A branched peptide targets virus and host to block influenza virus and rhinovirus entry

Abstract

The global burden of influenza virus and rhinovirus, along with significant mortality and severe case reports, underscores the urgent need for new antivirals. Human defensins serve as the first line of defense against viruses; however, the antiviral activity of defensin peptides is often sensitive to salt, which affects their effectiveness. This study investigates a branched human-defensin peptide H30 (4H30) that can more effectively inhibit influenza virus and rhinovirus compared to the linear form of H30. Mechanistic studies reveal that 4H30 binds to influenza HA to aggregate the virus, thereby blocking viral entry. 4H30 can also cross-link H1N1 virus with cell surface glycosaminoglycans to prevent viral release. The dual-functional peptide 4H30 protects mice from the lethal challenge of the A(H1N1)pdm09 virus, demonstrating a high barrier to viral resistance after 15 viral-culture passages in the presence of 4H30. Notably, 4H30 interferes with the low-density lipoprotein receptor (LDLR) to impede the entry of minor group rhinovirus and significantly inhibits rhinovirus replication in RD cells, nasal organoids, and stem cell-derived cardiomyocytes. These findings suggest that the branched peptide 4H30, targeting both the virus and host, can more effectively inhibit influenza and rhinovirus than the linear H30, providing a new avenue for antiviral peptide development.

Keywords: antiviral peptide; human defensin peptide; influenza virus; rhinovirus; viral entry.

Fig 1

4H30 cross-linked H1N1 and blocked viral entry. (a) The antiviral activity of H30 and 4H30 against H1N1 virus in MDCK cells. IC₅₀ of 4H30 against H1N1 is 1.1 µg mL⁻¹. (b) Compared to the MEM-treated virus, 4H30 (25 µg mL⁻¹) significantly inhibited H1N1 virus when the virus was treated before viral infection at 37°C, but 4H30 did not inhibit virus when cells were treated with 4H30 at 1 h before viral infection (1h-Pre) and cells treated with 4H30 at 1 h after viral infection (1h-Post). Viral RNA copies in cell lysates were measured at 6 hours post-infection (hpi). (c) 4H30 (25 µg mL⁻¹) cross-linked virus and blocked viral entry into cells. Nuclei were stained by DAPI (blue). Cross-linked viruses (green) stuck on the cell membrane (red) are shown in yellow. Scale bar = 20 µm. (d) 4H30 (25 µg mL⁻¹) increased viral binding to MDCK cells at 4°C. H1N1 neutralizing antibody (Ab) was used as a control. (e) 4H30 showed significant antiviral activity by targeting the virus. Virus was treated with 4H30 (50 µg mL⁻¹) and then was diluted 1000-fold to decrease the concentration of 4H30 to 0.05 µg mL⁻¹ for plaque assay. (f) The cytotoxicity of 4H30 in MDCK was evaluated by the MTT assay. * indicates P < 0.05. Data are presented as mean ± SD of indicated biological samples.

Fig 2

4H30 bound to H1N1 virus and cross-linked the virus with cellular GAGs to inhibit viral release. (a) 4H30 more efficiently bound to HA protein than NA. 4H30 (1 µg and 0 µg) was coated to an ELISA plate, and HA and NA (100 ng) binding to 4H30 was detected by anti-His antibody. (b) 4H30 (125.0 µg mL⁻¹) did not inhibit HA-mediated cell-cell fusion in 293T cells. Scale bar = 200 µm. (c) 4H30 inhibited H1N1 release from MDCK cells. H1N1-infected cells were treated with MEM, 4H30 (25.0 µg mL⁻¹), or zanamivir (5.0 µM) at 1 hour post-infection (hpi). The viral RNAs in supernatants were measured by RT-qPCR at 8 hpi. (d) Removing cell surface GAGs reduced the attachment of viruses treated with 4H30. MDCK cells treated with buffer (Buffer), and MDCK cells treated with Hase and ChABC to remove cellular GAGs were treated with MEM or 4H30 (25 µg mL⁻¹) for H1N1 virus attachment at 4°C. Viral RNAs from cell lysates were measured by RT-qPCR. (e) 4H30 inhibiting viral release was confirmed by anti-HA staining. H1N1-infected cells were treated with MEM, 4H30, or zanamivir at 14 hpi and fixed at 18 hpi for anti-HA staining. HA (green) overlapped with the cell membrane (red) is shown in yellow. Scale bar = 20 µm. (f) 4H30 inhibited viral release at 18 hpi. This experimental procedure was the same as described in (e). The supernatant virus at 18 hpi was measured by RT-qPCR. Data are presented as mean ± SD of indicated biological samples.

