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* Department of Neuro-Oncology, Brain Tumor Center and
Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA
1Correspondence: Department of Neuro-Oncology, Unit 1002, Brain Tumor Center, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA. E-mail: hjiang{at}mdanderson.org
SPECIFIC AIMS
Adenoviral E1A is a versatile protein that can reprogram host cells for efficient viral replication by interacting with numerous cellular proteins. However, there is a lack of knowledge of how E1A coordinately modulates these targets through its precise and timely subcellular localization. The specific aims of this study were to identify a CRM1-dependent nuclear export signal in E1A and to study the regulation of this signal by phosphorylation and the relevance of a potent adenoviral replication.
PRINCIPAL FINDINGS
1. E1A protein displays CRM1-dependent nuclear export activity
To test our hypothesis that the nuclear export of E1A depends on CRM1, we performed an immunoprecipitation assay with nuclear extract of 293 cells, which showed that E1A coprecipitated with CRM1 (Fig. 1
A). This finding was confirmed by our further observation that E1A accumulated in the nucleus of 293 cells treated with leptomycin B (LMB; Fig. 1B
). To facilitate our study on the nucleocytoplasmic transport of E1A, we added an enhanced green fluorescence protein (EGFP) tag at the N terminus of E1A. Because of the finding in a previous study showing that a K 
A mutation in the C-terminal nuclear location signal (NLS) changed the localization of the E1A protein from a nuclear to a pancellular localization and to further override the strong nuclear import activity of E1A, we generated and tested the effect of an EGFP-E1AK fusion protein with the same mutation (K285A in 13s E1A; Fig. 1C
). Treatment with LMB resulted in the relocation of EGFP-E1AK into the nucleus (Fig. 1C
), strongly indicating the existence of a nuclear export signal (NES) and other functional NLSs in E1A. Consistent with these observations, small interfering RNA (siRNA) targeting CRM1 induced the dramatic nuclear localization of EGFP-E1AK (Fig. 1D, E
). Taken together, these data show that E1A depends at least in part on CRM1 for nuclear export.
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2. E1A protein contains a functional NES element spanning amino acids 70–80 within the E1A protein
To identify the sequence within E1A responsible for the nuclear export activity, we performed systematic mutational analyses followed by examination of the subcellular localization of EGFP-E1AK mutants. The results indicated a functional NES (VMLAVQEGIDL) spanning amino acids 70–80 within the protein. Further analysis using alanine replacement to determine the contribution of the hydrophobic residues within the NES toward the nuclear export activity demonstrated that V74 and I78 were indispensable for the novel NES activity.
3. E1A NES activity is regulated by phosphorylation at S89
To determine whether the NES is regulated by phosphorylation, we replaced the adjacent S89 or S96 with alanine to abrogate phosphorylation or with glutamic acid to partially mimic the negative charge of phosphorylation in the EGFP-E1AK fusion protein. The results showed that S89, but not S96, was the phosphorylation site that affected the nuclear export activity of E1A. We next studied whether the inhibition of CDC2 modified the subcellular localization of EGFP-E1AK. We observed that the CDC2/CDK2-specific inhibitor roscovitine reduced the NES activity. Furthermore, although roscovitine did not modify the subcellular localization of the S89A mutant, the S89E mutant showed resistance to roscovitine. We conclude that phosphorylation at S89 by CDC2 enhances NES activity.
