Polyethylenimine is a strong inhibitor of human papillomavirus and cytomegalovirus infection

Abstract

Polyethylenimines are cationic polymers with potential as delivery vectors in gene therapy and with proven antimicrobial activity. However, the antiviral activity of polyethylenimines has not been addressed in detail thus far. We have studied the inhibitory effects of a linear 25-kDa polyethylenimine on infections with human papillomaviruses and human cytomegaloviruses. Preincubation of cells with polyethylenimine blocked primary attachment of both viruses to cells, resulting in a significant reduction of infection. In addition, the dissemination of human cytomegalovirus in culture cells was efficiently reduced by recurrent administration of polyethylenimine. Polyethylenimine concentrations required for inhibition of human papillomavirus and cytomegalovirus did not cause any cytotoxic effects. Polyethylenimines and their derivatives may thus be attractive molecules for the development of antiviral microbicides.

Fig 1

Analysis of the inhibitory effects of PEI on HPV16 infection and determination of the cytotoxic concentration of PEI on HeLa cells. (A) HeLa cells were preincubated with the indicated concentrations of PEI for 30 min and were subsequently infected with HPV16 pseudovirions. Infection rates were assessed at 24 h postinfection by measuring luciferase activity in infected cells. The infection rate of the control (0 nM PEI) was set to 100%. Bars represent four individual experiments ± SD. (B) HeLa cells were incubated with the indicated concentrations of PEI for 24 h. The LDH activity released from damaged cells into the culture supernatant was quantified as a measure for cytotoxicity. Data represent the mean of 3 individual experiments ± SD. OD, optical density.

Fig 2

Dependence of HPV inhibition by PEI on pseudovirion concentrations and on cell types. (A) Effect of PEI on different HPV16 titers. HeLa cells were preincubated with 52 nM PEI for 30 min and were then infected for 24 h with the indicated amounts of luciferase-positive HPV16 particles per cell. Infection inhibition through PEI was determined by correlating results from infected samples to mock-infected controls. Bars represent the mean of three individual experiments ± SD. (B) Inhibition of HPV infection in different cell lines. The indicated cell lines were preincubated with 52 nM PEI for 30 min and were then infected with HPV16, -18, or -31 pseudovirions for 24 h. Infection inhibition through PEI was determined by correlating results from infected samples to mock-infected controls. Bars represent the mean of three individual experiments ± SD.

Fig 3

Inhibition of HPV16 attachment by PEI. (A) HeLa cells were infected with HPV16 pseudovirions for 2 h (+PEI, HeLa cells preincubated with PEI; −PEI, untreated HeLa control cells). Subsequently, cells were washed extensively with PBS to remove unbound virions. Attached virions were detected by Western blotting, using a MAb directed against the HPV16 capsid protein L1. β-Actin was probed as a loading control. (B) Infection of GAG-deficient (pgsA-745) and parental CHO-K1 cells in absence or presence of PEI (52 nM). Infection was scored at 24 h p.i. Control infections (without PEI) were set to 100%. Bars represent the mean of 4 individual experiments ± SD.

Fig 4

Impact of the time point of PEI application on HPV16 infection. (A) HeLa cells were treated with PEI (52 nM) at the indicated times before and after infection with HPV16 pseudovirions. Infection was scored at 24 h postinfection. Control infections without PEI were set to 100%. Bars represent the mean of three individual experiments ± SD. (B) Effect of PEI on prebound pseudovirions. Cells were infected for 1 or 2 h and then incubated or not with PEI (52 nM) for 1 h. Cell-bound virions were detected by Western blotting, using an anti-L1 antibody. β-Actin was probed as a loading control.

Fig 5

Inhibitory effects of PEI on HCMV infection and cytotoxic effects of PEI on HFF cells. (A) Inhibition of HCMV infection by PEI. HCMV RV-ΔUS2-11_GFP particles (MOI, 1) and PEI were coadministered to HFF cells. Infection rates were determined after 24 h through quantification of GFP expressed by infected cells. Control infection (0 nM PEI) was set to 100%. Bars represent the mean of three individual experiments ± SD. (B) Toxicity of PEI on HFF cells. Cells were incubated with the indicated concentrations of PEI for 48 h. LDH activity in the culture supernatant was determined to be a measure for toxicity. Bars represent the mean of three individual experiments ± SD. (C) Inhibition of HCMV attachment by PEI. HFF cells were infected at an MOI of 5 with the HCMV strain RV-BADwt for 2 h (+PEI, HFFs preincubated with PEI; −PEI, untreated HFF controls). Subsequently, cells were washed extensively with PBS to remove unbound virions. Attached virions were detected by Western blotting, using a MAb directed against the HCMV tegument protein pp65. β-Actin was probed as a loading control.

Fig 6

Impact of PEI on infection and spread of HCMV in permissive HFFs. (A) Inhibition of HCMV dissemination in HFF cultures by PEI. Cells were infected with RV-ΔUS2-11_GFP (MOI, 0.003). At 10 h p.i., 13 or 16 nM PEI was added to the culture medium. Additional PEI was supplemented in 24-h intervals. Total viral load was quantified 10 days after infection by measuring GFP. Bars represent the mean of three individual experiments ± SD. Control infections (no PEI) were set to 100%. (B) Determination of PEI toxicity on HFFs after 10 days of recurrent administration. LDH activity in the supernatants and lysates of the HFF cells of the infection assay (A) was determined at 10 days of culture. Bars represent the mean of three individual experiments ± SD. (C) Impact of PEI (10 h p.i.) on initial HCMV infection in HFF cultures. Cells were infected with HCMV RV-ΔUS2-11_GFP (MOI, 0.03) or mock infected. At 10 h after infection, cells were washed with PBS. PEI at 16 nM was added to some of the cultures. At 48 h postinfection, GFP expression was measured as a means for determination of primary viral infection. Bars represent the mean of three individual experiments ± SD.

Fig 7

Impact of PEI on HCMV cell-cell spread and cell-free virus spread. Cells were infected with HCMV RV-ΔUS2-11_GFP (MOI, 0.003). Cells were either left untreated or treated with PEI at 10 h after infection; additional PEI was supplemented in 24-h intervals. At 10 days postinfection, viral spread was monitored by fluorescence microscopy. In untreated cells (−PEI), nearly 100% of cells were infected (light cells). In PEI-treated cells (+PEI), only clusters of infected cells could be observed, suggesting that in the presence of PEI, viral dissemination occurs exclusively by cell-cell spread.