Bovine Lactoferrin Inhibits Adenovirus Infection by Interacting with Viral Structural Polypeptides

Abstract

We recently demonstrated that lactoferrin, an antimicrobial glycoprotein, can inhibit adenovirus infection by competing for common glycosaminoglycan receptors. This study further characterizes the antiadenovirus activity of the protein, thus demonstrating that lactoferrin neutralizes infection by binding to adenovirus particles and that its targets are viral III and IIIa structural polypeptides.Lactoferrin is a multifunctional glycoprotein that possesses a variety of physiological roles, such as promotion of iron absorption (26), immunomodulation (16), and inhibiting activity toward different pathogens (14,16,24,26,(32)(33)(34), including viruses (1, 2, 7, 9-13, 17-19, 21, 25, 28-30, 38). For all viruses investigated to date, lactoferrin exerts its antiviral activity during the early phases of infection. Recently, we demonstrated a competition between bovine lactoferrin (bLf) and adenovirus for the attachment to cell membrane glycosaminoglycans, even though a binding between lactoferrin and viral particles could not be ruled out (6). The present study provides deeper insight into the mechanism of the antiadenoviral action of bLf, demonstrating that this glycoprotein also exerts its antiadenoviral activity by a direct interaction with adenovirus particles.Human epidermoid carcinoma larynx (HEp-2) cells, obtained from the American Type Culture Collection (ATCC), and human adenovirus type 2 were grown as previously described (2). Viral particles were purified according to the methods of Wadell and coworkers (35).bLf (Morinaga Milk Industry) and heparin (Sigma, 170 USP U/mg) were dissolved in phosphate-buffered saline (PBS; pH 7.2). bLf was biotinylated according to the manufacturer's instructions (Amersham). Dot blot assays were carried out by applying 50 l of purified adenovirus, heparin, or PBS to nitrocellulose paper and using a Bio-Dot apparatus according to the manufacturer's instructions (Bio-Rad). Air-dried membranes were blocked in block solution, washed with PBS containing 0.1% Tween 20, and then incubated with biotinylated bLf. After washings in PBS containing 0.1% Tween 20, streptavidin-horseradish peroxidase (streptavidin-HRP) conjugate was added to the blot. Blot was revealed by an enhanced chemiluminescence kit (Amersham Biosciences) and the 3,3Ј,5,5Ј-tetramethylbenzidine (TMB) substrate kit for peroxidase (Vector Laboratories) according to the manufacturer's instructions.Neutralization of adenovirus binding to cells was carried out by incubating lactoferrin with adenovirus for 1 h at 37 or 4°C.The suspensions were then added to cells for 1 h at 4°C. Viral antigen synthesis was monitored 24 h after infection (37°C) by immunofluorescence as previously described (2). Statistical analysis was performed using the Student's t test for unpaired data. P values of Ͻ0.05 were considered significant. Adenovirus proteins were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) (15) under denaturing conditions as described by Wadell and coworkers (36). Coxsackievirus proteins were...

Adenovirus serotype 5 infects human dendritic cells via a coxsackievirus–adenovirus receptor-independent receptor pathway mediated by lactoferrin and DC-SIGN

“…It is therefore likely that Lf is present at high concentration at anatomical sites where peripheral DCs encounter pathogens such as Ad5 in vivo. Lf has been demonstrated to interfere with cellular entry by viruses such as human immunodeficiency virus type 1 (Groot et al, 2005), Semliki Forest virus (Waarts et al, 2005), human papillomavirus (Drobni et al, 2004) and Ad2 Pietrantoni et al, 2003), indicating that Lf often acts to inhibit, not promote, viral entry (van der Strate et al, 2001) We found that Lf of both human and bovine origin enhanced rAd5 infection in a dose-dependent manner in all APC populations tested with the exception of PDCs. The mechanism by which this enhancement of infection occurs is probably initiated by Lf binding to rAd5, as pre-treating the cells with Lf had less effect than pre-treating the virus with Lf.…”

Effect of lactoferrin on enteric pathogens

“…However, there are no data evaluating the effect of lactoferrin on the human enteric caliciviruses (sopoviruses or noroviruses). Likewise there are data showing that lactoferrin prevents adenovirus replication [17] and that the antiviral activity is located in the N terminus [18,19]. However, these studies have focused on respiratory rather than enteric adenoviral serotypes.…”

Differential Modulation of Human Lactoferrin Activity against Both R5 and X4-HIV-1 Adsorption on Epithelial Cells and Dendritic Cells by Natural Antibodies

“…Lf is produced by epithelial cells and, as a result, is present in mucosal secretions (11) such as seminal and vaginal fluids (12). One of the functions (13,14) of Lf is the transport of metals, but Lf is also an important component of the nonspecific immune system since Lf has a broad spectrum of activity against a variety of bacteria, fungi, and viruses (15). The antiviral effect of Lf lies in the early phase of infection by inhibiting attachment of viruses as alphavirus (16) to heparan sulfate glycosaminoglycans expressed on cell surface.…”