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Drug 9AA reactivates p21/Waf1 and Inhibits HIV-1 progeny formation.

Wu W, Kehn-Hall K, Pedati C, Zweier L, Castro I, Klase Z, Dowd CS, Dubrovsky L, Bukrinsky M, Kashanchi F - Virol. J. (2008)

Bottom Line: Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery.A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene.Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated.

View Article: PubMed Central - HTML - PubMed

Affiliation: The George Washington University Medical Center, Department of Biochemistry and Molecular Biology, Washington, DC 20037, USA. bcmwxw@gwumc.edu

ABSTRACT
It has been demonstrated that the p53 pathway plays an important role in HIV-1 infection. Previous work from our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1. P21/waf1 is a well-known cdk inhibitor (CKI) that can lead to cell cycle arrest upon DNA damage. Most recently, the p21/waf1 function was further investigated as a molecular barrier for HIV-1 infection of stem cells. Therefore, we reason that the restoration of the p53 and p21/waf1 pathways could be a possible theraputical arsenal for combating HIV-1 infection. In this current study, we show that a small chemical molecule, 9-aminoacridine (9AA) at low concentrations, could efficiently reactivate p53 pathway and thereby restoring the p21/waf1 function. Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery. A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene. Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated.

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Phosphorylated p53ser15 doesn't interact with HIV-1 Tat protein. (A) ACH2 cells were transfected with FLAG-Tat and treated with 9AA or DMSO as a mock control. Expression of FLAG-Tat was detected by anti-FLAG (Sigma). Reactivation of p53ser15 was evaluated by anti-p53ser15 (Cell Signaling). (B) The cell lysates were then used for immunoprecipitation with anti-p53 or anti-p53ser15 (Cell Signaling). The immunopreciptated complexes were separated on 4–20% SDS-PAGE gels and then submitted to western blot to detect the presence of FLAG-Tat.
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Figure 4: Phosphorylated p53ser15 doesn't interact with HIV-1 Tat protein. (A) ACH2 cells were transfected with FLAG-Tat and treated with 9AA or DMSO as a mock control. Expression of FLAG-Tat was detected by anti-FLAG (Sigma). Reactivation of p53ser15 was evaluated by anti-p53ser15 (Cell Signaling). (B) The cell lysates were then used for immunoprecipitation with anti-p53 or anti-p53ser15 (Cell Signaling). The immunopreciptated complexes were separated on 4–20% SDS-PAGE gels and then submitted to western blot to detect the presence of FLAG-Tat.

Mentions: We have previously shown that unmodified Tat binds directly to p53 [12]. This is in agreement with a number of other publications that showed similar Tat p53 binding [12,13,35-39]. We now asked whether drug treatment which results in phosphorylation of p53 could still show binding to Tat. Therefore we transfected ACH2 cells with a Flag-Tat plasmid and looked for the presence of Flag-Tat and phosphorylated p53 in treated and untreated cells. Results in Figure 4A show that Flag-Tat and phospho p53 could be detected before drug treatment. Importantly, 9AA treatment of these cells did not alter the expression level of Flag-Tat but greatly increased serine 15 p53 levels. We next immunoprecipitated serine 15 p53 and asked if Tat was present in that complex after drug treatment. Results in panel B show that serine 15 phosphorylated p53 has been dissociated away from Tat and therefore may now be free to bind to endogenous promoters such as p21/waf1. In contrast, Tat was found to be associated with the p53 when the same lysates were incubated with anti-p53, which is in agreement with our previous work that p53 is inactivated though binding to HIV-1 Tat protein [12]. Collectively these results indicate that phosphorylation of p53 affects its release from Tat and its DNA-binding activity and ultimately induce gene expression on promoters such as p21/waf1.


Drug 9AA reactivates p21/Waf1 and Inhibits HIV-1 progeny formation.

Wu W, Kehn-Hall K, Pedati C, Zweier L, Castro I, Klase Z, Dowd CS, Dubrovsky L, Bukrinsky M, Kashanchi F - Virol. J. (2008)

Phosphorylated p53ser15 doesn't interact with HIV-1 Tat protein. (A) ACH2 cells were transfected with FLAG-Tat and treated with 9AA or DMSO as a mock control. Expression of FLAG-Tat was detected by anti-FLAG (Sigma). Reactivation of p53ser15 was evaluated by anti-p53ser15 (Cell Signaling). (B) The cell lysates were then used for immunoprecipitation with anti-p53 or anti-p53ser15 (Cell Signaling). The immunopreciptated complexes were separated on 4–20% SDS-PAGE gels and then submitted to western blot to detect the presence of FLAG-Tat.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2315641&req=5

Figure 4: Phosphorylated p53ser15 doesn't interact with HIV-1 Tat protein. (A) ACH2 cells were transfected with FLAG-Tat and treated with 9AA or DMSO as a mock control. Expression of FLAG-Tat was detected by anti-FLAG (Sigma). Reactivation of p53ser15 was evaluated by anti-p53ser15 (Cell Signaling). (B) The cell lysates were then used for immunoprecipitation with anti-p53 or anti-p53ser15 (Cell Signaling). The immunopreciptated complexes were separated on 4–20% SDS-PAGE gels and then submitted to western blot to detect the presence of FLAG-Tat.
Mentions: We have previously shown that unmodified Tat binds directly to p53 [12]. This is in agreement with a number of other publications that showed similar Tat p53 binding [12,13,35-39]. We now asked whether drug treatment which results in phosphorylation of p53 could still show binding to Tat. Therefore we transfected ACH2 cells with a Flag-Tat plasmid and looked for the presence of Flag-Tat and phosphorylated p53 in treated and untreated cells. Results in Figure 4A show that Flag-Tat and phospho p53 could be detected before drug treatment. Importantly, 9AA treatment of these cells did not alter the expression level of Flag-Tat but greatly increased serine 15 p53 levels. We next immunoprecipitated serine 15 p53 and asked if Tat was present in that complex after drug treatment. Results in panel B show that serine 15 phosphorylated p53 has been dissociated away from Tat and therefore may now be free to bind to endogenous promoters such as p21/waf1. In contrast, Tat was found to be associated with the p53 when the same lysates were incubated with anti-p53, which is in agreement with our previous work that p53 is inactivated though binding to HIV-1 Tat protein [12]. Collectively these results indicate that phosphorylation of p53 affects its release from Tat and its DNA-binding activity and ultimately induce gene expression on promoters such as p21/waf1.

Bottom Line: Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery.A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene.Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated.

View Article: PubMed Central - HTML - PubMed

Affiliation: The George Washington University Medical Center, Department of Biochemistry and Molecular Biology, Washington, DC 20037, USA. bcmwxw@gwumc.edu

ABSTRACT
It has been demonstrated that the p53 pathway plays an important role in HIV-1 infection. Previous work from our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1. P21/waf1 is a well-known cdk inhibitor (CKI) that can lead to cell cycle arrest upon DNA damage. Most recently, the p21/waf1 function was further investigated as a molecular barrier for HIV-1 infection of stem cells. Therefore, we reason that the restoration of the p53 and p21/waf1 pathways could be a possible theraputical arsenal for combating HIV-1 infection. In this current study, we show that a small chemical molecule, 9-aminoacridine (9AA) at low concentrations, could efficiently reactivate p53 pathway and thereby restoring the p21/waf1 function. Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery. A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene. Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated.

Show MeSH
Related in: MedlinePlus