Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex.

The exocyst is a hetero-octameric complex that has been proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst complexes from Saccharomyces cerevisiae and showed that they are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin induced-degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer and demonstrated that several known exocyst-binding partners are not necessary for exocyst assembly and stability.

Roles of the putative integrin-binding motif of the human metapneumovirus fusion (f) protein in cell-cell fusion, viral infectivity, and pathogenesis.

Unlabelled: Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin αvβ1 as a cellular receptor.

A mutation in the stalk of the newcastle disease virus hemagglutinin-neuraminidase (HN) protein prevents triggering of the F protein despite allowing efficient HN-F complex formation.

Newcastle disease virus (NDV)-induced membrane fusion requires formation of a complex between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. Substitutions for NDV HN stalk residues A89, L90, and L94 block fusion by modulating formation of the HN-F complex. Here, we demonstrate that a nearby L97A substitution, though previously shown to block fusion, allows efficient HN-F complex formation and likely acts by preventing changes in the HN stalk required for triggering of the bound F protein.

Individual N-glycans added at intervals along the stalk of the Nipah virus G protein prevent fusion but do not block the interaction with the homologous F protein.

The promotion of membrane fusion by most paramyxoviruses requires an interaction between the viral attachment and fusion (F) proteins to enable receptor binding by the former to trigger the activation of the latter for fusion. Numerous studies demonstrate that the F-interactive sites on the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) and measles virus (MV) hemagglutinin (H) proteins reside entirely within the stalk regions of those proteins. Indeed, stalk residues of NDV HN and MV H that likely mediate the F interaction have been identified.

Localization of a region in the fusion protein of avian metapneumovirus that modulates cell-cell fusion.

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH.

Role of the two sialic acid binding sites on the newcastle disease virus HN protein in triggering the interaction with the F protein required for the promotion of fusion.

Newcastle disease virus (NDV)-induced membrane fusion requires an interaction between the hemagglutinin-neuraminidase (HN) attachment and the fusion (F) proteins, triggered by HN's binding to receptors. NDV HN has two sialic acid binding sites: site I, which also mediates neuraminidase activity, and site II, which straddles the membrane-distal end of the dimer interface. By characterizing the effect on receptor binding avidity and F-interactive capability of HN dimer interface mutations, we present evidence consistent with (i) receptor engagement by site I triggering the interaction with F and (ii) site II functioning to maintain high-avidity receptor binding during the fusion process.

Triggering of the newcastle disease virus fusion protein by a chimeric attachment protein that binds to Nipah virus receptors.

The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein.

Engineered intermonomeric disulfide bonds in the globular domain of Newcastle disease virus hemagglutinin-neuraminidase protein: implications for the mechanism of fusion promotion.

The promotion of membrane fusion by Newcastle disease virus (NDV) requires an interaction between the viral hemagglutinin-neuraminidase (HN) and fusion (F) proteins, although the mechanism by which this interaction regulates fusion is not clear. The NDV HN protein exists as a tetramer composed of a pair of dimers. Based on X-ray crystallographic studies of the NDV HN globular domain (S.

Decreased dependence on receptor recognition for the fusion promotion activity of L289A-mutated newcastle disease virus fusion protein correlates with a monoclonal antibody-detected conformational change.

It has been shown that the L289A-mutated Newcastle disease virus (NDV) fusion (F) protein gains the ability to promote fusion of Cos-7 cells independent of the viral hemagglutinin-neuraminidase (HN) protein and exhibits a 50% enhancement in HN-dependent fusion over wild-type (wt) F protein. Here, we show that HN-independent fusion by L289A-F is not exhibited in BHK cells or in several other cell lines. However, similar to the results in Cos-7 cells, the mutated protein plus HN does promote 50 to 70% more fusion above wt levels in all of the cell lines tested.

An oligosaccharide at the C-terminus of the F-specific domain in the stalk of the human parainfluenza virus 3 hemagglutinin-neuraminidase modulates fusion.

The promotion of membrane fusion by the fusion (F) protein of human parainfluenza virus 3 (hPIV3) is dependent on a virus-specific contribution from the hemagglutinin-neuraminidase (HN) protein. By evaluation of chimeric hPIV3-Newcastle disease virus (NDV) HN proteins, we have previously shown that hPIV3-F-specificity is determined by a domain that extends from the middle of the membrane anchor to the 82nd residue in the ectodomain [Virology 209, (1995) 457; Arch. Virol.

Fusion deficiency induced by mutations at the dimer interface in the Newcastle disease virus hemagglutinin-neuraminidase is due to a temperature-dependent defect in receptor binding.

The tetrameric paramyxovirus hemagglutinin-neuraminidase (HN) protein mediates attachment to sialic acid-containing receptors as well as cleavage of the same moiety via its neuraminidase (NA) activity. The X-ray crystallographic structure of an HN dimer from Newcastle disease virus (NDV) suggests that a single site in two different conformations mediates both of these activities. This conformational change is predicted to involve an alteration in the association between monomers in each HN dimer and to be part of a series of changes in the structure of HN that link its recognition of receptors to the activation of the other viral surface glycoprotein, the fusion protein.