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HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR.

Vivarini Áde C, Pereira Rde M, Barreto-de-Souza V, Temerozo JR, Soares DC, Saraiva EM, Saliba AM, Bou-Habib DC, Lopes UG - Sci Rep (2015)

Bottom Line: The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication.Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages.In summary, we show that Tat enhances Leishmania growth through PKR signaling.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Parasitologia Molecular, Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.

No MeSH data available.


Related in: MedlinePlus

L. amazonensis reduces iNOS expression levels induced by treatment with Tat in a PKR-dependent manner.(A) RAW 264.7 cells stably transfected with either the empty vector (RAW-WT-Bla PKR) or the dominant-negative PKR K296R (RAW-DN-PKR) were infected with L. amazonensis. Non-internalized promastigotes were washed out and fresh medium was added, and then the cultures were treated for an additional three hours with Tat (100 ng/mL). Total RNA was extracted and a quantitative real-time RT-PCR for iNOS was performed. (B) RAW 264.7 cell lines were infected with L. amazonensis and/or treated with Tat (100 ng/mL) for 24 h. Then, the supernatant was analyzed for the presence of nitric oxide through the Griess reaction. (C) RAW 264.7 cell lines were infected for one hour with L. amazonensis. Non-internalized promastigotes were washed out and fresh medium was added, and then the cultures were treated for an additional five hours with Tat (100 ng/mL). Western blot analysis of iNOS protein expression was performed. RAW 264.7 cell lines were transiently transfected with the 3XNS-Luc (D) or 3XS-Luc (E) vectors. Then, 24 h post-transfection the cells were infected with L. amazonensis for 18 h and/or treated with Tat (100 ng/mL). After twenty-four hours, the whole cell lysates were analyzed for luciferase activity. The results are representative of three independent experiments. *P < 0.05.
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f4: L. amazonensis reduces iNOS expression levels induced by treatment with Tat in a PKR-dependent manner.(A) RAW 264.7 cells stably transfected with either the empty vector (RAW-WT-Bla PKR) or the dominant-negative PKR K296R (RAW-DN-PKR) were infected with L. amazonensis. Non-internalized promastigotes were washed out and fresh medium was added, and then the cultures were treated for an additional three hours with Tat (100 ng/mL). Total RNA was extracted and a quantitative real-time RT-PCR for iNOS was performed. (B) RAW 264.7 cell lines were infected with L. amazonensis and/or treated with Tat (100 ng/mL) for 24 h. Then, the supernatant was analyzed for the presence of nitric oxide through the Griess reaction. (C) RAW 264.7 cell lines were infected for one hour with L. amazonensis. Non-internalized promastigotes were washed out and fresh medium was added, and then the cultures were treated for an additional five hours with Tat (100 ng/mL). Western blot analysis of iNOS protein expression was performed. RAW 264.7 cell lines were transiently transfected with the 3XNS-Luc (D) or 3XS-Luc (E) vectors. Then, 24 h post-transfection the cells were infected with L. amazonensis for 18 h and/or treated with Tat (100 ng/mL). After twenty-four hours, the whole cell lysates were analyzed for luciferase activity. The results are representative of three independent experiments. *P < 0.05.

Mentions: The production of nitric oxide (NO) is associated with the activation of macrophages and can reduce the number of Leishmania amastigotes31. Because HIV-1 Tat has been demonstrated to induce iNOS32, we investigated whether L. amazonensis interferes with iNOS expression and NO production in the context of the Tat-PKR axis. We found that Tat induced iNOS expression in a PKR-dependent fashion (Fig. 4A). The presence of Leishmania reduced this effect, suggesting that Leishmania impaired the induction of iNOS by Tat. Accordingly, NO production followed the same pattern (Fig. 4B). These results were corroborated by immunoblot assays. Figure 4C clearly shows that Tat induced iNOS expression via PKR and that this effect was reduced in the presence of the parasite. To evaluate the effect of Tat on the NF-κB/STAT1 regulatory elements present in the iNOS promoter33, we performed luciferase reporter assays. Sole infection by L. amazonensis did not induce the constructs carrying either the NF-κB/STAT1 or STAT1 regulatory element (Fig. 4D,E, respectively). Importantly, Tat treatment resulted in the activation of both reporter constructs, but Leishmania infection reduced this effect on the NF-κB/STAT1 construct. Based on these results, we suggest that L. amazonensis reduced the expression of iNOS induced by HIV1 Tat.


HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR.

