Similar to the activity of most paramyxoviruses, the fusion promotion activity of the wt NDV F protein is completely dependent on contributions from the homologous HN protein. One of the functions of HN in fusion is its ability to recognize sialic acid receptors. Despite the conservation of this activity among members of the paramyxovirus genus, heterologous HN proteins cannot substitute for NDV HN in complementing NDV F in the promotion of fusion. This is consistent with the existence of a virus-specific interaction between the two NDV glycoproteins demonstrable at the surface of both infected and transfected cells (7
). The specificity of NDV HN-F-mediated fusion is determined by the stalk region of the HN ectodomain (8
It has previously been demonstrated that a single amino acid substitution, L289A, in the NDV F protein alters the requirement for HN in the promotion of fusion (26
). NDV F, carrying this substitution, is capable of promoting a significant level of syncytium formation in Cos-7 cells in the absence of HN. Here, we show that this HN-independent mode of fusion is apparently specific to this cell line. In several other cell types, L289A-F promotes less than 3% of wt fusion in the absence of HN.
The fact that HN-independent fusion is exhibited only in Cos-7 cells can potentially be explained in several ways. The possibility exists that only Cos-7 cells display an unidentified receptor molecule on their surfaces that is recognized by L289A-F. Alternatively, the unknown molecule may be present on the surfaces of all of the cells tested but may be present in significantly greater amounts on Cos-7 cells. Finally, HN-independent fusion may simply be a reflection of an inherently easier fusogenicity of Cos-7 cells.
We attempted to discriminate between these possibilities by performing the content-mixing fusion assay with HN and F coexpressed in one cell type (BHK or Cos-7) with the other cell type (BHK or Cos-7) serving as receptor-bearing target cells. We have been unable to detect significant fusion with either combination of cells. However, interpretation of these experiments is limited by the fact that Cos-7 cells do not tolerate the vaccinia virus well, necessitating the use of lower MOIs.
The combination of HN and L289A-mutated F consistently promotes a significantly greater amount of fusion than that promoted by the two wt proteins in all of the cell lines tested. Thus, we compared several parameters of the HN-dependent mode of fusion of this mutant F protein to that of wt F. In many ways, the mutated F protein is identical to the wt F protein. As we have shown previously for the wt F protein (8
), L289A-F-induced fusion is not complemented by the heterologous hPIV3 HN protein. Thus, the contribution of receptor recognition activity, even by a heterologous HN protein, is not sufficient for L289A-F to induce fusion. Related to this finding, the mutated F protein shows the same fusion specificity exhibited by the wt protein with respect to NDV-hPIV3 HN chimeras. The stalk region of the HN spike determines the fusion specificity of L289A-F, just as it does the wt F protein.
Finally, L289A-mutated F appears to interact more efficiently with HN than does the wt F protein. We have previously shown that wt HN and F can be coimmunoprecipitated from the surface of cells coexpressing the two proteins with an antibody to the F protein (21
). Approximately 42% of the HN present at the cell surface is coimmunoprecipitated through its interaction with L289A-F. This compares to the 21.5% of HN brought down with the wt F protein in the same experiment. This means that the changes induced in the F protein by the L289A substitution not only fail to interfere with its ability to interact with the HN protein at the cell surface, but rather may slightly enhance the interaction. The mutated F protein also displays a slightly different phenotype with respect to its ability to interact with a panel of NA-HAd- and fusion-deficient HN proteins. D198R- and K236R-mutated HN proteins are weakly coimmunoprecipitated with the L289A-F protein, though they are not with wt F. Also, Y526L- and E547Q-mutated HN proteins, which are not precipitated in significant amounts with the wt F protein, are precipitated in minimal amounts with L289A-F. Most importantly, I175E-HN, approximately 15% of which has been shown to coimmunoprecipitate with wt F despite its lack of receptor recognition activity, interacts more efficiently with L289A-F (approximately 23% of the total amount). Thus, not only does L289A-F interact with HN at the cell surface, but it does so more efficiently than the wt F protein.
However, L289A-F differs from the wt protein in one very important way. Although it requires the receptor recognition activity of the homologous HN protein in order to promote fusion (except in Cos-7 cells), it is much less dependent on this activity than the wt protein. This is demonstrated in several ways. First, the mutated F protein is capable of promoting significant levels of fusion with the I175E, D198R, K236R, Y526L, and E547Q HN mutants. Most notably, the combination of I175E-HN and L289A-F results in more than 50% of the level of fusion achieved with the two wt proteins. Fusion obtained with the other HN mutants and L289A-F ranges between 18 to 36% of that obtained with wt HN and F. This is in contrast to the negligible amount of fusion obtained by coexpression of these mutated HN proteins with wt F. The slight interaction detectable between these HN proteins and L289A-F does not appear to be sufficient to account for the significant level of fusion observed. Thus, L289A-F-induced fusion is still triggered under conditions where the HN-receptor interaction is undetectable by the HAd assay.
