Adenovirus type 5 E4orf3 protein targets the Mre11 complex to cytoplasmic aggresomes

Abstract

Virus infections have dramatic effects on structural and morphological characteristics of the host cell. The gene product of open reading frame 3 in the early region 4 (E4orf3) of adenovirus serotype 5 (Ad5) is involved in efficient replication and late protein synthesis. During infection with adenovirus mutants lacking the E4 region, the viral genomic DNA is joined into concatemers by cellular DNA repair factors, and this requires the Mre11/Rad50/Nbs1 complex. Concatemer formation can be prevented by the E4orf3 protein, which causes the cellular redistribution of the Mre11 complex. Here we show that E4orf3 colocalizes with components of the Mre11 complex in nuclear tracks and also in large cytoplasmic accumulations. Rearrangement of Mre11 and Rad50 by Ad5 E4orf3 is not dependent on interactions with Nbs1 or promyelocytic leukemia protein nuclear bodies. Late in infection the cytoplasmic inclusions appear as a distinct juxtanuclear accumulation at the centrosome and this requires an intact microtubule cytoskeleton. The large cytoplasmic accumulations meet the criteria defined for aggresomes, including gamma-tubulin colocalization and formation of a surrounding vimentin cage. E4orf3 also appears to alter the solubility of the cellular Mre11 complex. These data suggest that E4orf3 can target the Mre11 complex to an aggresome and may explain how the cellular repair complex is inactivated during adenovirus infection.

FIG. 1.

Localization of Ad5 E4orf3 in infected and transfected cells. (A) HeLa cells were infected with Ad5 and analyzed by immunofluorescence at the indicated times after infection. The E4orf3 protein is found in nuclear track structures and cytoplasmic accumulations. At later times the E4orf3 and E1b55K proteins are found colocalized in a large perinuclear cytoplasmic accumulation as indicated by arrows. (B) E1b55K is not required for cytoplasmic accumulation of E4orf3. Similar patterns of localization were observed for E4orf3 when cells were infected with the mutant virus dl1017, which does not express E1b55K and E4orf6. The PML protein partially colocalizes with E4orf3 in nuclear tracks but is not found at the cytoplasmic accumulations. (C) E4orf3 is excluded from viral replication centers (stained with an antibody to RPA32) during infections with wild-type Ad5 or dl1017. (D) Localization of E4orf3 and E1b55K proteins when expressed alone or together by transfection of expression plasmids.

FIG. 2.

E4orf3 protein of Ad5 partially colocalizes with the Mre11 complex. (A) Infection with wild-type Ad5 leads to redistribution of the Mre11 complex into nuclear tracks and cytoplasmic accumulations that partially colocalize with E4orf3. (B) Colocalization of E4orf3 with the Mre11 complex is not dependent upon E1b55K as it is still observed in cells infected with dl1017. (C) The Mre11 complex is redistributed by E4orf3 transfection into nuclear tracks and large cytoplasmic accumulations, in the absence and presence of E1b55K.

FIG. 3.

E4orf3 and the Mre11 complex accumulate at the centrosome. Cells were infected with wild-type Ad5 or the dl1017 mutant that lacks E1b55K. (A) The E4orf3 and Nbs1 proteins accumulate at the centrosome and costain with γ-tubulin independently of E1b55K. (B) The cytoplasmic accumulation of E4orf3 is surrounded by vimentin. (C) The cytoplasmic accumulation of Nbs1 induced by E4orf3 does not correspond to the Golgi apparatus as assessed by staining for giantin and β-COP.

FIG. 4.

Quantitation of viral and cellular proteins at a single juxtanuclear cytoplasmic aggregate during virus infections. (A) HeLa cells were infected with Ad5 for 24 h and analyzed by immunofluorescence for E4orf3, E1b55K, and Rad50. The presence of a single juxtanuclear aggregesome compared to multiple cytoplasmic aggregates was determined for 200 cells. (B) Nocodazole prevents the formation of a single large aggresome. HeLa cells were infected with dl1017 for 24 h in the presence or absence of nocodazole. E4orf3 and Rad50 were localized by immunofluorescence, and 200 cells were counted and quantitated for the presence of a single cytoplasmic aggresome.

FIG. 5.

Reorganization of Rad50 and Mre11 in NBS cells. NBS fibroblasts were infected with wild-type Ad5 or mutants and analyzed by immunofluorescence at 48 and 72 h postinfection. (A) In adenovirus-infected cells, Rad50 accumulates in cytoplasmic clusters and migrates progressively to the centrosome. Representative images are shown for progressive stages of the life cycle by DNA-binding protein staining. (B) Rad50 reorganization is dependent on E4orf3 expression. In uninfected (mock) cells, Rad50 localizes diffusely to the cytoplasm due to the NBS1 mutation. After 48 h of infection, adenovirus mutants that lack E4orf3 (dl1004) fail to relocate Rad50, while E4orf3-containing mutants (dl1017) display the same pattern as with wild-type Ad5. (C) Infection of NBS cells with dl1017 results in accumulation of Mre11 at the centrosome and is inhibited by nocodazole. (D) Quantitation of the effect of nocodazole on accumulation of Mre11 at a single cytoplasmic aggregate in NBS cells infected with dl1017.

FIG. 6.

Rad50 localization is altered by Ad5 infection in both the nucleus and cytoplasm of A-TLD cells. (A) The Rad50 protein is localized in the cytoplasm of A-TLD1 cells that express a truncated form of Mre11 (top panels). In A-TLD1 cells complemented with wild-type Mre11 from a retrovirus the Rad50 protein is recruited back into the nucleus (lower panels). In both cases the Nbs1 protein is nuclear. (B) In cells infected with the dl1017 virus for 48 h the E4orf3 protein is found in nuclear and cytoplasmic speckles, and these colocalize with rearranged Rad50 whether it is in the cytoplasm (in A-TLD1) or in the nucleus (in Mre11-complemented A-TLD1 cells). (C) Nbs1 localization is unaffected by virus infection in the absence ofMre11 (A-TLD1) but is disrupted into speckles in cells expressing wild-type Mre11, where it colocalizes with Rad50.

FIG. 7.

E4orf3 expression alters the solubility of the Mre11 complex. (A) The relative abundance of proteins in soluble (S) and insoluble pellet (P) fractions was assessed by immunoblotting of lysates from transfected cells. Mre11, Rad50, and Nbs1 proteins are found mostly in the soluble fraction in mock-transfected cells. Following E4orf3 expression Mre11, Rad50, and Nbs1 are found both in the soluble fraction and in the insoluble pellet. In contrast, the solubility of Ku86 and RPA32 is unchanged by E4orf3 expression. (B) HeLa cells that were either untreated (mock) or transfected with an E4orf3 expression vector were extracted with Triton X-100 prior to fixation. The detergent treatment extracts the soluble Mre11 complex, as indicatedby the diminished staining for Rad50 in the mock sample. In the presence of E4orf3 the Rad50 protein remains in nuclear tracks even after detergent treatment.