Fig 3

Protection of 4H30 on H1N1-infected mice and drug-resistance testing of 4H30 against H1N1 virus. (a) 4H30 significantly inhibited H1N1 replication in mouse lungs at 48 hpi (n = 6–9). (b) 4H30 increased the survival of mice infected with a lethal dose of H1N1 infection (n = 6). (c) The body weight changes of H1N1-infected mice. H1N1 virus was intranasally inoculated to mice, and then PBS or 4H30 (0.5 mg kg⁻¹) was intranasally inoculated to mice at 6 hpi. On the following day, two more doses were given to mice. (d) 4H30 did not induce a drug-resistant virus after 15 passages. H1N1 virus was passaged in the presence of 4H30 (2.5, 5.0, and 10.0 μg/mL⁻¹ for every five passages). The 15-passage virus (P15) was not resistant to 4H30 compared to the antiviral activity of 4H30 against the parental virus (P0). ** indicates P < 0.01. Data are presented as mean ± SD of indicated biological samples.

Fig 4

4H30 inhibited rhinovirus entry. (a) 4H30 (25.0 µg mL⁻¹) significantly inhibited rhinovirus replication in RD cells. Virus was treated with the indicated concentrations of peptides, and viral RNA in the supernatants was measured at 30 hpi. (b) 4H30 significantly inhibited rhinovirus A1 (RV-A1) replication when cells were treated with 4H30 at 33°C for 1 h before viral infection (1h-Pre), and the virus was treated before infecting cells (Pre-mix). When infected cells were treated with 4H30 at 1 hpi (1h-Post) or 2 hpi (2h-Post), it did not inhibit viral infection. Viral RNAs in supernatants were measured at 16 hpi. (c) 4H30 (25.0 µg mL⁻¹) cross-linked rhinovirus and blocked viral entry into RD cells. Nuclei were stained with DAPI (blue). Scale bar = 20 µm. (d) 4H30 reduced viral attachment on RD cells at 4°C when virus was treated with 4H30. (e) 4H30 (25.0 µg mL⁻¹) did not inhibit viral release from RD cells. RD cells were infected with RV-A1 and were treated with 4H30 at 20 hpi. The supernatant viral RNAs were measured at 30 hpi. (f) 4H30 did not inhibit rhinovirus A1 (RV-A1) by targeting the virus. Rhinovirus A1 was treated with 4H30 (100 µg mL⁻¹) in PBS for 1 h. After 1 hour of treatment, the treated virus was diluted in MEM with 200-fold to let the concentration of 4H30 decrease to 0.5 µg mL⁻¹. The diluted virus was used to infect RD cells at 33°C. Viral RNA copies in cell lysates were measured at 16 hpi. No viral infection was the mock control. (g) 4H30 (1.0 µg, 0.25 µg, and 0 µg) bound to LDLR in the ELISA assay. (h) 4H30 showed significant antiviral activity before the virus was bound to RD cells. The virus was added to cells in the presence of 4H30 (25 µg mL⁻¹) (4H30-Pre) or without 4H30 to infect cells at 4°C. After 1 h attachment, the virus was removed, and fresh MEM or MEM with 4H30 (4H30-Post) was added to cells for viral culture at 33°C. Viral RNA copies in cell lysates were measured at 16 hpi. * indicates P < 0.05. Data are presented as mean ± SD of indicated biological samples.

Fig 5

4H30 inhibited rhinovirus replication in nasal organoids and cardiomyocytes. (a) Rhinovirus A1 could not replicate efficiently in mice. Rhinovirus (1 × 10⁷ TCID₅₀) was intranasally inoculated to mice. Viral RNAs were measured on day 1, day 2, and day 3 post-infection. (b) Rhinovirus A1 (0.01 MOI) could efficiently replicate in nasal organoids, and 4H30 (25 µg mL⁻¹) could significantly inhibit viral replication. (c) Rhinovirus A1 (0.01 MOI) could effectively replicate in cardiomyocytes. (d) 4H30 (25 µg mL⁻¹) could significantly inhibit rhinovirus replication in cardiomyocytes at 48 phi. Viral RNAs in the cell supernatants were measured by RT-qPCR at the indicated time points. Data are presented as mean ± SD of indicated biological samples.