Synthetic sulfonated derivatives of poly(allylamine hydrochloride) as inhibitors of human metapneumovirus

Abstract

Human metapneumovirus (hMPV) is a widely distributed pathogen responsible for acute upper and lower respiratory infections of varying severity. Previously, we reported that N-sulfonated derivatives of poly(allylamine hydrochloride) (NSPAHs) efficiently inhibit replication of the influenza virus in vitro and ex vivo. Here, we show a dose dependent inhibition of hMPV infection by NSPAHs in LLC-MK2 cells. The results showed strong antiviral properties of NSPAHs. While the activity of NSPAHs is comparable to those of carrageenans, they show better physicochemical properties and may be delivered at high concentrations. The functional assays showed that tested polymers block hMPV release from infected cells and, consequently, constrain virus spread. Moreover, further studies on viruses utilizing different egress mechanisms suggest that observed antiviral effect depend on selective inhibition of viruses budding from the cell surface.

Fig 1. Sulfonated polymers are not toxic for cells at effective concentration range.

The cytotoxicity of the tested polymers (ι-car: ι-carrageenan, λ-car: λ-carrageenan) determined by XTT assay. Values that are significantly different (P < 0.05) from the control are indicated by an asterisk. All experiments were performed in triplicate. Average values with standard deviations (error bars) are presented.

Fig 2. Sulfonated polymers hamper hMPV B2 infection.

Inhibition of the infection was evaluated using RT-qPCR (A) and virus titration (B). Polymers were present during the whole infection process. Data are shown as log reduction values (LRVs) normalized to the control samples. All experiments were performed in triplicate. The results are presented as average values with standard deviations (error bars). An asterisk (P < 0.05) indicates values that are significantly different from the control. ι-car: ι-carrageenan, λ-car: λ-carrageenan.

Fig 3. Sulfonated polymers limit the number of infected cells.

NSPAHs and carrageenans inhibit viral infection by hampering virus release from infected cells. Fluorescence images of LLC-MK2 cells (nuclei in blue) infected with hMPV B2 (green) were collected 5 days p.i. (scale bar = 100 μm).

Fig 4. Sulfonated polymers hamper hMPV A1 infection.

Inhibition of the infection was evaluated using RT-qPCR (black bars) and virus titration (grey bars). Polymers (1000 μg/ml) were present during the entire hMPV replication cycle. Data are shown as log reduction values (LRVs) normalized to the control samples. All experiments were performed in triplicate. The results are presented as average values with standard deviations (error bars). An asterisk (P < 0.05) indicates values that are significantly different from the control. ι-car: ι-carrageenan, λ-car: λ-carrageenan.

Fig 5. Inhibition of hMPV B2 replication by sulfonated polymers during late steps of the virus replication cycle.

Inhibition of the infection was evaluated using RT-qPCR (black bars) and virus titration (grey bars). Polymers (1000 μg/ml) were added after infection of LLC-MK2 cells with hMPV for 2 h and supernatants were collected 6 days p.i. Data are shown as log reduction values (LRVs) normalized to the control samples. All experiments were performed in triplicate. The results are presented as average values with standard deviations (error bars). An asterisk (P < 0.05) indicates values that are significantly different from the control. ι-car: ι-carrageenan, λ-car: λ-carrageenan.

Fig 6. Sulfonated polymers do not inhibit intracellular hMPV B2 replication.

Inhibition of intracellular hMPV replication was tested using RT-qPCR (black bars) or titration (grey bars). Polymers (1000 μg/ml) were added 2 h after infection of LLC-MK2 with hMPV and lysates were prepared 6 days p.i. Data are shown as log reduction values (LRVs) normalized to the control samples. All experiments were performed in triplicate. The results are presented as average values with standard deviations (error bars). An asterisk (P < 0.05) indicates values that are significantly different from the control. ι-car: ι-carrageenan, λ-car: λ-carrageenan.

Fig 7. Sulfonated polymers do not inhibit HCoV-NL63 infection.

Inhibition of the infection was evaluated using RT-qPCR (black bars) and virus titration (grey bars). Polymers (1000 μg/ml) were present throughout the infection. Data are shown as log reduction values (LRVs) normalized to the control samples. All experiments were performed in triplicate. The results are presented as average values with standard deviations (error bars). An asterisk (P < 0.05) indicates values that are significantly different from the control. ι-car: ι-carrageenan, λ-car: λ-carrageenan.