Elsevier

Antiviral Research

Volume 51, Issue 2, August 2001, Pages 141-149
Antiviral Research

Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomegalovirus into human fibroblasts

https://doi.org/10.1016/S0166-3542(01)00146-2Get rights and content

Abstract

Lactoferrin is mainly produced by polymorphonuclear leukocytes and has been demonstrated in mammalian milk and external secretions. Lactoferrin is an iron-binding, multifunctional protein and may play an important role in immune regulation and in defense mechanisms against bacteria, fungi and viruses. Lactoferricin is a potent antimicrobial peptide generated from the N-terminal part of lactoferrin by pepsin cleavage. We demonstrate that lactoferrins from different species and its N-terminal peptide lactoferricin (particularly the cyclic form) inhibit expression of early and late antigens, as well as production of infectious viral progeny during human cytomegalovirus (HCMV) infection in vitro. Iron-saturated lactoferrin did not affect HCMV antigen expression. Heparin had the same effects as iron-depleted lactoferrin. Yet, mixtures of lactoferrin and heparin did not inhibit HCMV multiplication i.e. lactoferrin and heparin seemed to mutually block each other's antiviral activities. HCMV-infected cells exposed to lactoferrin and cyclic lactoferricin contained less intracellular virus than unexposed cells. The antiviral activity of cyclic lactoferricin was more than seven-fold weaker than that of the maternal molecule. Lactoferrin and cyclic lactoferricin prevented HCMV entrance into the host cell.

Introduction

Human cytomegalovirus (HCMV), classified within the Herpesviridae (Mocarski, 1995), may cause diseases like mononucleosis syndrome and hepatitis in the immunocompetent host. Between 0.2 and 2.2% of all newborn children are congenitally infected, and about 15% of these show clinical symptoms from the nervous and other organ systems. Furthermore, HCMV infection may complicate allograft transplantation and may contribute to progression of HIV infection to AIDS (Britt and Alford, 1995). Hence, preventive or therapeutical intervention in HCMV infections is important.

Lactoferrin (LF) is a protein found in milk, on mucosal surfaces and in polymorphonuclear leukocytes (PMN) (Masson et al., 1966). LF is a multifunctional protein of approximately 80 kDa and a part of the innate immune defense (Shau et al., 1992). Stimulation of PMNs leads to the release of LF, and, hence, LF is an early indicator of inflammatory state (Gutteberg et al., 1984, Gutteberg et al., 1988). The molecular structure of LF has been revealed (Anderson et al., 1989, Day et al., 1992, Day et al., 1993), and LFs from human, goat, mouse, cow, swine and sheep have been characterized (Metz-Boutigue et al., 1984, Le Provost et al., 1994, Cunningham et al., 1992, Pierce et al., 1991, Lydon et al., 1992, Qian et al., 1995). The N-terminal and C-terminal sequences of LF are symmetrical and each bind one Fe+++.

Heparin and heparan sulfate belong to a family of cell-membrane inserted glycosaminoglycans, and heparan sulfate participates in the attachment and internalization of HCMV (Neyts et al., 1992, Kari and Gehrz, 1992, Compton et al., 1993). LF is known to bind to heparan sulfate proteoglycans and to the low-density-lipoprotein receptor (Ji and Mahley, 1994). The first 33 amino acid residues in the N-terminal part of LF engages in binding to glycosaminoglycans like heparin and heparan sulfate (Zou et al., 1992, Ji and Mahley, 1994, Mann et al., 1994).

Lactoferricin is generated upon gastric cleavage of LF (Tomita et al., 1991) and corresponds to the N-terminal part of LF. Lactoferricin makes a cationic distorted β-sheet joined together by a disulfide bridge (Hwang et al., 1998). In LF, the peptide structure forms a loop with a cationic charge at the tip.

Lactoferricin derived from the bovine LF is active against a wide range of Gram-negative and Gram-positive bacteria, fungi, protozoa and tumors (Bellamy et al., 1992, Bellamy et al., 1993, Isamida et al., 1998, Turchany et al., 1995, Yoo et al., 1997). Bovine lactoferricin (lactoferricin B) consists of 25 amino acid residues (17–41 in bovine LF) and human lactoferricin (lactoferricin H) consist of 47 amino acid residues including a region homologous to lactoferricin B.

Earlier studies have established that LF exerts antiviral activity against HCMV, human immunodeficiency virus (HIV), herpes simplex virus (HSV) and hepatitis C virus (HCV) (Harmsen et al., 1995, Hasegawa et al., 1994, Fujihara and Hayashi, 1995, Ikeda et al., 1998). It has been suggested that LF inhibits an early step in viral infection (Hasegawa et al., 1994).

The aim of this study was to elucidate if LF and lactoferricin could prevent HCMV entry into human fibroblasts either by binding to the virion or by blocking host cell receptors.

In the present report, we describe the inhibitory effects of LFs and lactoferricin on HCMV infection in cell culture.

Section snippets

Reagents

Heparin (200 IE per mg) was purchased from LEO, Denmark. Human lactoferrin (55839) was a gift from Cappel/Organon Teknika Corporation, Durham, NC. The following items were purchased from Sigma Co, St. Louis, MO, human lactoferrin, human iron-saturated lactoferrin, bovine lactoferrin. Goat lactoferrin was purified from goat milk (gift from Tine Dairy, Tromsø, Norway) as earlier described (Gutteberg et al., 1984). Cyclic lactoferricin B was a gift from Morinaga Milk Industry, Zama City, Japan.

Results

Fig. 1 summarizes data from experiments comparing the relative HCMV inhibitory effect of lactoferrins from different species. It also compares the effects of the iron-saturated and apo-forms of human lactoferrin. The bovine version was the most effective, reducing the HCMV antigen expression by 90–100% at concentrations of 250–500 μg/ml (IC50 ∼55 μg/ml (0.7 μM)). The apo-form of human lactoferrin was slightly, and the goat version markedly, less inhibitory than the bovine, exhibiting IC50

Discussion

In this report, the antiviral effects of LF and lactoferricin were investigated. We demonstrate that goat LF and the N-terminal peptide of different LFs, lactoferricin inhibit HCMV multiplication in vitro. Earlier, it has been well established that both human and bovine LF inhibit HCMV infection (Hasegawa et al., 1994, Swart et al., 1998. Hasegawa et al. (1994) suggested that the antiviral activity resulted from interference with an early event of virus infection. In this study, we have tried

Acknowledgements

We thank Hilde Olsen, Liv Tone Eliassen, Jill Andersen and Nils-Peder Willasen for technical help and discussions. This work was supported by University Hospital of Tromsø, Norway.

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