Antiviral Activity of RhoA-Derived Peptides against Respiratory Syncytial Virus Is Dependent on Formation of Peptide Dimers

Abstract

A synthetic peptide containing amino acids 77 to 95 of the intracellular GTPase RhoA has previously been shown to inhibit replication of respiratory syncytial virus (RSV) in cultured cells. We show that residues 80 to 90 of RhoA are sufficient for this activity and that the cysteine residue at position 83 is critical. Further studies with an optimal peptide sequence containing amino acids 80 to 94 of RhoA revealed that the antiviral potency of the peptide is dependent on the oxidation of cysteine 83. Size-exclusion chromatography and sedimentation equilibrium studies of the peptide comprising residues 80 to 94 revealed that it is capable of forming aggregates in both reduced and oxidized states. A peptide (83A) in which the cysteine residue is replaced by an alanine does not form dimers or higher-order aggregates and did not inhibit RSV replication at any concentration tested. These data indicate that formation of peptide multimers is necessary for the antiviral activities of RhoA-derived peptides and suggest that the observed antiviral activities of these peptides may be unrelated to the biological functions of their parent molecule.Respiratory syncytial virus (RSV) is an enveloped, negativesense, single-stranded RNA virus of the Paramyxovirus family. It is the most important cause of lower respiratory tract infections in children worldwide and is a significant cause of morbidity and mortality among immunocompromised adults and the institutionalized elderly (4). RSV enters host cells by fusing its viral envelope with the host cell plasma membrane to allow penetration of the viral nucleocapsid. The processes of viral attachment and fusion are mediated by the viral glycoproteins, termed the fusion protein (F), glycoprotein (G), and the small hydrophobic protein (SH). While G and SH can increase the efficiency of the viral entry process, F alone is sufficient (10,13,27).It has previously been observed that RSV F can interact with the small GTPase RhoA (20) and that a peptide derived from the F-binding region of RhoA is able to block the entry of RSV into susceptible host cells (21). This peptide, which we termed peptide 77-95, comprises the linear peptide sequence corresponding to amino acids (aa) 77 to 95 of RhoA. On the basis of the observation that peptide 77-95 can interfere with binding of F to RhoA in an in vitro enzyme-linked immunosorbent assay (ELISA), it was originally hypothesized that peptide 77-95 may inhibit an interaction between RSV F and RhoA essential to F-mediated membrane fusion (21). However, an in vivo interaction between F and RhoA at the time of viral entry has not been demonstrated. In addition, other agents that should be capable of inhibiting a RhoA-F interaction, such as anti-RhoA antibodies and exogenous, purified RhoA, have no inhibitory effect on RSV entry (unpublished data). Thus, the ability of the RhoA-derived peptide to inhibit RSV entry may be unrelated to its ability to disrupt an in vitro F-RhoA interaction.The region from aa 77 to 95 of RhoA corresponds to an internal beta st...

In Silico Discovery of Antimicrobial Peptides as an Alternative to Control SARS-CoV-2

“…Similar to this study, in silico analyses showed the potent antiviral effects of AMPs against Betacoronavirus [ 27 , 39 ]. According to our results, the AMPs are attractive candidates as alternative to conventional antiviral drugs to control SARS-CoV-2 infection, because they offer several potential advantages, including specific anti-CoV effects, high selectivity, and do not be associated with severe adverse effects according to in vitro and in vivo assays [ 58 , 59 , 60 ].…”

RhoA-Derived Peptide Dimers Share Mechanistic Properties with Other Polyanionic Inhibitors of Respiratory Syncytial Virus (RSV), Including Disruption of Viral Attachment and Dependence on RSV G

“…In agreement with this, we have also observed that the addition of positively charged lysine residues to RhoA-derived peptides from the aa 77 to 95 region abolishes their antiviral activity. This observation was made when lysine residues were added to several previously tested peptide 77-95 derivatives (5) in an effort to simplify their elution from a reverse-phase high-performance liquid chromatography column. In each instance, the addition of three C-terminal lysines resulted in a loss of antiviral activity compared to the corresponding peptide without the C-terminal lysines (data not shown).…”

“…Based on truncation studies, it was shown that a slightly shorter peptide comprising amino acids (aa) 80 to 94 of RhoA was optimal for anti-RSV activity. Interestingly, the antiviral activity of this peptide was dependent upon the oxidation of an internal cysteine residue, resulting in the formation of peptide dimers (5). This dependence on an increased molecular weight, coupled with the anionic nature of the optimal peptide sequence, suggested that the antiviral activities of RhoA-derived peptides are unrelated to a specific in vivo F-RhoA interaction, but rather are a reflection of their similarities to other antiviral polyanions.…”

Induction of protective effector immunity to prevent pathogenesis caused by the respiratory syncytial virus. Implications on therapy and vaccine design

“…A similar pharmacological approach consisted of the peptide Rho‐A, which inhibits the syncytia formation that is characteristic of hRSV infection. RhoA is a small GTPase that is involved in the fusion process and the inhibitor of this protein has been tested in HEp‐2 cells and mice, with promising results …”

“…RhoA is a small GTPase that is involved in the fusion process and the inhibitor of this protein has been tested in HEp-2 cells and mice, with promising results. 56,61 Besides peptides that inhibit hRSV fusion, there are several other chemical compounds that impair the fusion process. The benzimidazole JNJ2408068 has shown a high antiviral activity, 100 000 times higher than ribavirin and acts by preventing virus fusion and syncytia formation.…”