Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly

Abstract

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites.IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly.

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

“…6 ). These observations disagree with the commonly believed model that the viral envelope glycoproteins coalesce to the matrix protein for the assembly of the nascent virions 7 , 8 , 31 , 32 . Previous studies using western blot analysis suggest that F may facilitate G to incorporate into VLPs 7 .…”

“…Many virus families, such as the togaviruses, the rhabdoviruses, the para- and orthomyxoviruses, and the retroviruses, undergo assembly at the plasma membrane 1 . For example, extensive electron microscopic and western blot analyses have been carried out for the paramyxoviruses and suggest that the matrix proteins recruit the nucleocapsid and glycoproteins to the assembly sites 2 – 8 . However, neither electron microscopic studies nor western blot analyses are done on intact cells 9 .…”

Nipah and Hendra Virus Glycoproteins Induce Comparable Homologous but Distinct Heterologous Fusion Phenotypes

“…For henipaviral Fs, both the transmembrane (TM) and cytoplasmic tail (CT) domains have been implicated as being important for membrane fusion as well as virus-like particle (VLP) formation (19,44,45). To understand the roles of these domains in heterologous fusion and budding, we created HF and NF chimeras with swapped transmembrane/cytoplasmic tail domains (TM/CT; amino acids 488 to 546).…”

Nano-based approach to combat emerging viral (NIPAH virus) infection

“…32,33 Unlike G, precursor form of F protein, F 0 get lodged within the membrane and activated through endocytosis followed by cathepsin L mediated endosomal degradation to form mature F 1/2 (Cifuentes-Muñoz et al 2017). 34 The local infection starts with the lateral spreading of virus occurred by basolaterally expressed protein (G, F) mediated cellcell merging that gives rise to a consequential configuration of syncytia which are pathological hallmarks for NiV infection of epithelial cells. This has been seen at 14 h of post infection (p.i) which is the earliest time needed for protein detection in in vivo study.…”