| Paramyxoviruses are a family of viral pathogens that represent a group of significant human diseases. These viruses are enveloped negative sense RNA viruses that include measles virus, mumps virus, respiratory syncytial virus, human metapneumovirus, and the zoonotic Hendra and Nipah viruses. Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F.;Our studies also examined the intracellular trafficking of both the Hendra virus fusion and attachment proteins. Despite the prevailing hypothesis in the field of paramyxoviral membrane fusion that these two proteins interact with one another after synthesis and are transported through the secretory pathway as a complex that arrives at the cellular surface, our data indicates that this model of fusion and attachment protein interaction is not correct. We demonstrate that the Hendra F and G proteins have very different trafficking rates with regard to their transport through the secretory pathway and when these proteins arrive at the cell surface. Hendra F is resistant to Endoglycosidase H digestion within 30 minutes, while it takes more than two hours for a significant amount of Hendra G to become Endo H resistant. We also examined the folding kinetics of these two proteins and found that Hendra F quickly folds into its mature trimer while monomeric Hendra G is detectable two hours after metabolic labeling. This indicter that the intrinsically slow folding of Hendra G is what allows for its slow transport.;Keywords: Paramyxovirus, viral membrane fusion, Hendra virus, attachment protein, cellular trafficking. |
