Herpes simplex virus 1 induces cytoplasmic accumulation of TIA-1/TIAR and both synthesis and cytoplasmic accumulation of tristetraprolin, two cellular proteins that bind and destabilize AU-rich RNAs

Abstract

Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the U(L)41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3'-to-5' degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking U(L)41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking U(L)41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.

FIG. 1.

Real-time PCR analysis of TTP transcripts in HSV-1-infected cells. Total RNAs were extracted at the indicated times after infection of HFFs (A) or HeLa cells (B) with HSV-1(F) or the Δα4 or ΔU_L41 mutant virus and then were analyzed by real-time PCR. The amount of total RNA was normalized with respect to reverse-transcribed 18S rRNA. The amount of TTP RNA that accumulated in infected cells was calculated as the fold change compared to that extracted from cells harvested 12 h after mock infection.

FIG. 2.

Accumulation of TTP RNA in cytoplasm of HeLa cells infected with HSV-1. HeLa cells were mock infected or exposed to 10 PFU of HSV-1(F) or ΔU_L41 mutant virus per cell. Cytoplasmic RNAs were purified from cells harvested at the indicated times after mock infection (lanes 1 to 5) or infection with HSV-1(F) (lanes 6 to 10), the ΔU_L41 mutant (lanes 11 to 15), or the Δα27 mutant (lanes 16 to 20). RNAs (8 μg/lane) were loaded onto a denaturing formaldehyde gel and probed with a ³²P-labeled fragment containing the entire coding sequence of TTP. The lower panel represents the ethidium bromide-stained gel showing the relative levels of rRNA. The arrows to the right of the upper panel indicate the positions of the full-length TTP mRNA transcript and the unspliced form of TTP.

FIG. 3.

Immunoblots of TTP and TIA-1 proteins after HSV-1 infection. (A and B) Accumulation of TTP after HSV-1 infection. (A) HeLa cells were mock infected (lane 1) or exposed to 10 PFU of HSV-1(F) per cell (lanes 2 to 4) at time zero and were collected at the indicated times after infection. Cell lysates were incubated with an antibody to TTP. (B) SK-N-SH cells were mock infected (lane 1) or exposed to 10 PFU of HSV-1(F) (lane 2) or Δα27 (lane 3), ΔU_L41 (lane 4), or Δα4 (lane 5) mutant virus per cell, collected 17 h after infection, and incubated with an antibody to TTP. (C and D) Cytoplasmic accumulation of TIA-1 after HSV-1 infection. (C) Immunoblot of uninfected or HSV-1(F)-infected SK-N-SH whole-cell lysates. The cells were harvested at the indicated times after infection. (D) Immunoblot of cytoplasmic (C) and nuclear (N) fractions of uninfected or HSV-1(F)-infected HeLa, HEp-2, and SK-N-SH cell lysates incubated with a goat polyclonal antibody to TIA-1. The cells were harvested at the indicated times after infection.

FIG. 4.

Localization of TTP and TIA-1 in HEp-2 cells infected with HSV-1. HEp-2 cells were mock infected (a, e, and i) or exposed to 10 PFU of HSV-1(F) (b, f, and j) or the Δα4 (c, g, and k) or ΔU_L41 (d, h, and l) mutant virus per cell and fixed by paraformaldehyde and then methanol 12 h after infection. The cells were labeled with an anti-TTP antibody, detected with a FITC-labeled secondary antibody (green), and counterstained with propidium iodide (PI; red), or they were labeled with anti-TIA-1/TIAR antibodies and detected with an Alexa fluor 594-labeled secondary antibody (red). Immunostaining of the monolayers was evaluated by confocal microscopy. Cells were exposed to 0.5 mM arsenite for 30 min.

FIG. 5.

Localization of TTP and TIA-1 in HFFs infected with HSV-1. HFFs were mock infected (a, g, and m) or infected with 10 PFU of HSV-1(F) (b to d, h to j, and n to p) or the Δα4 (e, k, and q) or ΔU_L41 (f, l, and r) mutant virus per cell and fixed with paraformaldehyde and then methanol at different times postinfection. The cells were labeled with an anti-TTP antibody, detected with a FITC-labeled secondary antibody (green), and counterstained with propidium iodide (PI; red), or they were labeled with anti-TIA-1/TIAR antibodies and detected with an Alexa fluor 594-labeled secondary antibody (red). Immunostaining of the monolayers was evaluated by confocal microscopy.

FIG. 6.

Lack of colocalization of induced TTP and aggregated TIA-1/TIAR in ΔU_L41 virus-infected cells. HFFs were infected with 10 PFU of ΔU_L41 virus per cell, incubated for 12 h, fixed, and then incubated with a rabbit anti-TTP antibody detected with a FITC-labeled secondary antibody (green) and with a mouse anti-TIA-1/TIAR antibody detected with an Alexa fluor 594-labeled secondary antibody (red).

FIG. 7.

Vhs interacts with TTP in cells infected by HSV-1. SK-N-SH cells were mock infected (lanes 1 and 3) or infected with HSV-1(F) (lanes 2 and 4). Sixteen hours after infection, the cells were harvested, solubilized, immunoprecipitated with a goat polyclonal anti-TTP serum, eluted from protein A-Sepharose, electrophoretically separated in an SDS-10% polyacrylamide gel, transferred to a nitrocellulose sheet, and incubated with a specific antibody directed to the Vhs protein (lanes 3 and 4). The whole-cell lysate was loaded as a control (lanes 1 and 2).

FIG. 8.

Schematic representation of a proposed model of NF-κB-dependent RNA synthesis and Vhs-dependent selective degradation in cells infected with HSV-1. Studies reported in this and earlier reports showed that PKR is activated in wild-type virus-infected cells and that in cells infected with HSV-1, NF-κB is translocated into the nucleus by activated PKR through the activation of IKK and the degradation of IκBα. A consequence of activated NF-κB is the upregulation of numerous cellular genes, resulting in the synthesis of both ARE-rich and non-ARE-containing RNAs. ARE-containing RNAs are degraded in a Vhs-dependent manner, and their protein products are either not made or accumulate only transiently in HSV-1-infected cells, whereas RNAs lacking AREs are translated. The degradation of ARE-containing RNAs correlates with the induction and translocation of TTP to the cytoplasm of wild-type virus-infected cells, and to a lesser extent, to that of ΔU_L41 virus-infected cells, whereas TIA-1 and TIAR appear to be activated in both wild-type and mutant virus-infected cells. TTP and TIA-1/TIAR represent families of proteins that bind and target ARE-containing mRNAs for degradation. As indicated schematically, it has been reported that the activation and translocation of TIA-1 and TIAR are mediated by the phosphorylation of eIF-2α. Even though eIF-2α-P is efficiently dephosphorylated by the γ₁-34.5-phosphatase 1α complex, sufficient eIF-2α-P may be present to activate TIA-1/TIAR.