Abstract
Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability.
Highlights
Enveloped viruses, including members of the paramyxovirus family, such as measles virus, mumps virus, Sendai virus, and the zoonotic Hendra virus (HeV),2 utilize surface membrane
To further define elements that drive TM-TM interactions in Hendra F, we analyzed the TM domain sequence for motifs involved in protein-protein association and identified a heptad repeat leucine-isoleucine zipper that continued through the TM domain in-frame with the leucine zipper of the upstream heptad repeat B (HRB) domain (Fig. 1A)
To determine whether this motif could be involved in TM-TM association in other viruses, the sequences of 140 paramyxovirus F proteins were analyzed for amino acid frequency in the predicted TM domain
Summary
To further define elements that drive TM-TM interactions in Hendra F, we analyzed the TM domain sequence for motifs involved in protein-protein association and identified a heptad repeat leucine-isoleucine zipper that continued through the TM domain in-frame with the leucine zipper of the upstream HRB domain (Fig. 1A). When normalized to WT at each spin speed, the LIZ 4A mutant displayed an approximately 1000-fold decrease in association constant, suggesting weaker TM-TM interactions (Table 1) These results implicate the predicted L/I zipper motif in TM-TM association for the HeV F TM domain. The analytical ultracentrifugation data suggested a shift in equilibrium upon mutation of the L/I zipper motif in the isolated TM domain, consistent with reduction in TM-TM association To determine how these mutations affected expression, intracellular transport, and function, the LIZ 4A mutant protein was analyzed for total and surface expression by cell surface biotinylation. The single point mutant L509A presented with a large reduction in total and cell surface protein expression levels, similar to that of LIZ 4A (Fig. 3B). The relative apparent association constant (Ka) is normalized to the WT SN-TM construct for each spin
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