Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

Abstract

Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry. membrane fusion | measles virus M embrane fusion is an essential process in eukaryotic cell biology. Examples range from fusion of lipid vesicles to sustain intracellular transport along secretory and endocytotic pathways to cell fusion in fertilization and development and to the fusion of viral with cellular membranes resulting in viral infection. Because there are several high-energy barriers (1), lipid membranes do not merge spontaneously under physiological conditions and therefore require the aid of auxiliary proteins to induce membrane stress and lower the activation energy for lipid merger. For vesicular transport, for instance, soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) proteins resident in both the target and donor membrane mediate the process (2). Two fundamental differences distinguish cellular fusion machineries from viral fusogenic membrane glycoproteins (vFMGs), which mediate the entry of enveloped viruses by the fusion of the envelope with cellular membranes: (i) vFMGs do not require adapter proteins but insert a fusion peptide domain into the target membrane; and (ii) vFMGs fold into metastable prefusion conformations, obviating the dependence on external energy sources. Three distinct types of viral fusion (F) proteins have been classified: class I vFMGs are found, among others, on influenza virus, retroviruses, and paramyxoviruses; class II proteins are present on alpha-and flaviviruses; and class III proteins mediate herpes-and rhabdovirus entry (reviewed in ref.3).Once the fusion p...

Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain

“…The weak yet helix-preserving FP-TMD interaction in the PIV5 fusion protein chimera gives insight into the mechanism of fusion-pore formation and constrains the possible postfusion structures of the intact protein. A recent study of the F protein of the measles virus, another member of the paramyxovirus family, found that an ectodomain 6HB was not absolutely required for complete fusion 68 . When disulfide bonds were engineered at the C-terminus of HR2 to prevent 6HB formation, virus-cell fusion still occurred 68 .…”

Differential Features of Fusion Activation within the Paramyxoviridae

“…Brindley et al (2014) demonstrated full closure of the 6HB is not necessary for MeV entry [131]. Covalent bonds were engineered into membrane-proximal positions of the HR2 domain that would be incompatible with the complete formation of a closed 6HB core.…”

Structure-guided design of small-molecule therapeutics against RSV disease

“…(F) Transmembrane domains and fusion peptides are brought into close proximity, leading to the opening of a fusion pore. Creation of a productive fusion pore by paramyxovirus F proteins is not mandatorily dependent on full closure of the 6HB [100]. Images of protein structures were generated in Pymol.…”