Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza

Abstract

Limited efficacy of current antivirals and antiviral-resistant mutations impairs anti-influenza treatment. Here, we evaluate the in vitro and in vivo antiviral effect of three defective interfering genes (DIG-3) of influenza virus. Viral replication is significantly reduced in cell lines transfected with DIG-3. Mice treated with DIG-3 encoded by jetPEI-vector, as prophylaxis and therapeutics against A(H7N7) virus, respectively, have significantly better survivals (80% and 50%) than control mice (0%). We further develop a dual-functional peptide TAT-P1, which delivers DIG-3 with high efficiency and concomitantly exerts antiviral activity by preventing endosomal acidification. TAT-P1/DIG-3 is more effective than jetPEI/DIG-3 in treating A(H7N7) or A(H1N1)pdm09-infected mice and shows potent prophylactic protection on A(H7N7) or A(H1N1)pdm09-infected mice. The addition of P1 peptide, which prevents endosomal acidification, can enhance the protection of TAT-P1/DIG-3 on A(H1N1)pdm09-infected mice. Dual-functional TAT-P1 with DIG-3 can effectively protect or treat mice infected by avian and seasonal influenza virus.

Fig. 1

Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b, c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e, f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P < 0.05. ** Indicates P < 0.01. P values were calculated by the two-tailed Student’s t test

Fig. 2

DIG could be packaged to generate defective interfering virus and competitively inhibit normal viral replication. a Antiviral activity of DIG-3 in A549-Dual KO-RIG-I cells. The A549-Dual KO-RIG-I cells were infected with A(H7N7) virus at 24 h post transfection of empty vector or DIG-3 (0.6 μg per well). Viral titers in cell supernatants of 40 h post infection were measured by plaque assay. b–d Full-length PA, full-length PB1, full-length PB2, DI-PA, DI-PB1, and DI-PB2 RNA levels in the supernatants of A(H7N7)-infected cells transfected with empty vector or DIG-3 before viral infection. e The percentage of DIG-3-treated virus compared with empty vector-treated virus in supernatants of 293T cells. Viral titers were measured by plaque assay and HA assay. f The passaged viral titers in supernatants of MDCK cells that were infected by the supernatant virus of A(H7N7)-infected 293T cells transfected with empty vector or DIG-3. g The virus titer ratio of DIG-3-treated virus compared with empty vector-treated virus in supernatants of MDCK cells. Viral titers were measured by plaque assay and HA assay. h The DI-PA RNA levels of passaged virus in the supernatants of MDCK cells. Dotted lines indicate the detection limit of RT-qPCR. Data were presented as mean ± SD of three independent experiments. ** Indicates P < 0.01. P values were calculated by the two-tailed Student’s t test

Fig. 3

The jetPEI/DIG-3 could provide potent anti-A(H7N7) virus efficacy in mice. a Experimental design for evaluating antiviral efficacy of jetPEI/DIG-3 in mice. b Expression of DI-PA, DI-PB1, and DI-PB2 RNAs in mouse lungs when jetPEI/DIG-3 (5 μg per mouse), DIG-3, jetPEI/empty vector were intratracheally inoculated to mouse lungs. Three mice in each group were included. c, d Prophylactic efficacy of jetPEI/DIG-3 against A(H7N7) virus. e, f Therapeutic efficacy of jetPEI/DIG-3 against A(H7N7) virus. For prophylactic experiment, 40 μl of PBS (n = 10), zanamivir (50 μg in PBS, n = 10), jetPEI (0.7 μl in 5% glucose solution, n = 5), jetPEI/empty vector (0.7 μl/5.0 μg in 5% glucose solution, n = 10), and jetPEI/DIG-3 (0.7 μl/5.0 μg in 5% glucose solution, n = 10) were intratracheally inoculated to corresponding mice at 48 and 24 h before viral inoculation. For therapeutic experiment, PBS, zanamivir, jetPEI, jetPEI/empty vector and jetPEI/DIG-3 were intratracheally inoculated to corresponding mice at 6 and 24 h after viral inoculation. Survivals and body weight data were generated from 5 to 10 mice in each group with mean ± SD. P values were calculated by Gehan–Breslow–Wilcoxon test