Vivarini Áde C, Pereira Rde M, Barreto-de-Souza V, Temerozo JR, Soares DC, Saraiva EM, Saliba AM, Bou-Habib DC, Lopes UG - Sci Rep (2015)

L. amazonensis reduces iNOS expression levels induced by treatment with Tat in a PKR-dependent manner.(A) RAW 264.7 cells stably transfected with either the empty vector (RAW-WT-Bla PKR) or the dominant-negative PKR K296R (RAW-DN-PKR) were infected with L. amazonensis. Non-internalized promastigotes were washed out and fresh medium was added, and then the cultures were treated for an additional three hours with Tat (100 ng/mL). Total RNA was extracted and a quantitative real-time RT-PCR for iNOS was performed. (B) RAW 264.7 cell lines were infected with L. amazonensis and/or treated with Tat (100 ng/mL) for 24 h. Then, the supernatant was analyzed for the presence of nitric oxide through the Griess reaction. (C) RAW 264.7 cell lines were infected for one hour with L. amazonensis. Non-internalized promastigotes were washed out and fresh medium was added, and then the cultures were treated for an additional five hours with Tat (100 ng/mL). Western blot analysis of iNOS protein expression was performed. RAW 264.7 cell lines were transiently transfected with the 3XNS-Luc (D) or 3XS-Luc (E) vectors. Then, 24 h post-transfection the cells were infected with L. amazonensis for 18 h and/or treated with Tat (100 ng/mL). After twenty-four hours, the whole cell lysates were analyzed for luciferase activity. The results are representative of three independent experiments. *P < 0.05.
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Related In: Results  -  Collection

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f4: L. amazonensis reduces iNOS expression levels induced by treatment with Tat in a PKR-dependent manner.(A) RAW 264.7 cells stably transfected with either the empty vector (RAW-WT-Bla PKR) or the dominant-negative PKR K296R (RAW-DN-PKR) were infected with L. amazonensis. Non-internalized promastigotes were washed out and fresh medium was added, and then the cultures were treated for an additional three hours with Tat (100 ng/mL). Total RNA was extracted and a quantitative real-time RT-PCR for iNOS was performed. (B) RAW 264.7 cell lines were infected with L. amazonensis and/or treated with Tat (100 ng/mL) for 24 h. Then, the supernatant was analyzed for the presence of nitric oxide through the Griess reaction. (C) RAW 264.7 cell lines were infected for one hour with L. amazonensis. Non-internalized promastigotes were washed out and fresh medium was added, and then the cultures were treated for an additional five hours with Tat (100 ng/mL). Western blot analysis of iNOS protein expression was performed. RAW 264.7 cell lines were transiently transfected with the 3XNS-Luc (D) or 3XS-Luc (E) vectors. Then, 24 h post-transfection the cells were infected with L. amazonensis for 18 h and/or treated with Tat (100 ng/mL). After twenty-four hours, the whole cell lysates were analyzed for luciferase activity. The results are representative of three independent experiments. *P < 0.05.
Mentions: The production of nitric oxide (NO) is associated with the activation of macrophages and can reduce the number of Leishmania amastigotes31. Because HIV-1 Tat has been demonstrated to induce iNOS32, we investigated whether L. amazonensis interferes with iNOS expression and NO production in the context of the Tat-PKR axis. We found that Tat induced iNOS expression in a PKR-dependent fashion (Fig. 4A). The presence of Leishmania reduced this effect, suggesting that Leishmania impaired the induction of iNOS by Tat. Accordingly, NO production followed the same pattern (Fig. 4B). These results were corroborated by immunoblot assays. Figure 4C clearly shows that Tat induced iNOS expression via PKR and that this effect was reduced in the presence of the parasite. To evaluate the effect of Tat on the NF-κB/STAT1 regulatory elements present in the iNOS promoter33, we performed luciferase reporter assays. Sole infection by L. amazonensis did not induce the constructs carrying either the NF-κB/STAT1 or STAT1 regulatory element (Fig. 4D,E, respectively). Importantly, Tat treatment resulted in the activation of both reporter constructs, but Leishmania infection reduced this effect on the NF-κB/STAT1 construct. Based on these results, we suggest that L. amazonensis reduced the expression of iNOS induced by HIV1 Tat.

Bottom Line: The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication.Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages.In summary, we show that Tat enhances Leishmania growth through PKR signaling.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Parasitologia Molecular, Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.

No MeSH data available.


Related in: MedlinePlus