Sulfated derivatives of Escherichia coli K5 capsular polysaccharide are potent inhibitors of human cytomegalovirus

Abstract

To date, there are few drugs licensed for the treatment of human cytomegalovirus (HCMV) infections, most of which target the viral DNA polymerase and suffer from many drawbacks. Thus, there is still a strong need for new anti-HCMV compounds with novel mechanisms of action. In this study, we investigated the anti-HCMV activity of chemically sulfated derivatives of Escherichia coli K5 capsular polysaccharide. These compounds are structurally related to cellular heparan sulfate and have been previously shown to be effective against some enveloped and nonenveloped viruses. We demonstrated that two derivatives, i.e., K5-N,OS(H) and K5-N,OS(L), are able to prevent cell infection by different strains of HCMV at concentrations in the nanomolar range while having no significant cytotoxicity. Studies performed to elucidate the mechanism of action of their anti-HCMV activity revealed that these compounds do not interact with either the host cell or the viral particle but need a virus-cell interaction to exert antiviral effects. Furthermore, these K5 derivatives were able to inhibit the attachment step of HCMV infection, as well as the viral cell-to-cell spread. Since the mode of inhibition of these compounds appears to differ from that of the available anti-HCMV drugs, sulfated K5 derivatives could represent the basis for the development of a novel class of potent anti-HCMV compounds. Interestingly, our studies highlight that small variations of the K5 derivatives structure can modulate the selectivity and potency of their activities against different viruses, including viruses belonging to the same family.

FIG. 1.

Effects of pretreatment of HCMV virions with sulfated K5 derivatives on virus infectivity. HCMV AD169 was incubated at 37°C for 1, 2, or 4 h with either K5-N,OS(H) (A) or K5-NS (B). The virus (2.5 × 10⁵ PFU) was then five times 10-fold diluted and titrated on fresh HFF monolayers. The data shown represent the means ± the standard deviations (SD) of data of three independent experiments performed in triplicate.

FIG. 2.

Effects of pretreatment of HFF cells with sulfated K5 derivatives on HCMV replication. HFFs were incubated with either sulfated K5 derivatives or heparin as a control at 37°C for either 2 (A) or 6 (B) h; the compounds were then removed from cell media, and cells were infected with HCMV AD169. The data shown are expressed as a percentage of the plaque number determined in treated samples with respect to the untreated mock-infected samples and represent the means ± the SD of three independent experiments done in duplicate.

FIG. 3.

Effects of sulfated K5 derivatives on cell-to-cell spread of HCMV. (A) HFFs were infected with HCMV AD169 (MOI, 0.003 PFU/cell) in the absence of compounds (HCMV) or not infected (Mock). Either K5-N,OS(H) or K5-NS was then added at different concentrations, and cells were incubated for 5 days at 37°C. Viral cell-to-cell spread was assessed by indirect immunofluorescence with an anti-IEA MAb. (B) Fifteen representative infected foci per concentration were analyzed with ImageJ software to determine the viral plaques size in the presence of the test compounds. The data shown represent the means ± the SD. The asterisks denote a statistically significant difference (*, P < 0.05; **, P < 0.001).