Rotavirus-encoded nonstructural protein 1 modulates cellular apoptotic machinery by targeting tumor suppressor protein p53

Abstract

p53, a member of the innate immune system, is triggered under stress to induce cell growth arrest and apoptosis. Thus, p53 is an important target for viruses, as efficient infection depends on modulation of the host apoptotic machinery. This study focuses on how rotaviruses manipulate intricate p53 signaling for their advantage. Analysis of p53 expression revealed degradation of p53 during initial stages of rotavirus infection. However, in nonstructural protein-1 (NSP1) mutant strain A5-16, p53 degradation was not observed, suggesting a role of NSP1 in this process. This function of NSP1 was independent of its interferon or phosphatidylinositol 3-kinase (PI3K)/AKT modulation activity since p53 degradation was observed in Vero cells as well as in the presence of PI3K inhibitor. p53 transcript levels remained the same in SA11-infected cells (at 2 to 14 h postinfection), but p53 protein was stabilized only in the presence of MG132, suggesting a posttranslational process. NSP1 interacted with the DNA binding domain of p53, resulting in ubiquitination and proteasomal degradation of p53. Degradation of p53 during initial stages of infection inhibited apoptosis, as the proapoptotic genes PUMA and Bax were downregulated. During late viral infection, when progeny dissemination is the main objective, the NSP1-p53 interaction was diminished, resulting in restoration of the p53 level, with initiation of proapoptotic signaling ensuing. Overall results highlight the multiple strategies evolved by NSP1 to combat the host immune response.

Fig 1

p53 level undergoes posttranscriptional depletion during SA11 infection. (A and B) MA104 cells (A) or Vero cells (B) were infected with SA11 at an MOI of 3 for 2 to 14 hpi, followed by immunoblotting with anti-p53, anti-NSP1, and anti-GAPDH antibody. The relative fold change in protein levels of p53 during SA11 infection in both MA104 cells (A) and Vero cells (B) was normalized relative to the level of the internal control, GAPDH. Results shown here are representative of triplicate immunoblotting experiments. (C) The level of p53 remains stable in mock-infected MA104 cells. Mock-infected MA104 cells were harvested at different time points, followed by immunoblotting with p53- and GAPDH-specific antibody. (D) Relative fold change in transcript levels of nsp4, bax, p53, and puma during SA11 infection compared with the level of the uninfected control normalized with respect to that of the internal control, the GAPDH gene. MA104 cells were infected with SA11 at an MOI of 3 for the indicated time points. RNA was isolated, and the nsp4, p53, bax, and puma mRNA levels were analyzed by quantitative reverse transcription-PCR. Fold changes were obtained by normalizing the relative gene expression to that of the GAPDH gene using the formula 2^−ΔΔCT (ΔΔC_T = ΔC_T for the sample minus ΔC_T for the untreated control).

Fig 2

NSP1 induces proteasomal degradation of p53. (A) In NSP1 mutant A5-16-infected MA104 cells, p53 is not degraded. MA104 cells were infected with A5-16 at an MOI of 3 for the indicated time points, followed by immunoblotting with anti-p53 antibody and anti-GAPDH antibody. (B) NSP1 expression in the absence of other rotaviral proteins is sufficient for p53 degradation. 293T cells were either transfected with pCMV-p53 alone or cotransfected with the pACGFP vector plus pCMV-p53 or pcDNSP1 or with pCMV-p53 plus pcDNSP1 for 36 h, followed by immunoblotting with anti-p53, anti-β-actin, and anti-NSP1 antibody. The relative fold change in protein levels of p53 was normalized with respect to the level of the internal control, β-actin. Results shown here are representative of triplicate immunoblotting experiments. (D) NSP1-induced p53 degradation is prevented in the presence of MG132. 293T cells were either transfected with pCMV-p53 or cotransfected with either pCMV-p53 or pcDNSP1 in the presence or absence of 10 μM MG132, followed by immunoblot analysis with anti-p53, anti-β-actin, and anti-NSP1 antibody. The relative fold change in protein levels of p53 was normalized with respect to that of the internal control, β-actin. Results shown here are representative of triplicate immunoblotting experiments.