Fig. 4

The antiviral mechanism and transfection efficiency of TAT-P1. a P1 and TAT-P1 could bind to H1N1 glycoprotein HA1 in an ELISA assay. OD values were normalized to P1 as 1. ** Indicates P < 0.01 when compared with BSA. b P1 and TAT-P1 could prevent endosomal acidification. Red dots (pHrodo^TM dextran) indicate pH lower than 6.0 in endosomes. P9-aci-1 (PA1), which did not prevent endosomal acidification, was a negative peptide control for P1. Blue color indicates nuclei. Live cell images were taken by confocal microscope (original magnification ×400, scale bar = 10 μm). c The quantification of red fluorescence of endosomal acidification in MDCK cells when cells were treated by BSA, bafilomycin A1 (A1), P1, TAT-P1, or PA1 corresponding to b. Ten microscope fields of each sample were included for quantification. ** Indicates P < 0.01 when compared with BSA + pH dye group. d P1 and TAT-P1 could prevent viral RNP release into nuclei. MDCK cells were infected with 1 MOI of A(H1N1) virus treated with or without drugs. Images of NP (green) and nuclei (blue) were taken at 3.5 h post infection (scale bar = 10 μm). e Percentages of NP colocalized to nuclei. The SD was determined from multiple microscope fields including ~ 200 cells for each sample. ** Indicates P < 0.01 when compared with BSA. f Transfection efficiency of peptide/pLuc in 293T cells. Numbers indicate weight ratios of corresponding peptide:pLuc. Mock means cells treated with TAT-P1 without DNA. Data were presented as mean ± SD of three independent experiments. g Transfection efficiency of pCMV-Luc transfected by TAT-P1 or jetPEI in mouse lungs. Luciferase expression in mouse lungs was normalized to 1 mg protein. Mock means mice treated with TAT-P1 or jetPEI without DNA. h Transfection efficiency of plasmid of DI-PA transfected by TAT-P1 or jetPEI in mouse lungs. Data were presented as mean ± SD of ≥3 mice in each group. * Indicates P < 0.05. ** Indicates P < 0.01 when compared with Mock in f and g or when compared with “0” (naked DNA) in h. P values were calculated by the two-tailed Student’s t test

Fig. 5

TAT-P1/DIG-3 could provide prophylactic and therapeutic protection against A(H7N7) virus infection in mice. a–d Prophylactic efficacy of TAT-P1/DIG-3 against A(H7N7) virus. e–h Therapeutic efficacy of TAT-P1/DIG-3 against A(H7N7) virus. For prevention, 40 μl of PBS (n = 10), zanamivir (50 μg in PBS, n = 10), TAT-P1 (20 μg in distilled water, n = 10), and TAT-P1/DIG (20 μg/5 μg in distilled water, n = 10) were intratracheally inoculated to corresponding mice at 48 and 24 h before viral inoculation. For therapeutic experiment, PBS (n = 10), zanamivir (n = 10), TAT-P1 (n = 10), TAT-P1/DIG (n = 10), or jetPEI/DIG (0.7 μl/5.0 μg in 5% glucose solution, n = 5) were intratracheally inoculated to corresponding mice at 6 and 24 h after viral inoculation. Viral titers and IL-6 in mouse lungs were detected at day 4 post infection with mean ± SD of three mice in each group. The expression of IL-6 was normalized to PBS group. * Indicates P < 0.05. ** Indicates P < 0.01 when compared with PBS group. P values were calculated by Gehan–Breslow–Wilcoxon test for survivals and by the two-tailed Student’s t test for viral titer and IL-6 analysis

Fig. 6

TAT-P1/DIG-3 could provide prophylactic and therapeutic protection against A(H1N1)pdm09 virus infection in mice. a–d Prophylactic efficacy of TAT-P1/DIG-3 against A(H1N1)pdm09 virus. e–h Therapeutic efficacy of TAT-P1/DIG-3 against A(H1N1)pdm09 virus. For prevention, 40 μl of PBS (n = 10), zanamivir (50 μg in PBS, n = 10), and TAT-P1/DIG-3 (20 μg/5 μg in distilled water, n = 10) were intratracheally inoculated to corresponding mice at 48 and 24 h before viral inoculation. For therapeutic experiment, PBS (n = 15), zanamivir (10), TAT-P1/DIG-3 (n = 15) or jetPEI/DIG-3 (20 μg/5 μg in 5% glucose solution, n = 5) were intratracheally inoculated to corresponding mice at 6 h and 24 h after viral inoculation. Viral titers and IL-6 in mouse lungs were detected at day 4 post infection with mean ± SD of >3 mice in each group. The expression of IL-6 was normalized to PBS group. * Indicates P < 0.05. ** Indicates P < 0.01 when compared with PBS group. P values were calculated by Gehan–Breslow–Wilcoxon test for survivals and by the two-tailed Student’s t test for viral titer and IL-6 analysis

