A System of Auxin-inducible Degradation of Human Ku70 Protein to Study Its Role in HIV-1 Replication

Sorkin, A.; Shadrina, O.; Kapitonova, M.; Gottikh, M.

Авторы: Sorkin Anton, Shadrina Olga, Kapitonova Maria, Gottikh Marina
Журнал: Protein Science
Том: 30
Номер: S1
Год издания: 2021
Издательство: Cold Spring Harbor Laboratory Press
Местоположение издательства: United States
Первая страница: 54
Последняя страница: 54
DOI: 10.1002/pro.4191
Аннотация: The human Ku70 protein is thought to be involved in several stages of the human immunodeficiency virus (HIV) replication. Thus, it is not easy to assess the Ku70 effect at individual steps. In this regard, a system of quick and reversible Ku70 degradation is needed. Previously we established the 293T-Ku70-AID cell line, in which the endogenous Ku70 is fused with the part of an auxininducible degron (AID). The second component of the auxin-inducible degradation system is an osTIR1 protein. Initially, the osTIR1 gene was introduced into cells under the CMV promoter. However, it led to Ku70 degradation even without auxin activation. To avoid background degradation,we created a cell line with osTIR1 under thecontrol of a doxycycline-inducible promoter. In these cells, Ku70 degradation was observed only after doxycycline and auxin addition.The study of degradation and recovery kinetics showed that the osTIR1 protein was detectable on a blot starting 2 h after induction and reached its maximum after 24 h. A gradual decrease of Ku70 level started at the 6 h mark, and after 24 h, the protein was not detectable. Removal of inductors caused the concentration of osTIR1 to decrease by 70% in 16 h. Meanwhile, the Ku70 level began to recover 20 h after inductor removal and reached its native level by 40 h. We compared these results with the kinetics of Ku70 knockdown by siRNAs in 293T cells: a significant decrease was observed only 48 h after transfection and continued for the next 96 h. Thus, the developed system of auxininducibledegradation of Ku70 allows to reduce and restore the protein level quickly and could be used toassess the effect of Ku70 on individual stages of HIV replication.
Добавил в систему: Готтих Марина Борисовна

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