Fig 3

NSP1 in the presence or absence of other viral proteins induces p53 degradation independently of PI3K and MDM2. (A and B) SA11 infection stimulates p53 degradation both in the presence and in the absence of PI3K inhibitor (LY294002) and MDM2 inhibitor (Nutlin-3). MA104 cells were infected with SA11 at an MOI of 3 for the indicated time points in the presence or absence of either LY294002 (A) or Nutlin-3 (B), followed by immunoblotting with anti-p53, anti-GAPDH, and anti-NSP1 antibody. (C and D) NSP1 stimulates p53 degradation both in presence and in the absence of PI3K inhibitor (LY294002) and MDM2 inhibitor (Nutlin-3). 293T cells were either cotransfected with pCMV-p53 and pcDNSP1, transfected with pCMV-p53, or kept mock transfected for 36 h in the presence or absence of either LY294002 (C) or Nutlin-3 (D), followed by immunoblotting with anti-p53, anti-β-actin, and anti-NSP1 antibody.

Fig 4

NSP1 interacts with p53 both during infection and in cells overexpressing NSP1. (A) NSP1 interacts with p53 in the absence of other viral proteins. 293T cells were either cotransfected with pCMV-p53 and pcDNSP1 or transfected with the individual constructs for 36 h and coimmunoprecipitated (IP) with either anti-p53 or anti-His antibody, followed by immunoblot analysis using the reciprocal antibodies. Whole-cell lysates were immunoblotted with anti-p53 and anti-NSP1 antibody. W.B., Western blot. (B) NSP1 interacts with p53 under in vitro conditions. IVT p53 or IVT NSP1 was immobilized with Ni⁺², and enterokinase-treated reciprocal IVT proteins were incubated with them, followed by immunoblot analysis for p53 and NSP1, respectively. (C) NSP1 induces ubiquitinylation of p53. 293T cells were either transfected with pCMV-p53 or cotransfected with pCMV-p53 plus pFLAG-CMVUb or pCMV-p53, pcDNSP1, and pFLAG-CMV-Ub for 36 h and coimmunoprecipitated with anti-p53 antibody, followed by immunoblotting with anti-FLAG antibody. Whole-cell lysates were immunoblotted with anti-p53 and anti-NSP1 antibody. (D and E) NSP1 interacts with p53 during SA11 infection. MA104 cells were infected with SA11 at an MOI of 3 for the indicated time points, and coimmunoprecipitation was done with either anti-p53 (D) or anti-NSP1 (E) antibody, followed by immunoblotting with the reciprocal antibody. Whole-cell lysates were immunoblotted with anti-p53 and anti-NSP1 antibody.

Fig 5

The DNA binding domain (amino acids 100 to 300) of p53 interacts with NSP1. (A) Analysis of expression of truncated constructs of p53. 293T cells were transfected with the region of pFLAG-CMV-p53 from amino acids 1 to 80, pFLAG-CMV-p53 from amino acids 100 to 300, or pFLAG-CMV-p53 from amino acids 300 to 393, followed by immunoblotting with anti-FLAG and anti-β-actin antibody. (B and C) The region from amino acids 100 to 300 interacts with NSP1. 293T cells were either cotransfected with pcDNSP1 and each of the p53 domains or transfected with the individual constructs for 36 h and coimmunoprecipitated with either anti-FLAG (B) or anti-His (C) antibody, followed by immunoblotting with reciprocal antibody. Whole-cell lysates were immunoblotted with anti-NSP1 and anti-FLAG antibody. (D and E) The DNA binding domain of p53 interacts with NSP1 in vitro. Different IVT products of p53 mutants or IVT NSP1 were immobilized with Ni⁺², and enterokinase-treated reciprocal IVT proteins were incubated with them, followed by immunoblot analysis of the proteins interacting with the immobilized proteins.

