Peptide inhibition of fusion.
Membrane fusion is thought to occur in a series of steps including attachment of the attack membrane to the target membrane, activation of a fusion protein, close approach of the attack and target membranes, hemifusion, pore formation, and pore expansion (24
). In most paramyxovirus systems, the coexpression of the HN and F proteins is both necessary and sufficient for these steps (reviewed in 24
). NDV attachment is minimally mediated by the HN protein (28
). Paramyxovirus fusion protein activation requires the cleavage of the F0
protein to form a disulfide-linked F1
). In addition, other changes in the F protein, probably mediated in some way by the HN protein, are required since a cleaved F protein alone will not direct fusion in most systems (2
). Close approach requires that the attack and target membranes be pulled together. How this step occurs is not clear; however, it has been proposed that the HIV env protein accomplishes this step by the interaction of two distant domains in the gp41 protein, an interaction thought to be initiated by the attachment of the env to its receptor (11
Candidate domains for such an interaction in the paramyxovirus fusion protein are two heptad repeat regions (HR domains) initially recognized by Chambers et al. (9
). HR1 is located just carboxyl terminal to the fusion peptide and may therefore be initially located near the target membrane. HR2 is adjacent to the transmembrane domain and is therefore located near the attack membrane. Thus, an interaction of these two domains during the fusion process could cause the close approach of the two membranes. Mutational analyses of both regions of the several F proteins including the NDV F protein have shown that alterations in the sequences of both these regions inhibit fusion, suggesting that they play a central role in the fusion process (4
We and others have previously shown that a peptide with a sequence from the F-protein HR2 region can inhibit fusion mediated by the coexpression of the HN and F proteins (4
). Here, we have shown that a peptide with a sequence from the NDV HR1 domain, from amino acids 150 to 174, will also inhibit fusion directed by the NDV F and HN proteins. The inhibitory activity of the peptide is comparable to that of the peptide derived from the HR2 region. Both peptides show 50% inhibition at approximately 2 μM. Previously, Rapaport et al. (37
) showed that a peptide with a sequence from the Sendai virus F HR1 region did not inhibit fusion. However, this peptide was derived from a sequence slightly more carboxyl terminal than the one characterized here. Lambert et al. reported that peptides from HR1 sequences in the respiratory syncytial virus, human parainfluenza virus 3, and measles virus HR1 regions inhibit fusion, but the inhibition was not further characterized (29
). Joshi et al. have recently reported that a much larger peptide, with a sequence from amino acids 129 to 184, inhibits SV5 fusion, although not as efficiently as a peptide from the HR2 region of the SV5 F protein (26
In contrast to results described for other paramyxovirus systems (26
), we have found that the NDV HR1 peptide will not inhibit fusion unless the peptide is added before the cleavage of surface-expressed F protein. Cleavage of our wild-type F protein (derived from virulent NDV) is mediated by the host cell protease furin, located in the trans
-Golgi membranes (22
). Thus, the protein expressed at the surface is cleaved. Fusion of cells expressing this F protein is not inhibited by the HR1 peptide. However, alteration of the F-protein furin recognition site by mutagenesis results in the expression of F0
at the cell surface, and the mutant F0
can be readily cleaved upon the addition of exogenous trypsin, resulting in synchronized fusion (30
). Addition of the HR1 peptide prior to trypsin results in inhibition of fusion. The HR1 peptide does not, however, block the cleavage of the F protein. This result suggests that the target of the HR1 peptide is accessible only prior to cleavage or, alternatively, that binding of the HR1 prior to F protein cleavage may inhibit a conformational change in a protein that occurs upon cleavage.
We have also found that the inhibition mediated by the HR1 peptide is virus specific. We were able to explore this question by using Sendai virus glycoproteins, since the Sendai virus F protein must also be cleaved by the addition of exogenous trypsin (28
). The peptide only minimally affects fusion directed by the Sendai virus HN and F proteins. Thus, it is likely that the HR1 peptide binds to a target protein via interactions with sequence motifs unique to a specific protein.
