The herpes simplex virus 1 UL41 gene-dependent destabilization of cellular RNAs is selective and may be sequence-specific

Abstract

In cells infected with herpes simplex virus 1, the RNA encoded by the stress-inducible immediate early response gene IEX-1 was up-regulated immediately after infection. However, the accumulated RNA was degraded 3'-5', and the protein was detectable only at very early times after infection. The degradation was dependent on the U(L)41 gene encoding the virion host shutoff (vhs) protein and resulted in the accumulation of truncated RNA containing the 5'-end portion of the transcript. IEX-1 contains an AU-rich element (ARE) in its 3'-untranslated domains known to regulate negatively the RNA lifespan. To examine the role of ARE in signaling the degradation, we compared the stability of several RNAs up-regulated during infection to WT virus. These were ARE-containing RNAs encoding IEX-1, c-fos, and IkappaBalpha and the non-ARE-containing RNAs GADD45beta and tristetraprolin. We report that the ARE-containing RNAs exemplified by IEX-1 RNA are deadenylated and cleaved in the ARE within the 3' UTR in a U(L)41-dependent manner. In contrast, Northern blot hybridizations and analyses of poly(A) tails revealed no evidence of degradation of GADD45beta RNA. GADD45beta protein was detected in WT virus-infected cells. These results indicate that the degradation of RNAs and the mechanism by which cellular RNAs are degraded are selective and may be sequence specific. The persistence of partially degraded ARE-containing RNAs may reflect specific targeting of the vhs proteins to the ARE and the modification of the RNA degradation machinery of the cell induced by the presence of the vhs protein.

Fig. 1.

RNA accumulations of c-fos, IκBα, TTP, and GADD45β in cells infected with HSV-1. Cells were mock-infected or infected with 10 plaque-forming units (pfu) of HSV-1(F) per cell. Cytoplasmic RNA was purified and analyzed as described in Materials and Methods. (A Left) Accumulation of c-fos mRNA in cytoplasmic RNA extracted from mock-infected (lanes 1 and 2) or HSV-1-infected (lanes 3-6) HeLa cells. (A Right) Accumulation of IκBα mRNA in cytoplasmic RNA extracted from mock-infected (lanes 7-9) or HSV-1-infected (lanes 10-12) SK-N-SH-infected cells. •, intact RNA; ○, degraded RNA. (B) Accumulation of TTP and GADD45β mRNAs in cytoplasmic RNA extracted from mock-infected (lanes 1-5) or HSV-1-infected (lanes 6-10) HeLa cells.

Fig. 2.

Accumulation of GADD45β protein in HeLa cells after HSV-1 infection. HeLa cells were mock-infected (lane 1) or infected with 10 pfu of HSV-1(F) per cell (lane 2) and were collected at 7 h after infection. Cell lysate from 293 cells stably transfected with a GADD45β-flag tagged construct or the empty vector was used as positive (lane 3) and negative (lane 4) controls, respectively.

Fig. 3.

Analysis of mRNA deadenylation. HeLa cells were mock-infected (lanes 1-10) or infected with 10 pfu of HSV-1(F) per cell (lanes 11-20), and cytoplasmic RNA was extracted at the indicated hours after infection. Poly(A)^- RNA was prepared in vitro by treating the RNA samples with oligo(dT) and RNase H as described in Materials and Methods. Treated (+) and untreated (-) RNA was probed with a ³²P-labeled fragment containing the entire coding sequences of IEX-1 or GADD45β.

Fig. 4.

Analysis of the 5′ and 3′ regions of c-fos and GADD45β mRNA by real-time PCR. (A Upper) The location of the primers used for real-time PCR along the c-fos RNA sequence. (A Lower) Total RNA was extracted at the indicated hours after mock or HSV-1(F) infection of human foreskin fibroblasts. (B Upper) Location along the GADD45β RNA sequence of the primers used for real-time PCR. (B Lower) Total RNA was extracted at the indicated hours after mock or HSV-1(F) infection of HeLa cells. The amount of RNA accumulating in infected cells was calculated as fold change compared with that of cells harvested 1 h after mock infection.

Fig. 5.

Involvement of U_L41 in the progressive 3′-5′ degradation of IEX-1 mRNA. (A) Location along the IEX-1 RNA sequence of the three sets of primers used for real-time PCR. (B) Total RNA was extracted at the indicated hours after mock or ΔU_L41-mutant virus infection of HeLa cells.

Fig. 6.

Mapping of the vhs-dependent endonucleolytic cleavage site in IEX-1 mRNA. (A) Location along the IEX-1 RNA sequence of Ribo1 and Ribo2, two riboprobes antisense to the 3′ UTR, used for Northern blot analysis. (B) HeLa cells were mock-infected or infected with 10 pfu of HSV-1(F) or ΔU_L41-mutant virus per cell. Cytoplasmic RNA was purified from cells harvested at the indicated times after mock infection (lanes 1 and 5), infection with HSV-1(F) (lanes 6-10), or ΔU_L41 mutant (lanes 11-15).