Inhibition of respiratory syncytial virus by RhoA-derived peptides: implications for the development of improved antiviral agents targeting heparin-binding viruses

Philip J. Budge1,2,* and Barney S. Graham2,{dagger}

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, 20892–3017, USA


    Abstract
 Top
 Abstract
 Characterization of RhoA-derived...
 Parallels to CD4-derived...
 Clinical potential of RhoA...
 General clinical implications
 Footnotes
 References
 
The respiratory syncytial virus (RSV) fusion glycoprotein (F) can interact with the small intracellular GTPase RhoA, and peptides derived from RhoA inhibit RSV replication. These observations initially suggested that RhoA-derived peptides might inhibit RSV replication by disrupting an in vivo interaction between RSV F and RhoA. However, recent data indicate that the antiviral activity of RhoA-derived peptides is not due to competitive inhibition of an hypothesized F–RhoA interaction, but is rather a function of the peptides' intrinsic biophysical properties. We summarize here what is known about the mechanism of RSV inhibition by these peptides and give our opinion regarding the potential implications of this work with regards to RSV biology, and to the development of antiviral agents targeting RSV and other enveloped viruses.

Keywords: RSV , polyanions , fusion inhibition , dextran sulphate , sulphated polysaccharides , antiviral agents , post-attachment neutralization , human immunodeficiency virus , HIV , heparin , heparan sulphate


    Characterization of RhoA-derived peptides
 Top
 Abstract
 Characterization of RhoA-derived...
 Parallels to CD4-derived...
 Clinical potential of RhoA...
 General clinical implications
 Footnotes
 References
 
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory illness in infants worldwide and is also a significant cause of morbidity and mortality in adult populations.1,2 Because there is currently no licensed vaccine for RSV, there is substantial interest in the identification and development of RSV-specific antiviral agents.35 Agents that target the process of viral entry are of particular interest, since these might be used for prophylaxis against RSV as well as for treatment of early infections.

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 protein–protein 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 67–105 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 77–95 of RhoA (peptide 77–95) 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 77–95 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 77–95 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 80–94 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 80–94 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 80–94 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 80–94 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 80–94 is not (P. J. Budge, unpublished observations). This suggests that in addition to disrupting viral attachment, peptide 80–94 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 80–94 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


    Parallels to CD4-derived peptides
 Top
 Abstract
 Characterization of RhoA-derived...
 Parallels to CD4-derived...
 Clinical potential of RhoA...
 General clinical implications
 Footnotes
 References
 
It is interesting to note that inhibition of RSV by RhoA-derived peptides contains many parallels to inhibition of HIV by peptides derived from the CDR3-like region of CD4 (first reported by Lifson et al.22 in 1988). Like the RhoA-derived peptides, these peptides contain multiple acidic residues, and their antiviral activity depends upon modifications that increase their hydrophobicity, such as benzylation, sequence scrambling or dimerization.2325 Early work with these peptides was based upon the assumption that they specifically blocked the interaction between CD4 and gp120. However, the co-crystal structure of a portion of gp120 bound to a portion of CD4 conclusively showed that the interaction between these two molecules did not involve the region from which the peptides were derived.26 Like the RhoA peptides described above, the antiviral activity of peptides derived from the CDR3-like region of CD4 appears to be based on non-specific masking of important charged regions of the viral envelope protein. In this regard, it is not surprising that RhoA-derived peptide 77–95 also inhibits HIV replication in vitro (P. J. Budge & B. S. Graham, unpublished data).

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?


    Clinical potential of RhoA-derived peptides
 Top
 Abstract
 Characterization of RhoA-derived...
 Parallels to CD4-derived...
 Clinical potential of RhoA...
 General clinical implications
 Footnotes
 References
 
Oxidized RhoA-derived peptide 80–94 inhibits a variety of RSV strains at sub-micromolar concentrations in vitro (P. J. Budge & B. S. Graham, unpublished data). Whether this might translate into in vivo efficacy is unclear. Pastey et al.14 showed that RhoA-derived peptide 77–95 inhibited RSV replication in the lungs of mice when given intranasally prior to, or shortly after, virus challenge. Based on this observation, it seems that RhoA-derived peptides might be able to prevent or reduce RSV infection when given intranasally as therapy, or as a prophylactic measure. Because viral clearance in typical RSV infections has already begun before the peak of clinical symptoms, any antiviral therapy would need to be initiated very early to have an effect on disease. In this light, perhaps the most likely use for RhoA-derived peptides would be prophylactic administration to decrease nosocomial spread of RSV. Whether adequate concentrations can be attained on respiratory surfaces following intranasal administration, without accompanying toxic effects, are issues that would need to be addressed in subsequent studies. Even if peptides should prove safe and effective in such a setting, the cost of synthetic peptide synthesis and purification would have to be competitive with antibodies or small molecules being developed for the same application.2729 The demonstration of clinical antiviral efficacy by the HIV fusion inhibitor enfuvirtide (T-20) shows that synthetic peptides can be developed into effective antiviral drugs.30,31 However, much more needs to be known about the safety, bioavailability and in vivo stability of the RhoA-derived peptides before their potential for therapeutic use can be estimated.


    General clinical implications
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 Abstract
 Characterization of RhoA-derived...
 Parallels to CD4-derived...
 Clinical potential of RhoA...
 General clinical implications
 Footnotes
 References
 
Many viruses expedite their entry into host cells via initial interactions with cell surface heparan sulphate or other sulphated glycosaminoglycan (GAG) molecules.32,33 In most cases, heparan sulphate is not the final viral receptor, but plays a role in facilitating attachment of viruses to one or more specific receptors.32 For these viruses, low-affinity, charge-mediated interactions with heparan sulphate may attract and keep the virus at the cell surface, yet still allow significant off-rates. This may give the virus the opportunity to ‘sample’ the cell surface for its specific receptor without being subject to the limiting effects of diffusion in the extracellular fluid or displacement by movement of the mucociliary blanket. In this way, heparin-binding viruses may have evolved to take advantage of low-affinity, charge-mediated interactions with cellular heparan sulphate, or other sulphated GAGs, to increase the efficiency of their entry into host cells.

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.


    Footnotes
 
{dagger} Corresponding author. Tel: +1-301-594-8468; Fax: +1-301-480-2771; Email: bgraham{at}nih.gov

* Present address. Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, 37232, USA. Back


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 Abstract
 Characterization of RhoA-derived...
 Parallels to CD4-derived...
 Clinical potential of RhoA...
 General clinical implications
 Footnotes
 References
 
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