The target of the HR1 peptide is potentially the lipid bilayer, host cell proteins, HN protein, or F protein. Indeed, Ghosh et al. have reported that peptides from the HR1 and HR2 regions of the Sendai virus F protein will bind to lipid bilayers (18
). However, such an interaction is not likely to be directly responsible for fusion inhibition, since the inhibition is virus specific. Similar arguments can be made in ruling out a host cell protein as primary target. While our data cannot exclude the HN protein as a target for the peptide, we explored potential interactions of the HR1 peptide with the HR2 peptide because of results with gp41-derived peptides (31
). Our results clearly show that the HR1 peptide can interact specifically with the HR2 sequence. This result was obtained in two different assays. One directly measured the binding of the HR2 peptide tagged with biotin to the HR1 peptide. This interaction was specific for the HR1 peptide and was competed by the addition of untagged HR2 peptide. The second method was a functional assay of the interaction of the two peptides. Importantly, our results show that equimolar mixture of the two peptides minimally inhibited fusion. Furthermore, relief of inhibition was maximal when the peptides were present in equimolar amounts, suggesting a 1:1 complex. These results are consistent with the notion that the two peptides form a complex which inhibits the interaction of either peptide with the intact protein and therefore relieves the inhibition observed with each peptide when added alone.
Using very different methods, Ghosh et al. (19
) have also reported data suggesting that peptides from the HR1 and HR2 regions of Sendai virus interact. Joshi et al. (26
) have also reported that peptides from HR1 and HR2 regions of the SV5 F protein coassemble. However, their complex still inhibits fusion, in contrast to the results reported here for the NDV system. The reasons for this different result are unclear, although the difference may indicate that the SV5 complex may be somewhat different than the NDV complex described here. Furthermore, the peptides used by Joshi et al. were much larger than those described here, and the two peptides were of different lengths, in contrast to those described here, which differed in length by only 4 amino acids.
Our results do not address the precise form of the complex between the two peptides. By direct analogy to the HIV env structure, Joshi et al. (26
) proposed that the HR1 and HR2 peptides form a six-stranded complex, with the HR1 forming a trimer and three HR2 peptides lying on the outside. Indeed, because of the hydrophobic face of the HR1 peptide, it is likely to form an oligomeric structure in aqueous solution, and, by gel filtration, we have obtained results consistent with the proposal that HR1 forms an oligomer (unpublished data). However, the NDV HR2 peptide may also form an oligomer in an aqueous environment. The results of NMR analyses suggest an ordered helical structure with a hydrophobic face which should promote oligomer formation. Indeed, gel filtration analysis has yielded results consistent with a trimer (unpublished data). Furthermore, Ghosh et al. have reported that an HR2 peptide with sequences from the Sendai virus HR2 region self-assembles in aqueous solution (19
). Thus the HR1-HR2 complex described here may result in an association of an oligomer of HR1 and an oligomer of HR2. Indeed, our choice of a sequence for the HR1 peptide was governed by a potential interaction which could occur between the charged surfaces of the two peptides (Fig. C), surfaces that would be exposed if the peptides formed oligomeric structures with their hydrophobic faces in the interior.
Predictions of the structures of the inhibitory forms of the peptides and their potential interactions in the context of the cell membrane are considerably complicated by the report that peptides with sequences from both the HR1 and HR2 regions of the Sendai virus F protein can associate with lipid (18
). The results presented in this paper are consistent with the idea that the peptides bind, as monomers, parallel to the bilayer, with the hydrophobic face of the peptides buried in the membrane. Such an interaction would promote the dissociation of any oligomers in the presence of the cell membrane and allow for alternative associations of the peptides with each other as well as with the intact protein. Indeed, our results reported here show that the HR1 peptide retains an ordered α-helical structure in a hydrophobic environment. Furthermore, any conclusions about structures in the fusion-active form of the intact F protein as well as any prefusion conformation should also be tempered by the presence of other possible HR domains in the F protein that may be involved in fusion. For example, Ghosh et al. (18
) reported that peptides from other HR domains within the Sendai virus F protein sequence had inhibitory activity and potential to interact with peptides from the HR1 and HR2 domains (18