Effects of the Natural Flavonoid Quercetin on Arenavirus Junín Infection

Abstract

There is no specific chemotherapy approved for the treatment of pathogenic arenaviruses that cause severe hemorrhagic fever (HF) in the population of endemic regions in America and Africa. The present study reports the effects of the natural flavonoid quercetin (QUER) on the infection of A549 and Vero cells with Junín virus (JUNV), agent of the Argentine HF. By infectivity assays, a very effective dose-dependent reduction of JUNV multiplication was shown by cell pretreatment at 2-6 h prior to the infection at non-cytotoxic concentrations, with 50% effective concentration values in the range of 6.1-7.5 µg/mL. QUER was also active by post-infection treatment but with minor efficacy. Mechanistic studies indicated that QUER mainly affected the early steps of virus adsorption and internalization in the multiplication cycle of JUNV. Treatment with QUER blocked the phosphorylation of Akt without changes in the total protein expression, detected by Western blot, and the consequent perturbation of the PI3K/Akt pathway was also associated with the fluorescence redistribution from membrane to cytoplasm of TfR1, the cell receptor recognized by JUNV. Then, it appears that the cellular antiviral state, induced by QUER treatment, leads to the prevention of JUNV entry into the cell.

Keywords: Argentine hemorrhagic fever; Junín virus; PI3K/Akt pathway; antiviral cell state; arenavirus; natural flavonoid; pretreatment; quercetin; transferrin receptor; virus entry.

Figure 1

Cytotoxicity and anti-proliferative effect of QUER. To measure the effects on (A) confluent or (B) actively growing A549 or Vero cells, two-fold dilutions of QUER were added 24 or 2.5 h after cell seeding in 96-well plates, respectively. After 48 h of incubation, the ratio of viable cells in drug-treated and mock-treated cultures was determined by MTT assay and expressed as % cell viability. (C) Colony forming cell assay (CFC): Actively growing A549 (upper panel) and Vero (lower panel) cells were treated with QUER as in (B), after 48 h at 37 °C cells were fixed and stained with 1% crystal violet solution. Images were obtained using optical microscope Nikon Eclipse TS100. Magnification 40×. (D) Stained cell colonies were quantified by absorbance of solubilized crystal violet. Viability values (% respect to non-treated cell control) are presented as the mean ± SD obtained from three independent experiments. * p < 0.05; ** p < 0.001; *** p < 0.0001; **** p < 0.00001.

Figure 2

Antiviral activity of QUER against JUNV. The antiviral activity of the compound was determined after 48 h of infection (m.o.i. 0.1) by plaque formation. (A) Cells were infected in the absence of compound and, after 1 h adsorption at 37 °C, refed with MM containing different dilutions of QUER. (B) After incubation with different dilutions of QUER in MM for 2 h at 37 °C, cells were infected with JUNV and refed with compound-free MM. (C) A549 or (D) Vero cells were treated as in (B) for 6 or 24 h before viral infection. Inhibition values (% respect to non-treated viral control) are presented as the mean ± SD obtained from three independent experiments. * p < 0.05; ** p < 0.001; *** p < 0.0001.

Figure 3

Virucidal effect of QUER. The inactivating effect of the compound was determined by plaque formation assay in Vero cells, after 1.5 h exposure (37 or 4 °C) to MM containing different dilutions of QUER. The remaining infectivity was determined by PFU. Inhibition values (% respect to non-treated viral control) are presented as the mean ± SD obtained from three independent experiments. * p < 0.05; ** p < 0.001; *** p < 0.0001.

Figure 4

Effect of time of treatment with QUER on antiviral activity. (A) A549 or (B) Vero cells were infected with JUNV (m.o.i 2) in the presence (0 h p.i.) or absence of compound (1 to 12 h p.i.). After 1 h at 4 °C, cell cultures were refed with MM and incubated at 37 °C. At different times p.i. cells were treated with 50 µg/mL of QUER. Extracellular virus yields were determined by plaque formation at 24 h p.i. (C) A549 or (D) Vero cells were infected with JUNV (m.o.i 2) under different treatment conditions. Adsorption: cells were infected in MM containing or not 50 µg/mL of QUER, after 1 h at 4 °C, cells were refed with compound-free MM. Internalization: virus was adsorbed to cells for 1 h at 4 °C in MM, then MM with or without 50 µg/mL of QUER was added, after 2 h incubation at 37 °C supernatant was removed and cells were covered with compound-free MM. For both treatments, extracellular virus yields were determined after 48 h p.i. by PFU. Virus titers are presented as the mean ± SD obtained from three independent experiments. ** p < 0.001; *** p < 0.0001.

Figure 5

Antiviral activity of QUER against TCRV (•) and LCMV (▪). Vero cells were incubated with different dilutions of QUER in MM for 2 h at 37 °C, then washed, infected, unadsorbed virus removed and refed with compound-free MM. Viral yields were determined at 48 h p.i. and inhibition values (% respect to non-treated viral control) are presented as the mean ± SD obtained from three independent experiments. * p < 0.05.

Figure 6

Effect of QUER on Akt and TfR1. (A) A549 cells were treated (QUER) or not (CC: cell control) with QUER 50 µg/mL during 2 h. Then, cells were lysated and proteins were separated, transferred onto a PVDF membrane, blocked and revealed by using anti-phospho-Akt, anti-Akt, anti-TfR1 and anti-β-actin antibodies. Chemiluminescence detection was performed and protein bands were quantified. (B) A549 cells were treated with (QUER) or without (CC) QUER 50 µg/mL during 2 h and then were processed to immunofluoescence assay. Cells were fixed, permeabilized, incubated with anti-TfR1 and then incubated with anti-mouse Alexa 488 conjugated antibodies. Cell nuclei were stained using DAPI. Coverslips were mounted, cells were visualized and images were captured by confocal microscopy (63×). (C,E) A549 cells were treated with (QUER) or without (CC) QUER 50 µg/mL during 2 h and then were processed to immunofluorescence assay without permeabilization (C) or permeabilized with Triton X-100 (E). Cells were stained for TfR1 as in B, and cell membrane was labelled using WGA-TRITC. Cells were then visualized using the epifluorescence microscope Olympus IX71, magnification 60×. (D,F) The analysis of the data was performed using the free software ImageJ, and statistical analysis was performed counting 30 cells per treatment using the t-student test. *** p < 0.0001.