virus differ in their mode of F cleavage activation, we employed a whole genome CRISPR activation screen and extracellular serine protease inhibitors to reveal that F0s of HPIV3 field strains are cleaved by TMPRSS2 and TMPRSS13, in distinction to laboratory adapted strains F0s that are cleaved by intracellular furin.

These findings support an alternative mechanism of F activation in vivo, reliant on host factors expressed in a select subset of cells, thereby opening the possibility that HPIV3 virions with differentially cleaved F may be produced and contribute to viral fitness. In support of this theory, we observed that about half of the fusion proteins on virions collected directly from infected human subjects remain uncleaved. We showed that relative to cleaved F, uncleaved F is triggered more slowly, increases the distance virions travel from the site of inoculation, and limits cell-cell fusion associated cell death.

Based on these results, we theorize that virions are produced in vivo by cells with varying degrees of F cleaving proteases and thus a range of propensities to infect rapidly or travel further from their origin. Conserved dependence on narrowly expressed serine proteases may therefore permit a single genome to produce a spectrum of virions with F cleavage-regulated fusion machinery optimized to spread fast or far.