Rapamycin modulation of p70 S6 kinase signaling inhibits Rift Valley fever virus pathogenesis

Abstract

Despite over 60 years of research on antiviral drugs, very few are FDA approved to treat acute viral infections. Rift Valley fever virus (RVFV), an arthropod borne virus that causes hemorrhagic fever in severe cases, currently lacks effective treatments. Existing as obligate intracellular parasites, viruses have evolved to manipulate host cell signaling pathways to meet their replication needs. Specifically, translation modulation is often necessary for viruses to establish infection in their host. Here we demonstrated phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eIF4G following RVFV infection in vitro through western blot analysis and in a mouse model of infection through reverse phase protein microarrays (RPPA). Inhibition of p70 S6 kinase through rapamycin treatment reduced viral titers in vitro and increased survival and mitigated clinical disease in RVFV challenged mice. Additionally, the phosphorylation of p70 S6 kinase was decreased following rapamycin treatment in vivo. Collectively these data demonstrate modulating p70 S6 kinase can be an effective antiviral strategy.

Figure 1. Growth kinetics of MP12 and its effect on p70 S6K signaling in H2.35 cells

(A) H2.35 cells were infected with MP12 at an MOI 5.0. Cell lysates were collected 3, 9, 18 and 24 hpi and viral titers were measured by standard plaque assay (B) H2.35 cells were serum starved for 72 hours and then either mock-infected or infected with MP12 at an MOI 5.0. Cell lysates were collected at 3, 9, 18 and 24 hours post-infection and analyzed by western blot. Membranes were probed p70 S6K (Thr389), S6 ribosomal protein Ser235/236 [noted as S6 rp (Ser235/236)], eIF4G (Ser1108), RVFV nucleoprotein (NP), and β-actin as a loading control. (C) Data plotted represents fold change over mock from blots in (B). Mean values and standard deviations of the chemiluminescent signal intensity of three biological replicates are represented. All values were normalized to the loading control, β-actin, with concomitant background signal subtraction. Error bars represent the means ± SD; n=3.

Figure 2. Growth kinetics of ZH501 and its effect on p70 S6K signaling in H2.35 cells

(A) H2.35 cells were infected with ZH501 at an MOI 5.0. Cell lysates were collected 3, 9, 18 and 24 hpi and viral titers were measured by standard plaque assay. (B) H2.35 cells were serum starved for 48–72 hours and then either mock-infected or infected with ZH501 at an MOI 5.0 for one hour. Cell lysates were collected at 3, 9, 18 and 24 hours post-infection and analyzed by western blot. Membranes were probed p70 S6K (Thr389), S6 rp (Ser235/236), eIF4G (Ser1108), RVFV NP, and β-actin as a loading control. (C) Data plotted represents fold change over mock from blots in (B). Mean values and standard deviations of the chemiluminescent signal intensity of three biological replicates are represented. All values were normalized to the loading control, β-actin, with concomitant background signal subtraction. Error bars represent the means ± SD; n=3.

Figure 3. Rapamycin inhibits p70 S6K signaling and RVFV infection in vitro

(A) To determine the EC₅₀ of rapamycin (RAP), H2.35 cells were allowed to incubate at 33°C and 10% CO₂ overnight. H2.35 cells were pre-treated for 1 hour with drug concentrations starting at 100 μM with 1:2 serial dilutions down to 1.56 μM. Cells were then infected with MP12 virus at a MOI of 1. Infectious media was removed, cells washed once and drug was re-applied. Cells were analyzed at 18 hpi using the Renilla Glo Assay. Luminescence is presented calculated relative to the DMSO control. (B) H2.35 cells were serum starved for 72 hours and then were pre-treated with either DMSO or 10 μM RAP. Cells were infected with MP12 (MOI 5) for one hour, followed by removal of viral inoculum, and addition of growth medium containing DMSO and 10 uM RAP. At 18 hpi, cell lysates were collected for western blot analysis. (C–D) H2.35 cells were serum starved for 72 hours and then were pre-treated with either DMSO, 10 μM RAP, or 30 μM RAP. Cells were infected at an MOI of 5 with (C) MP12 or (D) ZH501 for one hour, followed by removal of viral inoculum, and addition of growth medium containing DMSO, 10 μM RAP, or 30 μM RAP. At 18 hpi, supernatants were collected for standard plaque assay. The mean and SD (N=4) are plotted. For ZH501, 30 μM treatments, N=2. *p-value ≤ 0.05 (E) At 18 hpi, cell lysates were collected in RLT buffer for RNA extraction and qRT-PCR was performed to determine viral genomic copies (F); At 18 hpi, supernatants were collected andviral RNA extracted and quantified by qRT-PCR.(F). The mean and SD (N=5) are plotted for panels E and F.