Second, L289A-F gives markedly enhanced levels of fusion with HN proteins carrying substitutions for residues F220 and S222. For example, coexpression of either F220N-HN or S222T-HN with wt F results in more than 80% of wt fusion. This is a dramatic increase over the less than 10% of wt fusion obtained when these mutated HN proteins are coexpressed with the wt F protein. The diminished fusogenic activity of these mutated HN proteins correlates with an unstable interaction between HN and receptors on target cells. Evidently, this interaction, although apparently too transient to enable the wt F protein to promote fusion, is strong enough to trigger fusion by L289A-F. The mutated F protein is able to promote fusion even with a weaker HN-receptor interaction.
Third, fusion induced by L289A-F is significantly more resistant to receptor removal than that induced by the wt F protein. Target cells were treated with exogenous NA in an attempt to deplete them of sialic acid receptors. This treatment did not completely eliminate fusion by wt HN and F; 18% of wt fusion survives this treatment. The remaining fusion is likely due to a very low level of receptors replenished during the incubation period of the content-mixing fusion assay. Still, the VCNA-treated monolayers coexpressing L289A-F and wt HN promote a level of fusion that is not significantly different from that obtained with untreated monolayers coexpressing the two wt proteins. Similarly, although fusion induced by wt F and NDV-hPIV3 HN chimeras CH3, CH6, and CH11 is completely blocked by VCNA treatment; monolayers coexpressing these chimeras and L289A-F also promote a level of fusion comparable to that exhibited by untreated monolayers coexpressing the respective chimera and the wt F protein. Again, these findings are consistent with a decreased dependence of L289A-F induced fusion on receptor binding by HN. In fact, the mutated F protein is capable of promoting fusion, albeit to a relatively minimal extent, in sialic acid-deficient Lec2 cells. This minimal amount of fusion is likely due to the fact that Lec2 cells still retain 5 to 10% of the wt amount of sialic acid (9
). Although this amount of sialic acid is insufficient for the wt F protein to promote fusion, it is enough for the L289A-mutated protein.
There are several examples of membrane fusion induced by expression of only the fusion protein. Most notably, the F protein of the W3A strain of another paramyxovirus, simian virus 5, is capable of promoting HN-independent fusion. Based on sequence differences with the F protein of the highly homologous WR strain, which requires HN for fusion, it was shown that two amino acids, proline 22 and serine 443, are responsible for the HN-independent phenotype (23
). Other examples of attachment protein-independent fusion include the F proteins of several respiratory syncytial viruses in the Pneumovirus
) and that of peste-des-petits-ruminants virus, a morbillivirus (27
). However, it should be noted that the fusion promotion activity of each these attachment protein-independent F proteins is significantly enhanced by coexpression of the homologous receptor binding protein, as it is for L289A-mutated NDV F.
Attachment protein-independent fusion has also been induced by site-directed amino acid substitutions in important domains of various F proteins. In this way, it was shown that residues E132 in HR1 and A290 in HR3 also contribute to the HN-independent fusion activity of simian virus 5 F (18
). Similarly, mutations in the cytoplasmic domain of the SER virus F protein rescue syncytium formation and eliminate the HN protein requirement for membrane fusion (28
The mechanism of attachment protein independent fusion is poorly understood. Perhaps, the best-characterized system is that of the respiratory syncytial virus F protein. This protein can bind cellular heparin and heparan sulfate receptors and trigger a conformational change in the F protein, leading to extensive fusion in the absence of the viral attachment protein (1
). Recently, it has been shown that heparan sulfate also acts as a receptor for two other paramyxoviruses, Sendai virus and hPIV3 (2
). Consistent with this, the F proteins of these viruses have several highly basic heparan sufate binding motifs (2
). However, it seems unlikely that heparan sulfate serves as a receptor for NDV F. The only such motif in the protein is the F cleavage site itself, which is not conserved in avirulent strains of the virus (13
). Also, depletion of heparan sulfate from the cell surface with heparinase I has no significant effect on the extent of fusion induced by either wt F or L289A-F (data not shown).
The structural importance of HR3, of which L289 is a part, is most likely related to the fact that it is situated between HR1 and HR2. The L289A substitution may weaken the helical structure of HR3 and thereby lower the energy needed for the conformational change that results in HR1 and HR2 coming together in the fusion-active form of F. In addition, the reduced energy of activation of the L289A-mutated NDV F protein correlates with a conformational change in the protein detectable by conformation-specific antibodies.