Temporin G, an amphibian antimicrobial peptide against influenza and parainfluenza respiratory viruses: Insights into biological activity and mechanism of action

Abstract

Treatment of respiratory viral infections remains a global health concern, mainly due to the inefficacy of available drugs. Therefore, the discovery of novel antiviral compounds is needed; in this context, antimicrobial peptides (AMPs) like temporins hold great promise. Here, we discovered that the harmless temporin G (TG) significantly inhibited the early life-cycle phases of influenza virus. The in vitro hemagglutinating test revealed the existence of TG interaction with the viral hemagglutinin (HA) protein. Furthermore, the hemolysis inhibition assay and the molecular docking studies confirmed a TG/HA complex formation at the level of the conserved hydrophobic stem groove of HA. Remarkably, these findings highlight the ability of TG to block the conformational rearrangements of HA2 subunit, which are essential for the viral envelope fusion with intracellular endocytic vesicles, thereby neutralizing the virus entry into the host cell. In comparison, in the case of parainfluenza virus, which penetrates host cells upon a membrane-fusion process, addition of TG to infected cells provoked ~1.2 log reduction of viral titer released in the supernatant. Nevertheless, at the same condition, an immunofluorescent assay showed that the expression of viral hemagglutinin/neuraminidase protein was not significantly reduced. This suggested a peptide-mediated block of some late steps of viral replication and therefore the impairment of the extracellular release of viral particles. Overall, our results are the first demonstration of the ability of an AMP to interfere with the replication of respiratory viruses with a different mechanism of cell entry and will open a new avenue for the development of novel therapeutic approaches against a large variety of respiratory viruses, including the recent SARS-CoV2.

Keywords: antimicrobial peptides; antiviral agents; influenza; parainfluenza; viral infection.

FIGURE 1

TG does not affect the viability of A549 cells compared to TA and TB. A, Cell monolayers were treated or not with increasing concentrations (0.1, 7.5, 15, 30, 40, 60 µM corresponding to −1, 0.87, 1.18, 1.48, 1.6, 1.78 log(dose)) of each peptide for 24 hours and the amount of metabolically active cells was determined by the MTT assay and expressed as a percentage compared to untreated control cells. The CC50 of the three peptides was calculated by regression analysis of the dose‐response curve. Values are expressed as mean ± SD from two independent experiments, each performed in duplicate (n = 4). B, Influenza virus (PR8)‐infected cells were treated with TG at different concentrations (0.1, 7.5, 15, 30, 40 µM), and viral titer inhibition was determined by HAU assay. The IC₅₀ of the compound was calculated by regression analysis as described in Materials and Methods

FIGURE 2

TG inhibits PR8 replication at early steps of the virus life‐cycle. A549 cells were infected with PR8 and treated or not with the peptide at different phases of the virus life‐cycle: (i) during viral adsorption for 1 hour (ADS); (ii) immediately after viral adsorption for 24 hours (POST); (iii) during viral adsorption and for 24 hours (ADS + POST). A, Representative images of the virus‐induced cytopathic effect on A549 cells at the different phases of the virus life‐cycle upon treatment with TG, compared to uninfected control samples. B, The viral titer in the supernatants of infected cells was analyzed by TCID₅₀ and HAU assay and compared to that of untreated infected cells (dashed line). Values are the mean ± SD of one representative experiment out of three, each performed in quadruplicate (n = 4). Statistical significance of the data vs untreated‐infected samples was defined as *P < .05 and **P ≤ .01

FIGURE 3

TG reduces the expression of viral NP at early phases of the virus‐life cycle. A, The expression of NP protein was analyzed on monolayers of A549 cells by ICW assay using LI‐COR Image Studio Software to measure the RFI (left side). The graph (right side) represents the percentage of fluorescence intensity calculated in comparison to that of untreated‐infected cells (dashed line corresponding to 100%). The values represent the mean ± SD of four experiments each performed in duplicate (n = 8). Statistical significance of data vs untreated‐infected cells was defined as **P < .001 and ***P < .0001. B, Cell lysates were also analyzed by western blot to evaluate the expression of the three viral proteins HA, NP, and M1. Actin was used as a loading control. Results are representative of one out of three performed experiments. C, Densitometry analysis of viral proteins. The expression of each protein was normalized to that of actin and expressed as a percentage with respect to that obtained from untreated‐infected cells which are indicated by the dashed black line (100%). The results are the mean ± SD of three independent experiments, (n = 3). Statistical significance of data vs untreated‐infected cells was defined as *P < .05; **P < .001 and ***P < .0001

