Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins

Abstract

The paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane proteinprotein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions. IMPORTANCENipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in virus-host cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on coimmunoprecipitation, a technique with some pitfalls. We developed a flow-cytometric assay to study membrane protein-protein interactions, and using this assay we report a bidentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family.T he paramyxovirus family includes many important negativesense, single-stranded enveloped RNA viruses, such as measles (MeV), mumps (MuV), respiratory syncytial (RSV), Newcastle disease (NDV), parainfluenza (PIV) Nipah (NiV), and Hendra (HeV) viruses and human metapneumovirus (hMPV) (1). With very few exceptions, paramyxoviruses require two di...

A stochastic assembly model for Nipah virus revealed by super-resolution microscopy

“…To investigate the organization of the viral envelope glycoproteins on the plasma membrane, we used FLAG-tagged F 21 and hemagglutinin (HA)-tagged G 22 constructs (Supplementary Fig. 1 ).…”

Sortase-mediated chemical protein synthesis reveals the bidentate binding of bisphosphorylated p62 with K63 diubiquitin

“…3H ). 23 One typical previous example of the bidentate effect is that deubiquitinase hOtu1 specifically recognizes K48 diUb through binding to both the proximal and the distal Ub of K48 diUb at different binding sites simultaneously. 24 …”

Differential Features of Fusion Activation within the Paramyxoviridae

“…Instead, a bidentate interaction exists between NiV-F and both the globular head and 4HB stalk of NiV-RBP ( Figure 7B, Panel 1). Indeed, flow cytometric strategies with various truncated versions of soluble NiV-G and full length NiV-F expressed on cells confirmed this bidentate interaction [151]. This study also showed the soluble ectodomain version of NiV-G is not sufficient to induce cell-cell fusion, and only membrane-bound NiV-G triggers F. The data suggests anchoring of NiV-G is necessary for F activation or the transmembrane domain and/or cytoplasmic tail of G is involved in the fusion process.…”

“…Interaction is bidendate, involves head and stalk [151]; in the context of full length NiV-G, both head and stalk are necessary for productive NiV-F triggering [152] Interaction and triggering occur through the stalk domain [104,[147][148][149] The C-term of the stalk can be elongated by 41-residues, adding a pitch of~75 Å above residue 118, and triggering will still occur [108]; results suggest that interaction between…”