TY - JOUR
T1 - Functional Analysis of the Transmembrane Domain in Paramyxovirus F Protein-Mediated Membrane Fusion
AU - Bissonnette, Mei Lin Z.
AU - Donald, Jason E.
AU - DeGrado, William F.
AU - Jardetzky, Theodore S.
AU - Lamb, Robert A.
N1 - Funding Information:
We thank Charles Russell and Sarah Connolly for helpful discussions. The N-1 HA-tag peptide construct was kindly provided by Sarah Connolly. This work was supported in part by Research Grant R01 AI 23173 from the National Institute of Allergy and Infectious Diseases. M.L.Z.B and J.E.D. were supported by National Institutes of Health Medical Scientist Training Program Grants T32 GM08152-18 and HL107971, respectively. RAL is an investigator of the Howard Hughes Medical Institute.
PY - 2009/2/13
Y1 - 2009/2/13
N2 - To enter cells, enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single-cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the paramyxovirus parainfluenza virus 5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion.
AB - To enter cells, enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single-cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the paramyxovirus parainfluenza virus 5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion.
KW - modeling a transmembrane domain
KW - oxidative cross-linking
KW - protein refolding intermediates
KW - transmembrane domain function
KW - viral membrane fusion
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U2 - 10.1016/j.jmb.2008.12.029
DO - 10.1016/j.jmb.2008.12.029
M3 - Article
C2 - 19121325
AN - SCOPUS:58549102514
SN - 0022-2836
VL - 386
SP - 14
EP - 36
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -