Mutational analysis of conserved regions in paramyxovirus fusion proteins

Paramyxoviruses comprise a diverse family of enveloped viruses, which utilize an attachment protein to bind target cell receptors and a fusion (F) protein to enter cells via fusion of their lipid bilayer with that of a target cell membrane. Paramyxovirus fusion proteins exist as inactive precursors (F0) which must trimerize and be proteolytically cleaved into two disulfide-linked subunits: F1 and F2. Although regions of F are clearly critical for promotion of membrane fusion, the role of much of the protein remains unclear. Alignment and analysis of a set of paramyxovirus F protein sequences utilizing the Block Maker program identified three conserved blocks: one in the fusion peptide/heptad repeat A domain of F1, one in F1 N-terminal to heptad repeat B (CBF1), and one in F2 (CBF2). Single point mutations of conserved residues in CBF1 of both (simian virus 5) SV5 F and Hendra virus F resulted in proteins which did not fold and trimerize properly. A number of the CBF2 point mutations resulted in folding defects, though the residues critical for folding differed between SV5 and Hendra F. CBF2 mutants that were properly expressed and trafficked were found to have altered fusion-promotion activity, with mutant I49A in SV5 exhibiting a pronounced hyperfusogenic phenotype. The recently published prefusogenic structure of SV5 F (Yin et al. 2006. Nature 439:38-44) suggests that CBF1 is critical for proper folding of the fusion peptide in SV5 F, while CBF2 is closely associated with the heptad repeat A region and may be involved in triggering of membrane fusion. My results support the hypothesis that sequence conservation within regions of paramyxovirus F proteins can be attributed to a critical function in either F protein folding or promotion of membrane fusion.