The human adenovirus E4orf4 protein is toxic in both human tumor cells and Saccharomyces cerevisiae. Previous studies indicated that most of this toxicity is dependent on an interaction of E4orf4 protein with the B55 class of regulatory subunits of protein phosphatase 2A (PP2A) and in yeast with the B55 homolog Cdc55. We have found previously that E4orf4 inhibits PP2A activity against at least some substrates. In an attempt to understand the mechanism of this inhibition, we used a genetic approach to identify residues in the seven-bladed ␤-propeller proteins B55␣ and Cdc55 required for E4orf4 binding. In both cases, amino-terminal polypeptides composed only of blade 1 and at least part of blade 2 were found to bind E4orf4 and overexpression blocked E4orf4 toxicity in yeast. Furthermore, certain amino acid substitutions in blades 1 and 2 within full-length B55␣ and Cdc55 resulted in loss of E4orf4 binding. Recent mutational analysis has suggested that segments of blades 1 and 2 present on the top face of B55␣ form part of the "substrate-binding groove." Additionally, these segments are in close proximity to the catalytic C subunit of the PP2A holoenzyme. Thus, our results are consistent with the hypothesis that E4orf4 binding could affect the access of substrates, resulting in the failure to dephosphorylate some PP2A substrates.

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Ten-week-old C57B1/6J mice were given an intraperitoneal injection of 100 microCi radiostrontium (89Sr). One and two weeks following injection, the cellular composition of marrow and spleen was examined for cellularity, types of lymphoid cells, and incidences of small lymphocytes bearing sIgM, FcR, and CR. Strontium-89 treatment yielded a significant cell depletion in marrow, whereas spleen cellularity initially increased before returning to normal level in the second week. The increase in spleen cellularity was showed by pulse DNA labeling to be due to local production and not to influx from elsewhere. The percentage of medium and large lymphocytes, precursors of small lymphocytes, increased from 4% in the control spleen to 22% in the 89Sr-treated spleen. The incidences of small lymphocytes bearing sIgM, FcR, and CR were lower than normal levels with the exception of a substantial increase of FcR+ small lymphocytes in the spleen 2 weeks following 89Sr injection. Double-labeling studies, combining rosetting for surface receptors and DNA labeling for cell age, showed that small lymphocytes bearing sIgM, FcR, and CR form part of the major population of indigenous rapidly renewed cells in 89Sr-treated spleen.