Figure 4. RVFV model validation

(A) BALB/c mice were randomly assigned to groups of 3 and infected with 1,000 PFU of RVFV ZH501 subcutaneously. Three uninfected control mice were sacrificed on day 0 with infected mice being sacrificed from days 1–8. Liver, spleen, brain, serum, and lymph node were harvested from each mouse and sections were either placed in 10% neutral buffered formalin for histologic evaluation or were used to determine viral titers in each organ via plaque assay. (B) Liver, Infected Mouse, Day 4: Histologic examination revealed groups of hepatocytes with shrunken, hypereosinophilic, cytoplasm and condensed, eccentrically placed nuclei (red arrow) consistent with groups of cells undergoing apoptosis. Adjacent to these areas were nuclei containing 3 to 4 um, hypereosinophilic, intranuclear inclusions (blue arrow/red circle) consistent with RVFV protein inclusions. (C) Uninfected Liver – Day 4, RVFV Immunohistochemistry. (D) Day 3 PI – rare hepatocytes (circle) displayed intracytoplasmic immunohistochemical staining. (E) Day 4 PI – positive staining throughout the liver to include large groups of cells displaying strong, specific, intracytoplasmic immunohistochemical staining.

Figure 5. p70 S6K pathway is activated following RVFV infection in vivo.

(A) BALB/c mice (groups of 3) were subcutaneously infected with RVFV ZH501 (1000 PFU/mouse). Control mice were sacrificed on day 0 and infected mice on days 3, 4, and 5. Liver and spleen were collected and placed in blue lysis buffer. Tissues were homogenized, spun, transferred to new tubes, heated at 100°C for 15 minutes, and then frozen at −80°C prior to RPPA analysis. Error bars represent standard error of the mean; n=3. (B) and (C) Immunohistochemistry for p70S 6K (Thr421) was performed as an additional determinant of activation of p70 S6K within the liver of infected mice. Panel B is an uninfected control and panel C is an infected day 4 sample. Arrows indicate an increase in p70 S6K staining.

Figure 6. In vivo efficacy of rapamycin, low dose study

Female BALB/c mice, 6–8 weeks of age, were randomly distributed into treatment groups consisting of 10 mice per group. Mice were infected via the subcutaneous route with a low dose of RVFV ZH501 (150 PFU/mouse). Health scores and weights were assessed daily. The endpoint was day 16, death, or euthanasia. The survival curves are shown in panels A, % of weight maintained over the course of the study is shown in panel B, and the clinical scoring chart for the vehicle control group and rapamycin treated group are shown in panel C. Log-rank (Mantel-Cox) test was used to determine statistical significance of the survival curve and a two-way ANOVA analysis was used to evaluate the change in weight loss, *p-value ≤ 0.05.

Figure 7. In vivo efficacy of rapamycin, high dose study

Female BALB/c mice, 6–8 weeks of age, were randomly distributed into treatment groups consisting of 10 mice per group. Mice were infected via the subcutaneous route with a high dose of RVFV ZH501 (1,500 PFU/mouse). Health scores and weights were assessed daily. The endpoints were day 21, death, or euthanasia. The survival curve is shown in panels A, % of weight maintained over the course of the study is shown in panel B, and the clinical scoring chart for the vehicle control group and rapamycin treated group are shown in panel C. Log-rank (Mantel-Cox) test was used to determine statistical significance of the survival curve and a two-way ANOVA analysis was used to evaluate the change in weight loss, *p-value ≤ 0.05.

Figure 8. Pathway modulation screening in the liver and spleen following rapamycin treatment

Female BALB/c mice, 6–8 weeks of age, were infected via the subcutaneous route with a high dose of RVFV ZH501 (1,500 PFU/mouse). Mice were randomly distributed into groups of 5 with subsequent serial sacrifice of vehicle treated control mice and rapamycin (10mg/kg) treated mice on days 4 and 6. Liver and spleen were collected and placed in blue lysis buffer for downstream RPPA analysis. Data plotted represents means and standard deviations from 5 animals per condition. Black circles and red squares represent vehicle treated or rapamycin treated mice, respectively. Phospho-protein levels in the liver (A, C, E) or spleen (B, D, F) of both vehicle control and rapamycin treated groups on days 4 and 6 post-infection are displayed. A two-way ANOVA analysis was used to evaluate changes in protein phosphorylation, *p-value ≤ 0.05.