Fig 6

The RING domain of NSP1 is necessary for ubiquitinylation and degradation, but it does not bind to p53. (A) Schematic representation of NSP1, the RING domain (amino acids 1 to 82) of NSP1, and RING domain mutant NSP1 (amino acids 83 to 482) cloned in pcDNA. (B) Ectopic expression of fragments of NSP1. 293T cells were transfected with pcDRING-NSP1 and pcDΔRING-NSP1 for 36 h, followed by immunoblotting with anti-His and anti-β-actin antibody. (C and D) The RING domain is not necessary for interaction with p53. 293T cells were either transfected with pCMV-p53, pcDRING-NSP1, or pcDΔRING-NSP1 individually or cotransfected with either pCMV-p53 and pcDRING-NSP1 or pCMV-p53 and pcDΔRING-NSP1 separately for 36 h and coimmunoprecipitated with either anti-His (C) or anti-p53 (D) antibody, followed by immunoblotting with the reciprocal antibody. Whole-cell lysates were immunoblotted with anti-p53 anti-His antibody. (E) The RING domain is necessary for degradation of p53. 293T cells were either transfected with pCMV-p53 or cotransfected with either pCMV-p53 and pcDNSP1 or pCMV-p53 and pcDΔRING-NSP1 separately for 36 h, followed by immunoblotting with anti-p53 and anti-β-actin antibody. (F) The RING domain is necessary for ubiquitinylation of p53. 293T cells were either transfected with pCMV-p53 or cotransfected with pCMV-p53, pcDΔRING-NSP1, and pFLAG-CMV-Ub or pCMV-p53, pcDNSP1, and pFLAG-CMV-Ub for 36 h, followed by coimmunoprecipitation with anti-p53 antibody and immunoblotting with anti-FLAG antibody.

Fig 7

NSP1-mediated p53 degradation prevents p53-activated apoptosis initiation in SA11 infection during early infection independently of PI3K. (A and B) The levels of PUMA and activation of Bax remain downregulated at initial time points of SA11 infection but not during NSP1 mutant A5-16 infection. MA104 cells were infected with either SA11 (A) at an MOI of 3 or A5-16 (B) at an MOI of 3 or kept mock infected for the indicated time points, followed by immunoblot analysis of either the whole-cell lysates with anti-PUMA and anti-β-actin antibody or the mitochondrial fraction with anti-Bax and anti-Cox4 antibody. (C and D) p53-induced modulation of apoptosis during SA11 (NSP1 positive) or A5-16 (NSP1 negative) infection is independent of NSP1-mediated PI3K activation. MA104 cells were infected with either SA11 (C) or A5-16 (D) at an MOI of 3 in the presence of LY294002 for the indicated time points, followed by immune blot analysis of either the whole-cell lysates with anti-PUMA and anti-β-actin antibody or the mitochondrial fraction with anti-Bax and anti-Cox4 antibody. (E) NSP1 counteracts the proapoptotic function of p53. 293T cells were either transfected with pCMV-p53, cotransfected with either pcDNSP1 and pCMV-p53 or pCMV-p53-mt135 and pcDNSP1, or kept mock transfected for 36 h, followed by immunoblot analysis of the whole-cell lysate (PUMA, caspase-9, caspase-3, PARP, and β-actin antibodies) and the mitochondrial fraction (anti-Bax, and anti-Cox4 antibodies).

Fig 8

Apoptosis induced during later stages of rotavirus infection (SA11) is partly dependent on p53 expression. (A and B) Inhibition of p53 function during SA11 infection inhibits upregulation of the PUMA level and Bax activation. MA104 cells were transfected with pCMV-p53-mt135 (A) or a nonspecific vector, pACGFP (B), for 24 h and then infected with SA11 for the indicated time points, followed by immunoblot analysis of whole-cell lysates (anti-PUMA, anti-β-actin) and the mitochondrial fraction (anti-Bax, anti-Cox4). (C and D) In the absence of functional p53, rotavirus-induced caspase activation was decreased. MA104 cells were either infected with SA11 in the presence or absence of caspase-9 inhibitor benzyloxycarbonyl-Leu-Glu-His-Asp-methyl-fluoromethylketone (Z-LEHD-FMK) (C) or caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK) (D) or transfected with pCMV-p53-mt135 or a nonspecific vector, pACGFP, for 24 h and then infected with SA11 for the indicated time points, followed by caspase activity assay using synthetic substrates for caspase-9 (LEHD-AMC) and caspase-3 (DEVD-AFC). Results are representative of three independent experiments. Values represent the means ± SDs of one experiment with three measurements taken.