The stabilization of HIV-1 IN by Ku70 does not require a direct interaction between two proteins

Anisenko, A.; Knyazhanskaya, E.; Gottikh, M.

Авторы: Anisenko A., Knyazhanskaya E., Gottikh M.
Сборник: FEBS Open Bio
Серия: Suppl. 1
Том: 9
Тезисы
Год издания: 2019
Место издания: John Wiley & Sons Inc United States
Первая страница: 422
Последняя страница: 423
DOI: 10.1002/2211-5463.12675
Аннотация: The integration of HIV DNA into host cell genome is an essential step in retroviral life cycle. The major player in this process is viral enzyme integrase (IN), and it requires binding of some cellular proteins for its functioning. Recently, some reports showed that Ku70, which is a part of the DNA-PK complex, binds HIV-1 IN and probably protects it from proteasomal degradation. The Ku70 protective effect may arise from a direct shielding of HIV-1 IN in its complex with Ku70. We have shown that HIV-1 IN bearing substitutions E212A/L213A (IN_mut) shows a weaker binding towards Ku70 in vitro and in vivo. To verify if IN binding to Ku70 indeed protects it from proteasomal degradation, we expressed wild type IN (IN_wt) or IN_mut tagged with HA epitope in 293T cells, analyzed protein expression and found no differences in the expression levels of IN with or without mutation. When Ku70 was transiently overexpressed in cells, the amounts of both IN_wt and IN_mut were elevated. Conversely, siRNA mediated knockdownor CRISPR/Cas9 based heterozygous knockout of Ku70 led to a significant decrease in the levels of both variants of IN. Also, we measured the dynamics of IN degradation in the presence of translation inhibitor cycloheximide. IN_wt degraded quickly with a half-life of 65+/-10 min that is consisting with previous reports (A. Mousnier et al., 2007). The half-life of IN_mut was similar to that determined for IN_wt. Ku70 overexpression increased the half-life of both IN_wt and IN_mut to a similar extent (120+/-15 min), where as heterozygous knockout of Ku70 resultedin a 6-fold decrease in their expression. Interestingly, that the IN mRNA level remains constant in cells with different amounts ofKu70. Therefore, HIV-1 IN is indeed stabilized by Ku70 but this effect does not depend on a direct binding between these two proteins. This work was supported by RFBR grant 17-04-01178.
Добавил в систему: Анисенко Андрей Николаевич

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