Pseudoviruses were produced from 293T cells by transfecting at 1:1:1 ratio of plasmids expressing murine leukaemia virus gag/pol, arenaviral GP and pQCXIX transduction vector (BD Biosciences) expressing enhanced green fluorescent protein (EGFP), as described previously30 (link). Virus-containing culture supernatant was harvested two days later, and filtered through 0.45-μm filter disks. Anti-human TfR1 and anti-HLA-A,B,C antibodies were dialysed against PBS. Cells were incubated with each of these antibodies at the indicated concentrations for 30 min at 37 °C. Pseudoviruses were added, cells were washed 16 h after infection, and entry level was measured by flow cytometry. To study the effect of tranferrin on viral entry, pseudoviruses were produced in serum-free medium (FreeStyle; Invitrogen). The role of human TfR2 was assessed in BHK cells transfected with pCAGGS-human TfR2 plasmid complexed with Lipofectamine 2000 (Invitrogen). Transfected cells were infected the next day, and the infection level was assessed two days later.
To study the role of iron in arenaviral infection, cells were incubated in complete medium containing indicated concentrations (1–3 μM) of the iron chelator deferoxamine (Sigma) for 24 h, or ferric ammonium citrate (30–100 μg ml-1) for 1 h. Cells were cooled on ice and infected with pseudoviruses by centrifugation (2,000g) at 4 °C for 30 min. Cells were washed, and GFP expression level was assessed 24 h (293T and HeLa cells) or 48 h (SLK cells) after infection.
To study the role of iron in arenaviral infection, cells were incubated in complete medium containing indicated concentrations (1–3 μM) of the iron chelator deferoxamine (Sigma) for 24 h, or ferric ammonium citrate (30–100 μg ml-1) for 1 h. Cells were cooled on ice and infected with pseudoviruses by centrifugation (2,000g) at 4 °C for 30 min. Cells were washed, and GFP expression level was assessed 24 h (293T and HeLa cells) or 48 h (SLK cells) after infection.
