1 Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232; 2 Viral Pathogenesis Laboratory, Vaccine Research Center-NIAID, National Institutes of Health, MSC 3017, Bldg. 40 Room 2502, 40 Convent Dr., Bethesda, MD, 208923017, USA
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Abstract |
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Keywords: RSV , polyanions , fusion inhibition , dextran sulphate , sulphated polysaccharides , antiviral agents , post-attachment neutralization , human immunodeficiency virus , HIV , heparin , heparan sulphate
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Characterization of RhoA-derived peptides |
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RSV entry depends upon the action of the F glycoprotein, which shares structural homology with the fusion-mediating proteins of several other viruses, including the well-studied HIV gp160 and influenza HA glycoproteins.6,7 Like that of many other enveloped viruses, the attachment of RSV to host cells is facilitated by charge-mediated interactions between the RSV envelope glycoproteins and heparan sulphate or other sulphated proteoglycans on the cell surface.812 This initial attachment is probably followed by one or more specific proteinprotein interactions between the RSV surface glycoproteins and unidentified cell surface receptors. These specific interactions would trigger the conformational changes in F required to bring about membrane fusion. In an attempt to identify host cell proteins that might interact with RSV F, Pastey et al.13 conducted a yeast-two-hybrid (Y2H) screen that identified the small intracellular GTPase RhoA as a potential F-binding protein. Although the intracellular location of RhoA made it a topologically unlikely candidate to interact with RSV F, epitope mapping in the Y2H system showed that the region of RSV F that mediates this interaction localizes to the N-terminal portion of the F1 subunit. This region overlaps the hydrophobic fusion peptide of RSV F, which inserts into the host cell membrane during the process of membrane fusion. Since RhoA localizes to the cytoplasmic face of the plasma membrane when activated, the possibility that the RSV F fusion peptide might in some way contact RhoA subsequent to membrane penetration warranted further investigation.
The Y2H system was further used to identify amino acids 67105 as the region of RhoA responsible for F binding in this system.13 Studies of peptides derived from this region revealed that a 19-amino-acid peptide comprising residues 7795 of RhoA (peptide 7795) could inhibit replication of RSV and a related virus, PIV3, in cultured cells. This peptide also reduced illness and viral replication in a mouse model of RSV infection.14 Because peptide 7795 interferes with binding of purified F to RhoA in an in vitro ELISA assay, it was hypothesized that competition with an in vivo interaction between RSV F and RhoA might be the basis for the peptide's antiviral effect.14 However, several recent observations suggest that this is not the case. Truncation studies have shown that the region of peptide 7795 most critical for inhibition of RSV is not surface exposed (based on several crystal structures of whole RhoA). In addition, optimal antiviral activity of a slightly truncated peptide comprising amino acids 8094 of RhoA requires oxidation of an internal cysteine residue, resulting in the formation of peptide dimers.15 This optimal peptide sequence, ILMCFSIDSPDSLEN, has a hydrophobic and anionic character, and the net negative charge of the peptide is important for its antiviral activity.16 Dependence on charge and molecular weight is characteristic of broadly antiviral anionic molecules, including sulphated polysaccharides (such as heparan sulphate or dextran sulphate) and numerous synthetic anionic polymers.17
In agreement with the physical characteristics of the peptide, inhibition of RSV by oxidized peptide 8094 appears similar to that of soluble heparin or dextran sulphate. This can be mainly attributed to inhibition of viral attachment, presumably by competing with cell surface heparan sulphate for binding to the RSV surface glycoproteins.16 The effect of oxidized peptide 8094 on RSV entry is also highly dependent on the presence of the RSV G glycoprotein, further indicating that effects of this peptide should not be attributed to the disruption of a hypothetical interaction between RSV F and RhoA. In addition to blocking viral attachment, peptide 8094 also prevents viral entry when added to virus that has been pre-bound to host cells at 4°C prior to warming to 37°C.16 This is also the case for soluble heparin and dextran sulphate;16,18 however, the latter inhibitors are capable of displacing virus-bound virus from the host cell surface, whereas peptide 8094 is not (P. J. Budge, unpublished observations). This suggests that in addition to disrupting viral attachment, peptide 8094 may also directly affect viral fusion. Hydrophobic peptides have long been known to affect paramyxovirus fusion,19 and it may be that the hydrophobic portion of the 8094 peptide contributes to fusion inhibition, whereas the anionic portion of the peptide allows for its effects on viral attachment. The combination of hydrophobicity and negative charge appears to be an effective pattern for many antiviral molecules that target RSV,20,21 as well as many other enveloped viruses.17
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Parallels to CD4-derived peptides |
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Implications
There are two important questions regarding the clinical relevance of RhoA-derived peptides. First, might the peptides themselves be used as antiviral drugs in a clinical setting? Second, has work with RhoA-derived peptides provided insights that might be applied to the development of other clinically relevant antiviral agents?
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Clinical potential of RhoA-derived peptides |
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General clinical implications |
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Inhibition of RSV and HIV by anionic peptides suggests that the low-affinity, ionic interactions these viruses use to facilitate their binding to host cells may be exploited to enhance the activity of antiviral agents. Indeed, heparin-binding sites might generally facilitate the binding of negatively-charged inhibitors to other susceptible sites on the virus surface. For example, attractions between CDR3-derived peptides and the V3 loop of gp1203436 may result in their concentration around the CD4-binding site of gp120. One possible explanation for inhibition of HIV by these peptides would be that their attraction to the V3 loop facilitates and enhances a hydrophobic interaction between the derivatized peptides and the hydrophobic Phe-43 cavity of gp120.26 In this way, the combination of negative charge and hydrophobicity of these peptides may allow for efficient binding of CDR3-derived peptides to the CD4-binding site despite the fact that this does not mimic a physiologically relevant interaction. Similarly, RhoA-derived peptides may be attracted to important hydrophobic regions of RSV F or G based on ionic interactions with neighbouring positively-charged regions of these molecules. Even if the initial ionic attractions do not directly contribute to a specific fit [i.e. might not be part of the specific contact surface between the peptide and its binding target(s)], they may attract the peptide to sites on F or G where more specific binding contacts may be formed. This would effectively enhance an otherwise low affinity binding of the peptide to these regions. Thus, ionic attractions may improve the observed affinity of some interactions even if they do not directly contribute to a specific fit. This may be one reason for the recently observed enhancement of HIV neutralization by antibodies containing acidic CDR3 loops and sulphated tyrosine residues,37 or for the observed importance of the core sulphate moieties to the RSV-specific fusion inhibitor RFI-641.5
It would be interesting to assess whether adding negative charges to specific fusion inhibitors of RSV or other heparin-binding viruses might increase their antiviral activity. For example, the addition of negative charges to C-terminal heptad repeat peptides38 or neutralizing antibodies should not affect the specific affinity of these molecules for their targets. However, it may increase their overall antiviral efficacy, by increasing their initial attraction to (i.e. local concentration at) the viral surface. As mentioned above, tyrosine sulphation contributes to the antiviral efficacy of some neutralizing anti-HIV antibodies.37 While the mechanism of this enhancement is not known, it is possible that sulphation may lead to charge-mediated concentration of antibody at or around the positively charged V3 loop of HIV gp120, as suggested above. In a similar manner, chemical sulphation of anti-RSV neutralizing antibodies currently marketed for RSV prophylaxis might lead to enhancement of their antiviral activity. Investigating the effects of adding negative charge to specific antiviral agents may lead to more potent and more broadly neutralizing antiviral therapies for RSV and other viruses.
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Footnotes |
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* Present address. Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, 37232, USA.
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References |
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