Role of the Simian Virus 5 Fusion Protein N-Terminal Coiled-Coil Domain in Folding and Promotion of Membrane Fusion

Abstract

Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt ␣-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiledcoil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion.

A Conserved Region in the F 2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

“…5A). This hyperfusogenic phenotype was even more apparent at 30°C (data not shown), a finding similar to results published for other hyperfusogenic SV5 F mutants (42,56); at this temperature, the level of fusion promoted by wt SV5 F was much more drastically reduced than that promoted by the I49A mutant. These data indicate that surface expression alone does not dictate the fusogenic potential of F proteins and that SV5 F I49 may be a key residue in fusion promotion.…”

“…6). SV5 F residues I49 and V50 fall within this third ␤-strand and are located in close proximity to L161, a residue displaying a hyperfusogenic phenotype when mutated to me- thionine (56). We hypothesized that the SV5 F V50A mutant may act to stabilize HRA, and hence the F protein, in the prefusion conformation, while the I49A mutant (and the L161M mutant) promotes triggering to the postfusion conformation.…”

“…Using two separate algorithms for sequence alignment, the Block Maker program (19) identified three conserved regions: one in the fusion peptide/HRA region, known to be involved in promotion of F-mediated membrane fusion; a second in the F 2 subunit, designated the conserved block in F 2 (CBF 2 ); and a third within a large stretch of amino acids between the heptad repeats of F 1 , designated CBF 1 . Previous research in multiple laboratories has indicated a role for HRA in folding and fusion (47,49,56). We have recently demonstrated that there is a role for CBF 1 in F protein folding and homotrimerization (15).…”

Fusogenic Variants of a Noncytopathic Paramyxovirus

“…It was also reported by Russell et al (24) that mutation of Ser 443 to Pro and mutation of Leu 447 and Ile 449 to aromatic residues (residues proximal to heptad repeat B) in the SV5 F protein resulted in increased levels of fusion as well as fusion promotion at lower temperatures. Previous studies showed that mutations Leu140Met and Leu161Met (residues in the heptad repeat A) abolish the ␣-helical structure required for the formation of a coiled-coil domain but enhance syncytium formation (34). In the 6-1N mutant, Leu 447 can interact with residues Ile 444, Thr 357, and Gln 304 of the neighboring chain (data not shown) but, when mutated to Trp, as shown in Fig.…”

A Conserved Region between the Heptad Repeats of Paramyxovirus Fusion Proteins Is Critical for Proper F Protein Folding

“…To identify regions of conserved amino acid sequence within these proteins, sequence alignment of the F proteins from six paramyxovirus family members, representing four genera, was performed and analyzed for blocks of conservation. One conserved block was identified in the fusion peptide/heptad repeat A region, known to play critical roles in F-mediated membrane fusion and to be affected by mutagenesis (22)(23)(24). Two other blocks of conservation were identified: one in the F 2 subunit (Gardner and Dutch, manuscript submitted) and the other in a large intervening region between the heptad repeats of F 1 , designated as the Conserved Block in F 1 /CBF 1 ( Figure 1).…”