Fig. 7

The transfection efficiency and antiviral activity of TAT-P1 with DIG-3 could be increased by P1 peptide. a Transfection efficiency of TAT-P1/pLuc increased by additional ATPase inhibitor (bafilomycin A1) or P1 peptide. Before TAT-P1/pLuc (2.0 μg/0.5 μg) complex was added to 293T cells for transfection, the indicated concentrations of bafilomycin A1 (A1, nM), P1 peptide (μg per ml), or PA1 peptide (μg per ml) were added to cell culture media. Mock indicates cells treated with A1 or TAT-P1 without DNA. Data were presented as mean ± SD of three independent experiments. b Transfection efficiency of TAT-P1/pCMV-Luc increased by P1 in mouse lungs. c Representative In Vivo Imaging System image showed increased luciferase expression by P1 in mouse lungs. TAT-P1/pCMV-Luc (20 μg/5 μg) with additional P1 (20 μg, 10 μg, or 0 μg) or PA1 (20 μg) were inoculated to mouse lungs at 48 and 24 h before measuring bioluminescence signal or taking bioluminescence image. Mock indicates mouse lungs inoculated with TAT-P1 + P1 without DNA. d The RNA expression of DI-PA increased by P1 in mouse lungs. TAT-P1/DI-PA (20 μg/5 μg) with additional P1 (20 μg, 10 μg, or 0 μg) were inoculated to mouse lungs at 48 and 24 h before detecting RNA expression. Data were presented as mean ± SD of ≥3 mice. * Indicates P < 0.05 and ** Indicates P < 0.01 when normalized to Mock (a, b) or naked plasmid of DI-PA (d). e, f The prophylactic efficacy of TAT-P1/DIG-3 + P1 against A(H1N1)pdm09 virus. PBS (n = 10), TAT-P1 + P1(n = 10) or TAT-P1 + PA1 (20 μg + 20 μg, n = 5), TAT-P1/DIG-3 (20 μg/5 μg, n = 10), TAT-P1/DIG-3 + PA1 (20 μg/5 μg + 20 μg, n = 5), or TAT-P1/DIG-3 + P1 (20 μg/5 μg + 20 μg, n = 10) were intratracheally inoculated to corresponding mice at 48 and 24 h before viral inoculation. ** Indicates P < 0.01 when compared with PBS. P values were calculated by Gehan–Breslow–Wilcoxon test for survival analysis and by the two-tailed Student’s t test for analysis in a, b, d and f

Fig. 8

Schematic model of antiviral mechanism of TAT-P1/DIG-3 in one viral life cycle. When cells are transfected with TAT-P1/DIG-3 and infected with influenza virus, a TAT/P1-DIG-3 are internalized into cells by endocytosis; b Virus can be bound with TAT-P1 and internalized into cells by endocytosis. TAT-P1 can prevent endosomal acidification to block the release of viral RNP into the cytoplasm  and then  nuclei; c Some virus particles that are not bound by TAT-P1 might be internalized into cells by endocytosis and undergo endosomal acidification. d TAT-P1/DIG-3 released from endosomes are imported into nuclei (d1) and viral RNPs released from endosomes are imported into nuclei (d2). e Viral DIG and wild-type genes (WTG) replicate in nuclei. f Newly produced DIG and WTG are exported to cytoplasm. g Viral DIG and WTG are reassorted in cytoplasm and move to the cell membrane to be incorporated into new virions. Seven types of DIV and one type of wild-type virus (WTV) are released from cells to start new virus life cycle. The newly generated DIV can have sustained antiviral activity to competitively inhibit WTV replication in non-transfected cells