Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

Abstract

Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H, or G and the "triggering" interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region, and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active "headless" mumps and Newcastle disease virus HN proteins, provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed "stalk exposure model" to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion. HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes. We describe here the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed "stalk exposure model" first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirusmediated membrane fusion. IMPORTANCE Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [

Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly

“…This F-triggering function of the attachment protein can be dissociated from its other functions, such as intracellular transport, association with F and receptor binding, which indicates that it resides in a distinct region. Indeed, the attachment protein stalk domain is still able to trigger the F protein even when the globular head is removed (Bose et al, 2012(Bose et al, , 2014Brindley et al, 2013;Liu et al, 2013).…”

Probing the Functions of the Paramyxovirus Glycoproteins F and HN with a Panel of Synthetic Antibodies

“…7C and D). Interestingly, we recently showed that these residues could tolerate a variety of mutations while maintaining the ability to trigger F but that directly adjacent hydrophobic residues V81 and L85, critical for triggering PIV5 F, were much less tolerant of substitution (63). This may partially explain why A82 and Q86 were identified when we were selecting for viable sAb resistance mutations.…”

“…This movement exposes critical residues in the stalk domain that interact with and trigger F to initiate membrane fusion (41,62,63). This model is based on the following observations: (i) mutations in HN that affect only fusion activity map to the stalk region of the attachment protein of several paramyxoviruses (12,18,42,43,64,65); (ii) headless PIV5 and NDV HN, MeV H, and NiV G are sufficient to trigger fusion (41,63,66,67); (iii) the head domains of HN have been observed by EM to be in various orientations relative to the stalk for NDV (12) and PIV5 (41); (iv) a crystal structure of the tetrameric NDV HN ectodomain reveals the heads in the down position, forming an interface with the stalk that overlaps with the critical fusion (69) was used to morph high-resolution structures of HN, F, and the humanized anti-HER2 Fab, 4D5 (which the sAb phage library was based on), to 15-Å surface representations. Protein Data Bank (PDB) entries 3T1E and 3TSI were overlaid and used for the HN four-heads-down model.…”

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

“…pCAGGS-F and pCAGGS-HN expression constructs harboring the PIV5 W3A F and HN or NDV F and HN (strain Australia-Victoria) genes were used as described previously (19,50). Mutations in pCAGGS-F were constructed by four-primer PCR.…”

“…4). Together with biochemical and structural data of paramyxovirus attachment proteins (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), a general model of paramyxovirus fusion has been proposed: upon activation by the attachment protein, metastable prefusion F undergoes a series of large-scale, ATP-independent conformational changes, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. The energy released during F refolding is believed to facilitate membrane fusion to create a pore between the virus and host cell through which the viral ribonucleoprotein complex can enter the target cell.…”