Inhibition of Epstein-Barr virus infection by lactoferrin

Abstract

Lactoferrin (LF) is a multifunctional glycoprotein that plays an important role in native immune defense against infections, including human herpetic viruses, such as cytomegalovirus and herpes simplex virus types 1 and 2. However, its anti-Epstein-Barr virus (EBV, a γ-herpesvirus) function has not been reported in the literature. EBV is widespread in all human populations and is believed to be linked to tumorigenesis, such as lymphomas and nasopharyngeal carcinoma (NPC). We previously reported that LF expressed a significantly lower level in NPC tissues and was a likely tumor suppressor. Since EBV infection is a major carcinogen of NPC development, we investigated the effect of LF on EBV infection and found that LF could protect human primary B lymphocytes and nasopharyngeal epithelial cells from EBV infection, but had no effect on EBV genome DNA replication. LF prevented EBV infection of primary B cells mediated by its direct binding to the EBV receptor (CD21) on the B-cell surface. Tissue array immunohistochemistry revealed that LF expression was significantly downregulated in NPC specimens, in which high EBV viral capsid antigen-IgA levels were observed. These data suggest that LF may inhibit EBV infection and that its downregulation could contribute to NPC development.

Fig. 1

LF inhibits EBV binding to host cells. a Primary B cells were preincubated with hLF (50 µg/ml) or BSA as control at 4°C for 1 h; hLF was washed away with cold PBS, and then the cells were exposed to EBV (MOI 50) at 4°C for 3 h with gentle rocking. To remove unbound viruses, cells were washed extensively in PBS. Bound EBV particles on the cell membrane were detected by immunofluorescence with anti-gp350 antibody (red). b Primary B cells were preincubated with different concentrations of hLF (0–50 µg/ml) or with BSA as control (data not shown). The percentage of stained gp350-positive cells was counted by FACS as an index of EBV binding ability. B1 = Cells were not exposed to EBV; B2–B5 = cells were exposed to EBV; B2 = no hLF added; B3 = 12.5 µg/ml hLF; B4 = 25 µg/ml hLF; B5 = 50 µg/ml hLF. The colors refer to the online version of the figure.

Fig. 2

LF inhibits EBV entry. Primary B cells were incubated with hLF (50 µg/ml) or BSA (50 µg/ml) as control at 4°C for 1 h and hLF was washed away. After that, the cells were infected with EBV (MOI 50) at 4°C for 3 h and were then washed with PBS to remove unbound virus. a Total cellular and bound viral DNA was extracted, and the EBV copy number per cell (i.e. EBV titers) was determined using the Q-PCR assay of the EBV BamHI-W fragment. The EBV titer of the control treatment group (first lane) was adjusted as 1. b Total cellular RNA of the treated primary B cells was extracted, and mRNA expression levels of EBV gene EBER were measured by Q-PCR assay. The expression level of the control group was adjusted as 1. Two-way ANOVA was used to evaluate differences between the three groups. ** p < 0.01. c Detection of EBV genomes by FISH. Primary B cells were treated as above and then fixed; intracellular viral genomes were detected by green fluorescence, and nuclei were identified by DAPI staining.

Fig. 3

LF interacts with CD21. a Immunofluorescence colocalization of LF with CD21. Lymphoblastoid cells were incubated with hLF (10 µg/ml) at 4°C for 1 h. After washing, bound LF was detected by anti-LF antibody (red), and CD21 was detected by anti-CD21 antibody (green). The cells were observed using confocal fluorescent microscopy. The yellow color in the merged image represents the extent of the colocalization of LF with CD21 on the cell surface. The colors refer to the online version of the figure. b Coimmunoprecipitation of LF with CD21. 293T cells were cotransfected with pcDNA3.1-Flag-CD21 and pcDNA3.1-LF plasmids. The cell lysates were subjected to coimmunoprecipitation with either anti-LF or anti-Flag antibodies [an unrelated IgG antibody was used as negative immunoprecipitation (IP) control]. Immunoprecipitates were analyzed by Western blot with anti-CD21 or anti-LF antibodies. The left panel shows whole cell lysate from cotransfected cells (lane 2) or untransfected cells (lane 1, as negative control). The right panel shows immunoprecipitates with anti-CD21 or anti-LF or with unrelated IgG antibody as immunoprecipitation control.

Fig. 4

LF does not inhibit EBV replication. Lymphoblastoid cells (a) and P3HR1 cells (b) were treated with either hLF (10 or 100 µg/ml) or BSA (as control) for 3, 6, 15 or 21 days. hLF was presented throughout the reproduction process. The cells were harvested, and viral DNA copies (EBV titers) were determined by Q-PCR assay of the EBV BamHI-W fragment. The EBV titer of the control treatment group (first lane) was adjusted as 1 in both cell types.

Fig. 5

LF inhibits EBV transfer infection from resting B cells to nasopharyngeal epithelial cells. NP69 cells were pretreated with hLF (10 µg/ml) or BSA as control at 4°C for 1 h, then cocultured for 24 h with primary B cells (which had been pre-exposed to EBV), followed by washing extensively with PBS to remove the B cells. hLF was presented both before and throughout the transfer infection. a DNA of NP69 cells was isolated and viral DNA copy numbers (i.e. EBV titers) were determined by Q-PCR assay of the EBV BamHI-W fragment. The EBV titer of the control treatment group (i.e. EBV-LC group) was adjusted as 1. b Total cellular RNA of the NP69 cells was extracted, and mRNA expression levels of EBV gene EBER were measured by Q-PCR assay. The expression level of the control group was adjusted as 1. EBV-LC = NP69 cells cocultured with EBV-positive B lymphocytes. Two-way ANOVA was used to evaluate differences between the three groups. * p < 0.05; ** p < 0.01. c Detection of EBV genomes by FISH. NP69 cells were treated as above and then fixed; intracellular viral genomes were detected by green fluorescence, and nuclei were identified by DAPI staining.