Human parainfluenza virus fusion complex glycoproteins imaged in action on authentic viral surfaces

Abstract

Infection by human parainfluenza viruses (HPIVs) causes widespread lower respiratory diseases, including croup, bronchiolitis, and pneumonia, and there are no vaccines or effective treatments for these viruses. HPIV3 is a member of theRespirovirus speciesof theParamyxoviridae family. These viruses are pleomorphic, enveloped viruses with genomes composed of single-stranded negative-sense RNA. During viral entry, the first step of infection, the viral fusion complex, comprised of the receptor-binding glycoprotein hemagglutinin-neuraminidase (HN) and the fusion glycoprotein (F), mediates fusion upon receptor binding. The HPIV3 transmembrane protein HN, like the receptor-binding proteins of other related viruses that enter host cells using membrane fusion, binds to a receptor molecule on the host cell plasma membrane, which triggers the F glycoprotein to undergo major conformational rearrangements, promoting viral entry. Subsequent fusion of the viral and host membranes allows delivery of the viral genetic material into the host cell. The intermediate states in viral entry are transient and thermodynamically unstable, making it impossible to understand these transitions using standard methods, yet understanding these transition states is important for expanding our knowledge of the viral entry process. In this study, we use cryo-electron tomography (cryo-ET) to dissect the stepwise process by which the receptor-binding protein triggers F-mediated fusion, when forming a complex with receptor-bearing membranes. Using an on-grid antibody capture method that facilitates examination of fresh, biologically active strains of virus directly from supernatant fluids and a series of biological tools that permit the capture of intermediate states in the fusion process, we visualize the series of events that occur when a pristine, authentic viral particle interacts with target receptors and proceeds from the viral entry steps of receptor engagement to membrane fusion. Author summary Human respiratory parainfluenza viruses (HPIVs) cause the majority of childhood cases of croup, bronchiolitis, and pneumonia. HPIV3, like most other paramyxoviruses, uses two specialized proteins to mediate cell entry: the fusion protein (F) and the receptor-binding protein, hemagglutinin-neuraminidase (HN). F is only activated to mediate membrane fusion during entry when it is triggered by a signal from the separate receptor-binding molecule, HN-a mechanism first elucidated for HPIV3 and later extended to this entire group of human pathogenic viruses. These two distinct membrane glycoproteins work in synchrony as a molecular machine, exquisitely tuned to enter the right cells at the right time. Much has been learned about the functional sites on the receptor-binding and F molecules for this group of viruses, and mechanisms have been proposed to explain molecular, biochemical, and crystal structure data. But the key intermediate steps, from receptor binding to membrane fusion, are fleeting and unstable. Furthermore, it is unclear how HN ultimately promotes F-mediated fusion. Thus, it is impossible to understand intermediate fusion states, using standard methods and static observations. Here, we use an on-grid antibody capture method that facilitates examination of fresh, biologically active strains of virus directly from supernatant fluids by cryo-electron tomography and new biological tools that permit capture of the intermediate states in the fusion process. It allows us to visualize the series of events that occur as an authentic HPIV3 viral particle interacts with target receptors and proceeds through intermediate stages from receptor engagement to membrane fusion.