The PI3K/Akt pathway contributes to arenavirus budding

Abstract

Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose a significant public health concern in regions where they are endemic. On the other hand, evidence indicates that the globally distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway participates in many cellular processes, including cell survival and differentiation, and also has been shown to play important roles in different steps of the life cycles of a variety of viruses. Here we report that the inhibition of the PI3K/Akt pathway inhibited budding and to a lesser extent RNA synthesis, but not cell entry, of LCMV. Accordingly, BEZ-235, a PI3K inhibitor currently in cancer clinical trials, inhibited LCMV multiplication in cultured cells. These findings, together with those previously reported for Junin virus (JUNV), indicate that targeting the PI3K/Akt pathway could represent a novel antiviral strategy to combat human-pathogenic arenaviruses.

Fig 1

Effect of PI3K/Akt inhibitors on multiplication of LCMV and Akt phosphorylation. (A) LY inhibits multiplication of LCMV in cultured cells. 293T cells were infected with LCMV (MOI, 0.001 or 1.0). After 90 min of adsorption time, the inoculum was removed, cell monolayers were washed, and fresh medium containing Akt-IV (0.5 μM or 2 μM), Akt-VIII (2 μM), or LY (20 or 50 μM) was added. At the indicated times p.i., virus titers were determined in TCS using an immune focus-forming (IFF) assay (see Materials and Methods). (B) Effect of LY on Akt phosphorylation. (Bi) 293T cells were treated with Akt-IV (0.5 or 2 μM), Akt-VIII (2 μM), LY (20 or 50 μM), or DMSO as a control. At 4 or 24 h posttreatment, cell lysates were prepared and total Akt and phosphorylated Akt (S473) were detected by Western blotting (WB). (Bii) Cell toxicity associated with the indicated drug treatments was assessed by determining cell viability after 24 h of treatment using the CellTiter-Glo luminescent cell viability assay (Promega). DMSO treatment was adjusted to 1.0. Data are averages and standard deviations from three independent experiments normalized with respect to DMSO treatment.

Fig 2

Effect of BEZ-235 on Akt phosphorylation in 293T cells. (A) 293T cells were treated with BEZ-235 (1, 5, 50, or 500 nM or 5 μM) or DMSO as a control. At 4 or 24 h posttreatment, cell lysates were prepared and total Akt and phosphorylated Akt (S473) were detected by Western blotting (WB). (B) Cell toxicity associated with BEZ-235 treatment. 293T cells were treated with the indicated BEZ-235 concentrations or with DMSO for 24 h, and then cell viability was determined using the CellTiter-Glo luminescent cell viability assay (Promega). Survival in DMSO-treated cells was adjusted to 1.0. Data are averages and standard deviations from three independent experiments.

Fig 3

BEZ-235 inhibits LCMV multiplication in cultured cells. 293T cells were infected with LCMV (MOI, 0.01). After 90 min of adsorption time, the inoculum was removed, cell monolayers were washed, and fresh medium containing the indicated BEZ-235 concentration was added. At the indicated times p.i., virus titers were determined in TCS using an IFF assay (Materials and Methods).

Fig 4

LY and BEZ-235 do not inhibit LCMV GP-mediated cell entry. 293T cells were pretreated with LY (50 μM) (A) or BEZ-235 (5 μM) (B) for 1 h prior to infection with either VSVΔG-GFP/VSV-G or rVSVΔG-GFP/LCMV-GP (MOI, 1.0). Infections were done in the presence of drugs. At 12 h p.i., cells were fixed and for each sample numbers of GFP-positive cells in four different fields determined by epifluorescence. Averages and standard deviations were obtained. Numbers of GFP-positive cells were normalized with respect to values obtained in nontreated cells, which were adjusted to 100%.

Fig 5

Effects of commercially available PI3K/Akt inhibitors and BEZ-235 on LCMV MG-derived reporter gene expression. (A) Drug effects on MG-derived reporter gene expression. 293T cells were transfected with pC-T7, pMG-CAT, pC-NP, and pC-L as described previously (18, 19, 29, 31, 32). After 5 h of transfection, medium was replaced with fresh medium containing Akt-IV (0.5 μM), Akt-VIII (2.0 μM), LY (20 or 50 μM), or BEZ-235 (0.05, 0.5, or 5 μM). At 24 h posttransfection, cell lysates were prepared for CAT ELISA. CAT expression levels from vehicle (DMSO)-treated cells were set to 1.0 to normalize CAT expression levels from the other samples. (B) Drug effects on Pol-II-based transcription. 293T cells (8 × 10⁴/96-well plate) were transfected with pC-Fluc using Lipofectamine 2000 (Invitrogen), and 12 h later, medium was replaced with fresh medium containing the indicated compounds at the indicated concentrations. At 24 h post-compound treatment, levels of Fluc were determined using the Steady Glo assay (Promega) and a luminometer (Centro LB 960; Berthold Technologies). The viability of DMSO-treated control cells was set at 1.0. Data are averages and standard deviations from three independent experiments.

Fig 6

Effects of LY and BEZ-235 on LCMV-MG-derived RNA synthesis. 293T cells were transfected with pC-T7, pMG-CAT, pC-NP, and pC-L as described for Fig. 5. After 5 h, the transfection medium was replaced with fresh medium containing the indicated drugs and concentrations, and 24 h later, total cell RNA was isolated and analyzed by Northern blotting using a ³²P-RNA probe that specifically hybridized to the CAT mRNA and the recombinant S antigenome (rSag) RNA species.

Fig 7

Effect of LY and BEZ-235 on Z-mediated budding. 293T cells were transfected with 0.25 μg of either pC-LCMV-Z-FLAG or pC-LASV-Z-FLAG, and 12 h posttransfection, the medium was replaced with fresh medium containing the indicated concentration of LY (A) or BEZ-235 (B); 24 h later, TCS were collected and total cell lysates prepared. VLPs were isolated from TCS as described previously (40). Levels of Z protein in total cell lysates and VLPs were detected by WB using an antibody to FLAG (product number 162150; Cayman).

Fig 8

Effect of Rpm on LCMV multiplication. BHK-21 cells were infected (MOI, 0.1) with r3LCMV/GFP (12) and treated with Rpm at the indicated concentrations. At the indicated h p.i., TCS were collected and cell monolayers fixed in 4% PFA/PBS. In addition, total cellular RNA was isolated from duplicate infections treated with the indicated concentration of Rpm. (A) Infectious progeny in TCS was determined using an IFF assay. (B) Numbers of virus-infected cells in each case were determined based on GFP expression. (C) Levels of viral RNA synthesis, both replication and transcription, were assessed by Northern blot hybridization using an NP-specific double-strand DNA probe that hybridized to the rS (replication) and NP mRNA (transcription) RNA species.