lls (Fig 7). An ~80% reduction in infection was observed with herpes simplex virus (HSV) 1. However, reduction of HSV 2 infection was less remarkable (~40% reduction).
Cytomegalovirus infection is a significant clinical problem in infants and immunodeficient populations. There are two major problems with current anti-CMV treatments. First, current anti-CMV therapies have significant organ toxicity. Secondly, resistance to current therapies is increasing. In this study we examined a panel of synthetic peptides that bind MLN-8237 hypersulfated GAGs for their ability to inhibit herpesvirus infection, using MCMV as a model system. Of the seven peptides evaluated in this study, peptide p5+14 demonstrated effective inhibition of MCMV infection and reduced infection of both HCMV and HSV (HSV-1 and 2) in vitro (Figs 1A and 7). This suggests a broader applicability of GAG-binding synthetic peptides for inhibiting virus-cell interactions. We established that the peptide effectively competed for adsorption of CMV to susceptible cells, thereby reducing infection. We also demonstrated that the peptide does not have a direct neutralizing effect on the virus itself. The p5-related peptides are synthetic polybasic reagents with a predicted -helical secondary structure. The heptad amino acid repeat-KAQKAQA- positions the Lys residues along one face of the helix. This structural feature was engineered and intended to facilitate an interaction with linear sulfated GAG molecules, notably heparin [39, 42]. Due to their ability to preferentially bind hypersulfated GAGs these peptides have been used to effectively target and image tissue amyloid deposits [25, 27], which contain hypersulfated HS and possibly CS proteoglycans [43]. Remarkably, when radiolabeled the p5 and p5+14 peptides were injected in diseasefree mice, peptide did not bind to GAGs expressed in healthy organs or tissues [25]. This lead us to hypothesize that the linear positive charge on peptide p5+14 facilitates binding to negatively charged PGs on the cell surface, which mediates antiviral activity. This is supported by the fact that peptides with the same net positive charge exhibit differential anti-viral effects that are consistent with the peptide affinity for the GAGs and subsequently amyloid. Thus, peptide p5R (+8 charge), which has a higher affinity for heparin [39] and amyloid [44] as compared to peptide p5 (+8 charge), blocks viral infection 2-fold better (Fig 1A). These data suggest that the secondary structure of the peptides, as well as the overall net charge, affects binding to specific GAGs on the cell surface and their subsequent anti-viral activity. Based on the known restricted reactivity of peptides p5 [25] and p5+14 in vivo, our data using p5+14 suggests that CMV may preferentially bind hypersulfated GAGs, such as 6-O-sulfated GAGs [40] on the cell surface of cultured fibroblasts. This may differ from the ubiquitously expressed GAGs found in tissue HSPG and CSPG proteins in vivo. This is similar to the proposed mechanism for HSV that uses multiple different interactions for entry including 3-O sulfated GAGs, which differ between cells grown in vitro and in vivo [45].
Peptide p5+14 inhibits HCMV and HSV infections in vitro. Peptide p5+14 (100 M) was added in a plaque reduction assay using HCMV (TB40/E) on different cell types (HFF, HAEC, and ARPE-19) and HSV-1 or HSV-2 on VERO cells. Bars represent the average of the percent reduction in infection compared to PBStreated control from t
