Large (≥5 kDa) polyanionic molecules have been shown to be excellent in vitro inhibitors of attachment and entry for several enveloped viruses, including herpesviruses, retroviruses, orthomyxoviruses, and paramyxoviruses (11
). For many of these viruses, this has been shown to be due to the blocking of important charge-charge interactions involved in the binding of the virus to its cellular receptor(s). Such an inhibition is generally dependent upon the molecular weight and extent of the negative charge of the polyanions used and is somewhat nonspecific, in the sense that the interactions are thought to be largely charge mediated and of low affinity and may involve several sites on a particular viral or cellular protein (32
). The inhibition of human immunodeficiency virus (HIV) by dextran sulfate, for example, has been shown to involve the association of dextran sulfate with several positively charged regions of the surface glycoprotein gp120 (7
) and to interfere with the binding of gp160 to its receptor (30
), coreceptor (39
), and cell surface heparan sulfate (38
). Although not as thoroughly studied, RSV is also inhibited by large polyanions, and for RSV this inhibition was shown to occur at the levels of both binding and fusion (25
When the antiviral activity of a RhoA-derived peptide was originally described, it was in the context of a recently described interaction between RSV F and RhoA (43
), and the possibility of a direct interference upon F-RhoA interaction was considered as a mechanism of action (44
). While our present data do not directly address this hypothesis, they do indicate that the antiviral activity of the RhoA-derived peptide 80-94 is likely to be more general in nature. Previous work indicated that the antiviral potency of RhoA peptides is not correlated with their structural similarity to RhoA (5
), nor does their mechanism of activity seem to be specific for RSV F, given the relative insensitivity of the rgRSV-F virus to inhibition by peptide 80-94. In this light, the ability of RhoA-derived peptides to inhibit RSV entry should be viewed as a phenomenon separate from and perhaps unrelated to the previously described interaction between F and RhoA.
A more plausible explanation for the antiviral effects of the 80-94 peptide is suggested by the polyanionic nature of the peptide itself and its dependence on multimerization for its antiviral activity, both of which are hallmarks of previously described polyanionic inhibitors of enveloped viruses. The RhoA-derived peptide 80-94, like other polyanions, blocks the binding of RSV to host cells with approximately the same effective concentration at which it inhibits viral replication. This inhibition is dependent on the G protein of RSV, in agreement with its role as the primary attachment protein. Interestingly, the peptide was also effective at inhibiting the infectivity of a virus that had been prebound at 4°C. This agrees with the previous observation that polyanions can block both binding and postbinding entry events of RSV (25
) and with the observation of a fusion-inhibiting activity of the 77-95 peptide in previous studies (44
). The basis for the inhibition of RSV entry at a postbinding step by heparin, oxidized peptide 80-94, and other polyanions (25
) is not clear and is the object of ongoing research. It has previously been shown that the N-terminal fusion peptide of gp41, the analogous viral fusion protein of HIV, can interact with polyanions (17
), suggesting that polyanions may (at least for HIV) have a direct effect on viral fusion. However, the relative ineffectiveness of peptide 80-94 in inhibiting replication of the F-only virus indicates that direct effects of peptide 80-94 on the F protein are minimal. Given the dependence of peptide 80-94 on G for its antiviral effect, it may be that the peptide binds to complexes of F and G at the virion surface or to a conformation of F that is dependent upon the coexpression of G. Alternatively, the binding of peptide or other large polyanions to G may sterically block important sites on F that are necessary for the process of fusion. A requirement for steric hindrance or multisite binding would be consistent with the observation that the peptide activity requires multimerization. The identification of the specific binding targets of the 80-94 peptide is an ongoing area of research.
The identification of an in vitro interaction between RhoA and RSV F (43
) and a subsequent report of antiviral activity by a RhoA-derived peptide (44
) have led to several investigations into the role of RhoA in RSV infections. The results presented here indicate that the inhibition by RhoA-derived peptides is a function of the intrinsic biophysical properties of the peptides themselves and has no bearing on an interaction between RSV glycoproteins and RhoA in an infected cell. While RhoA and RSV F do interact in vitro, there is no evidence that they directly interact during RSV infection, and the inhibition of RSV by RhoA-derived peptides should not be seen as substantiating this hypothesis.
Even though RhoA and RSV F may not directly interact in vivo, RhoA signaling pathways are involved in several important aspects of RSV biology. RhoA is activated during RSV infections of cultured cells (18
), and this activation is necessary for the production of viral filaments, although it is not essential for viral replication (36
). RhoA activation appears to be important for the budding of RSV from lipid rafts (36
), and cytoskeletal rearrangements mediated by RhoA contribute to, but are not essential for, the growth of RSV in some cell lines (3
). RhoA signaling may also contribute to RSV pathogenesis, since interference with RhoA signaling pathways reduces the airway hyperreactivity induced by RSV infection in sensitized mice (23
). Thus, RhoA appears to play an important role in RSV infection and disease, although this is likely due to the intrinsic importance of RhoA to cellular physiology (15
), rather than as a specific binding partner for RSV F, as was previously hypothesized (43
Although the antiviral activity of RhoA-derived peptides is likely unrelated to the role of RhoA in RSV biology, studies of these peptides have provided insights into the ways in which RSV entry can be inhibited. We have shown here that the RhoA-derived peptide 80-94 is able to block both the attachment of RSV to host cells and a postattachment step in the RSV entry process and that this activity is dependent on both molecular weight and the net negative charge. This activity is largely dependent upon the presence of the G protein of RSV and appears functionally similar to RSV inhibition by soluble heparin or dextran sulfate. A further investigation of the postattachment neutralization of RSV by these molecules should lead to a better understanding of the process of RSV entry and the respective roles of RSV F and G in this process.