4. E1A NES activity is relevant for viral life cycle
To examine whether E1A NES is active during the adenovirus replication cycle, we monitored the localization of E1A for a period of 24 h after viral infection in U-2 OS cells. We observed that E1A relocated to the cytoplasm at 24 h post viral infection, indicating the increased nuclear export activity caused by phosphorylation. Parallel experiments involving the examination of the cell-cycle profile of adenovirus-infected cells revealed that 24 h after infection, the cells accumulated in the S and G2/M phases characterized by high CDC2 kinase activity. Next, we were interested in examining whether NES plays a role in efficient viral replication. In serum-starved, normal human lung fibroblasts, titration of the progenies of the viruses demonstrated that 48 h after infection the replication efficiency of AdV74A, a virus with the V74A mutation in E1A NES, was up to 500-fold lower than wild-type (WT) adenovirus. In a parallel experiment, the difference between replication efficiency of the two viruses is much smaller in 293 cells where the WT E1A should compensate the functional alteration due to V74A mutation in AdV74A, indicating the attenuated replication of AdV74A in the fibroblasts was mainly caused by the abrogation of NES activity of E1A instead of the initial viral infection dose difference. Coincident with the change in replication efficiency, immunostaining of E1A proteins in the infected cells showed that 48 h after infection, in the majority of the infected cells, WT E1A relocated to the cytoplasm, resulting in equal staining of E1A (60% of the cells), whereas the mutant E1A still displayed predominant nuclear localization (75% of the cells). The subcellular localization of E1A proteins was correlated with the stages of viral infection rather than the expression levels of the proteins. Thus we concluded that NES activity is required for efficient adenoviral replication.
CONCLUSIONS AND SIGNIFICANCE
In this study, we first reported a functional CRM1-dependent NES in E1A protein of human adenovirus type 5 that is modulated by phosphorylation at an adjacent S89. We further demonstrated that this NES activity is up-regulated during the late phase of the virus life cycle and is important for potent viral replication. Our observations of E1A NES activity in combination with previous findings regarding the NLS of E1A suggest a model for the nucleocytoplasmic transport of E1A (Fig. 2
). In this model, the E1A protein binds to importin-
3 through NLS in the C-terminus to localize to the nucleus, a process that is tightly regulated by acetylation at the lysine residue (K285 in 13s E1A) within the NLS. We show here that E1A is exported to the cytoplasm through a CRM1-dependent pathway that is regulated by phosphorylation and is important for efficient viral replication and probably for E1A degradation.
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Although it has been repeatedly reported that S89 and S96 are phosphorylated in vivo, the two serines map to a region of E1A whose role is unclear. The function regulated by the phosphorylation of these sites is also not clear. The identification of a functional NES close to these sites clarified the functional relevance of the phosphorylation. Our data show that at the late phase of viral infection, E1A began to relocate to the cytoplasm, which correlated with the accumulation of the infected cells at the S and G2/M phases. The phenomenon is explained by the previous report that CDC2 phosphorylated E1A at S89 and that E1A was phosphorylated at the highest level in vivo in mitotic cells that display maximal levels of CDC2 kinase activity.
Our data show that the active nuclear export of E1A is required for efficient viral replication in serum-starved normal human lung fibroblast (Fig. 2A
). It is possible that the virus uses the nuclear export activity of E1A to move some of the E1A-binding proteins from the nucleus to the cytoplasm. Because the majority of the identified E1A-binding proteins execute their functions in the nucleus, moving them to the cytoplasm should further attenuate their control on transcription and on the cell cycle, and thus creates a favorable environment for viral replication. On the other hand, the active nuclear export of E1A indicates that E1A may also exert its pleiotropic effects on the regulation of viral replication in part by affecting cytoplasmic processes by interacting with the cytoplasmic targets. In addition, late in adenovirus infection, in the cytoplasm, the cytoskeleton is destroyed and thus translocation of E1A to the cytoplasm could be required for efficient cell lysis.
Finally, the critical hydrophobic backbone of the NES is preserved in human subgroup C adenoviruses (hAd2, 5, and 6) but not in other subgroups, such as Ad12, in which the oncogenic function of E1A is more aggressively manifested. Increased levels of phosphorylated signal transducer and activator of transcription-1 protein and expression of IFN regulatory factor-7 were found in Ad5- vs. Ad12-transformed BRK cells, which could be the critical alteration caused by the nuclear export activity of E1A that leads to the plethora of oncogenic differences. Thus, the functional NES within Ad5 E1A could be one of the negative modulators for tumorigenesis.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6433fje
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  A. Kulisz and H.-G. Simon An Evolutionarily Conserved Nuclear Export Signal Facilitates Cytoplasmic Localization of the Tbx5 Transcription Factor Mol. Cell. Biol., March 1, 2008; 28(5): 1553 - 1564. [Abstract] [Full Text] [PDF]
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