br Gaining access to the

Gaining access to the cytoplasm: viral membrane fusion proteins
Paramyxovirus glycoproteins F and HN, H or G are important for the initial infection step, as well as subsequent cell–cell spread. The latter mode of transmission has being suggested as the major clinical route of spread within tissues of a living host (Duprex et al., 1999; Ehrengruber et al., 2002; Sattentau, 2008). F and HN, H or G transiently expressed in MRS 2578 are able to cause cell–cell fusion, potentially creating a transmission route for the viral nucleocapsid between adjacent cells (McChesney et al., 1997). Additionally, a recent report shows a secondary route for cell–cell spread of PIV5 using actin-associated intercellular connections that may bypass membrane fusion requirements between some cells of a tissue (Roberts et al., 2015).
Paramyxovirus F proteins are Class I viral membrane fusion proteins which are structurally and functionally similar to other Class I viral membrane fusion proteins from viruses that include Ebola virus, human immunodeficiency virus (HIV), influenza virus and severe acute respiratory virus-coronavirus SARS-CoV among many others (Bartesaghi et al., 2013; Caffrey et al., 1999; Chan et al., 1997; Julien et al., 2013; Lee et al., 2008a; Li et al., 2005; Malashkevich et al., 1999; McLellan et al., 2013, 2011; Pancera et al., 2014; Swanson et al., 2010; Varghese and Colman, 1991; Weissenhorn et al., 1998; Wiley and Skehel, 1977, 1987; Wilson et al., 1981; Yin et al., 2005, 2006; Zhao et al., 2000), reviewed in (Lamb and Jardetzky, 2007). F proteins on synthesis fold into a metastable, prefusion trimer conformation (Fig. 2A–B). The transition of these metastable, higher energy prefusion trimers to stable, low energy post-fusion trimers drives the process of viral and cellular membrane merger down an energy gradient without requiring ATP hydrolysis, making this transition irreversible in nature (Lamb et al., 2006) (Fig. 2C). Ultimately F proteins are converted to their stable post-fusion trimeric form on completion of membrane merger (Fig. 2D–F). For the Paramyxovirinae subfamily, the attachment proteins are believed to provide the trigger for this refolding process by overcoming an activation energy barrier when they bind a cellular receptor (Heminway et al., 1994a; Horvath et al., 1992; Hu et al., 1992; Morrison and Portner, 1991; Yao et al., 1997). Heat acting as a surrogate can also be used to artificially overcome this thermodynamic barrier and convert prefusion F to its post-fusion form (Ader et al., 2013; Bose et al., 2012; Chan et al., 2012; Connolly et al., 2006).

Cleavage by cellular and tissue proteases converts F into an active, pre-triggered form
Paramyxovirus F proteins, like the other Class I fusion proteins are synthesized as a biologically inactive precursor (F0) that has to be cleaved to the biologically active form, F1 and F2, which are linked together by a disulfide bond. Cleavage releases a hydrophobic fusion peptide at the N-terminus of the membrane anchored F1 fragment. The cleaved F1 protein on activation by the attachment protein or heat undergoes a refolding process that results in the fusion peptide being inserted into the target membrane. Subsequent refolding, through a ‘hairpin-like’ intermediate brings together the viral and cellular membranes for merger (Jardetzky and Lamb, 2014; Lamb and Jardetzky, 2007; Lamb et al., 2006) (Fig. 2C). For most paramyxoviruses, the cleavage activation event is believed to occur in the trans Golgi network, through the action of cellular furin-like proteases during F protein transport to the cellular surface (Homma, 1971; Homma and Ohuchi, 1973; Muramatsu and Homma, 1980; Scheid and Choppin, 1974). For Henipaviruses, the F0 protein is recycled from the cell surface by endocytosis into endosomes, where it is cleaved by cathepsin L (Diederich et al., 2005; Pager et al., 2006; Pager and Dutch, 2005). Most paramyxovirus F0 proteins have a single cleavage site, but the RSV F protein is cleaved at two sites, releasing a short, soluble peptide fragment (Gonzâlez-Reyes et al., 2001). Recently, Krzyzaniak and colleagues demonstrated a sequential cleavage of RSV-F with the first cleavage occurring during its transport through the exocytic pathway to the cell surface and a second cleavage, by a furin-like protease, occurring after the virus particle is internalized into endosomes by macropinocytosis (Krzyzaniak et al., 2013). This second cleavage has been implicated to destabilize F and initiate refolding leading to membrane fusion (Gonzâlez-Reyes et al., 2001). Interestingly, a similar hypothesis has very recently been suggested for another Class I fusion protein – the Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) S (spike) protein, perhaps suggesting a convergence of molecular mechanisms of fusion (Burkard et al., 2014; Millet and Whittaker, 2014). Other examples of Class I fusion proteins like SARS-CoV S and Ebola virus GP, are likewise proteolytically activated in the endosomal compartment (Chandran et al., 2005; Simmons et al., 2004). Interestingly, transplanting the two RSV F cleavage sites into Sendai virus (a member of the Paramyxovirinae subfamily) F protein caused the Sendai F protein to lose its dependence on the Sendai HN protein for activation (Rawling et al., 2011, 2008) presumably because the sequential cleavage of the RSV F cleavage sites destabilized the chimeric F protein. Though the mechanism for F-activation is yet unclear for RSV, taken together, these data suggest that perhaps F proteins from some viruses of the Pneumovirinae subfamily, with their unique sequential cleavage and the minimal requirement for an attachment protein for fusion, might share the molecular mechanisms of F activation more closely with viruses that utilize a single Class I fusion protein for receptor binding and fusion.