p53 Activation following Rift Valley Fever Virus Infection Contributes to Cell Death and Viral Production

Rift Valley fever virus (RVFV) is an emerging viral zoonosis that is responsible for devastating outbreaks among livestock and is capable of causing potentially fatal disease in humans. Studies have shown that upon infection, certain viruses have the capability of utilizing particular cellular signaling pathways to propagate viral infection. Activation of p53 is important for the DNA damage signaling cascade, initiation of apoptosis, cell cycle arrest and transcriptional regulation of multiple genes. The current study focuses on the role of p53 signaling in RVFV infection and viral replication. These results show an up-regulation of p53 phosphorylation at several serine sites after RVFV MP-12 infection that is highly dependent on the viral protein NSs. qRT-PCR data showed a transcriptional up-regulation of several p53 targeted genes involved in cell cycle and apoptosis regulation following RVFV infection. Cell viability assays demonstrate that loss of p53 results in less RVFV induced cell death. Furthermore, decreased viral titers in p53 null cells indicate that RVFV utilizes p53 to enhance viral production. Collectively, these experiments indicate that the p53 signaling pathway is utilized during RVFV infection to induce cell death and increase viral production

p53 phosphorylation following RVFV infection is NSs dependent.

<p>A) Vero cells were either mock infected or infected with MP-12, rMP-12 ΔNSs or arMP-12 ΔNSm viruses at an MOI of 3.0. Cells were collected 24 hours post infection and western blot analysis was performed on the cell lysates using antibodies against p53 (Ser15) and total p53. B) Cells were infected and processed as described in panel A. Western blot analysis was performed for p53 (Ser20), and the N protein of RVFV. Actin was used as a loading control. C) Vero cells were infected with MP-12, rMP-12 ΔNSs or arMP-12 ΔNSm viruses at an MOI of 3.0. Viral supernatants were collected at 16, 24 and 48 hours post-infection and released virus determined by plaque assays. (*) indicates statistically significant difference (unpaired t-test) p<0.001. Error bars indicate standard deviation.</p

RVFV infection induces the Up-regulation of p53 target genes.

<p>HSAECs were either mock infected or infected with MP-12 (MOI 3.0). Cells were collected at 24 hours post infection. RNA was extracted using Qiagen’s RNeasy Mini Kit. After cDNA synthesis, qRT-PCR was performed on the samples using the primers shown (Bax, Puma, Noxa, GADD45, 14-3-3σ, p21, MDM2 and p62) (Panels A–H). Actin was used to normalize the samples. (*) indicates statistically significant difference (unpaired t-test of triplicates) p<0.05. Error bars indicate standard deviation.</p

p53 localizes to the nucleus following infection with RVFV.

<p>A) HSAECs were either mock infected or infected with MP-12 (MOI 3.0). At 24 hours post infection the cells were washed in PBS without Ca and Mg, permeabilized with Triton X-100 and immunostained with anti-p53 primary antibody using Alexa-Fluor 568 secondary antibody and with anti-RVFV N protein antibody using Alexa-Fluor 488 secondary antibody. The nucleus was stained with DAPI. B) HSAECs were mock infected or infected with MP-12 (MOI 3.0) and collected at 24 hours post-infection for the creation of cytoplasmic and nuclear extracts, CE and NE. Western blot analysis was performed with antibodies against lamin, GAPDH, total p53 and RVFV N protein.</p

RVFV production is decreased in p53 null cells.

<p>HCT-116 p53+/+ and −/− cells were mock infected or infected at the indicated MOIs (0.1 and 5.0). At 96 hours post infection viral supernatants were collected and cells were used in a Cell Viability Assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036327#pone-0036327-g004" target="_blank">Figure 4b</a>). Plaque assays were performed in triplicates using the supernatants and the average viral titers (pfu/ml) of the triplicates are shown. (*) Indicates statistically significant difference (unpaired t-test of triplicates) p<0.05, or (**) p<0.01. Error bars indicate standard deviation.</p

p53 is phosphorylated on several residues following infection with RVFV.

<p>A) HSAECs were either mock or MP-12 infected at an MOI of 3.0. Cells were collected 6, 24 and 48 hours post infection and lysates were analyzed by western blotting for antibodies against p53 at the residues indicated (Ser 9, Ser 20, Ser 37, Ser 46 and Ser 15). B) HSAECs infected in the same manner as Fig. 1A were collected at the time points indicated and analyzed by western blotting for antibodies against total p53 and p53 (Ser392). C) HSAECs were treated in the same manner as Fig. 1A and Fig. 1B and analyzed by western blotting for antibodies against the N protein of RVFV. D) HSAECs were infected with UV inactivated MP-12 (MOI 3.0) and collected at 24 and 48 hours post-infection. In parallel, HSAECs were treated with doxorubicin (1 µM) and collected at 24 hours. Cell lysates were analyzed by western blot analysis as describe in panels A and B. Actin is used as a loading control in all panels.</p

Loss of p53 provides resistance to RVFV induced cell death.

<p>A) HCT-116 p53+/+, +/−, and −/− cells were mock infected or infected with MP-12 (MOI 0.1, 0.5, 1.0, and 5.0). Cell viability was determined 96 hours post-infection by CellTiter-Glo Assay (Promega). Viability of the infected cells was calculated relative to the mock infected cells (100%) (average of triplicates shown). (*) Indicates statistically significant difference (unpaired t-test of triplicates) p<0.05. Error bars indicate standard deviation. B) Western blot analysis of uninfected whole cell lysates from HCT-116 p53+/+, +/−, and −/− cells probed with anti-p53 total and actin antibodies. C) HSAECs were either mock infected or infected with MP-12 (MOI 1, 3, or 10). Cell viability was determined 48 and 72 hours post-infection by CellTiter-Glo Assay (Promega). Viability of the infected cells was calculated relative to the mock infected cells (100%) (average of triplicates shown). D) HSAECs were transfected with negative control siRNA (siNEG) or siRNA targeting p53 (sip53) at 50, 100, or 200 nM using attractene’s fast-forward method. Twenty-four hours post-transfection, cells were infected with MP-12 and collected 24 hours post-infection. Cell lysates were analyzed by western blot analysis for total p53 and actin. Lane 1 is a mock infected and untransfected control. E) HSAECs were transfected with siNEG or sip53 (100 nM) by attractene’s fast-forward method. Twenty-four hours post-transfection cells were mock infected or infected with MP-12 (MOI 10). Cell viability was determined 72 hours post-infection by CellTiter-Glo Assay (Promega). Viability of the infected cells was calculated relative to the mock infected cells (100%) (average of triplicates shown). (*) Indicates statistically significant difference (unpaired t-test of triplicates) p<0.01. Error bars indicate standard deviation.</p

Primer Sequences.

<p>Primer Sequences.</p