FIGURE 4

TG interferes with the entry phase of the PR8 life‐cycle. A, Viral titer in the supernatant of infected cells after peptide treatment was analyzed during the attach and entry phases by HAU assay and compared to that of untreated infected cells (INF). The image shows representative results of one out of three independent experiments (left side). The graph (right side) represents mean values of HAU ± SD obtained from supernatants of attachment and entry assays compared to INF, of three independent experiments, each performed in duplicate (n = 6). B, Cell lysates were analyzed by western blot to evaluate the expression of HA, NP, and M1 viral proteins. Actin was used as a loading control. C, Densitometry analysis of viral proteins. Expression of each protein was normalized to that of actin and expressed as a percentage with respect to the amount obtained from untreated‐infected cells, which is indicated by the dashed line (100%). The results are the mean ± SD of three independent experiments (n = 3). Statistical significance of data vs untreated‐infected cells was defined as *P < .05; **P < .001 and ***P < .0001

FIGURE 5

Pre‐treatment of A549 cells with TG does not affect the replication of PR8. A549 cell monolayers were pre‐treated (PRE) or not (INF) with TG for 3 hours at 37°C and then infected with PR8 for 1 hour at 37°C. At 24 hours p.i. supernatants were collected for HAU assay, while cell monolayers were analyzed for NP expression by ICW assay (left side). The results of viral titers and NP protein expression are expressed as HAU and as a percentage of protein expression compared to untreated‐infected cells (dashed line), respectively, and are the mean ± SD of two independent experiments, each performed in duplicate, (n = 4) (right side)

FIGURE 6

TG inhibits viral agglutination and lysis of RBC. A, PR8 solution was twofold serially diluted and pre‐incubated or not with the peptide (30 µM) and then analyzed for its ability to agglutinate RBCs (left side). The graph (right side) represents the mean values ± SD of HAU obtained from two independent experiments, each performed in duplicate, (n = 4). B, PR8 and TG were mixed for 30 minutes at room temperature; afterward, an equal volume of 2% RBC was added to the mixture for other 30 minutes at 37°C. Subsequently, sodium acetate was added for 30 minutes at 37°C, to lower the pH and trigger hemolysis. The absorbance of the released hemoglobin was measured from the samples’ supernatant. Results are expressed as the percentage of hemolysis compared to that provoked by untreated PR8. Values are the mean ± SD from two independent experiments, each performed in duplicate, (n = 4). Statistical significance of data vs PR8 was defined as **P < .001 and ***P < .0001

FIGURE 7

TG recognizes the stem groove at the HA1/HA2 interface. A, Model complex of HA and TG: HA is shown in ribbon and surface representation (HA1, light orange; HA2, light pink), while TG is shown as green ribbon. B, TG within the stem groove of HA. Side chain of interacting residues is shown as sticks with carbon atoms in green (TG), light orange (HA1), and light pink (HA2). Residues labels follow the same color code. Nitrogen and oxygen atoms are in blue and red, respectively. Hydrogen atoms are not shown for clarity

FIGURE 8

TG inhibits the replication of SeV. A549 cells were infected with SeV and treated or not with TG at different phases of the parainfluenza virus life‐cycle, as described for PR8. A, The supernatants of infected cells were analyzed by TCID₅₀ and HAU assays. Dashed black line indicates the viral titer of untreated‐infected cells. The results are the mean ± SD from two independent experiments, each performed in duplicate, (n = 4). Statistical significance of data vs untreated‐infected cells was defined as *P < .05 and **P < .01. B, The expression of the viral HN protein, in various phases of treatment with TG, was analyzed by ICW assay, using LI‐COR Image Studio Software to measure the RFI. The percentage of fluorescence intensity was calculated in comparison to that of untreated‐infected cells (dashed line which corresponds to 100%). The results are the mean ± SD from two independent experiments, each performed in duplicate, (n = 4)