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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.


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L. amazonensis and Tat induced NF-kB activation downstream of PKR signaling.(A) THP-1 and (B) RAW 264.7 cells stably transfected with either the empty vector (RAW-WT-Bla PKR cells) or the dominant-negative PKR K296R (RAW-DN-PKR cells) were transiently transfected with the 6kB-Luciferase consensus vector. 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, whole-cell lysates were analyzed for luciferase activity. (C) Western blot was performed for THP-1 cell nuclear extracts. The cells were treated with Tat and/or PKR two hours post-infection. Nuclear proteins were extracted and incubated with the anti-p65 antibody in PVDF membranes. The results are representative of three independent experiments. *P < 0.05.
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f3: L. amazonensis and Tat induced NF-kB activation downstream of PKR signaling.(A) THP-1 and (B) RAW 264.7 cells stably transfected with either the empty vector (RAW-WT-Bla PKR cells) or the dominant-negative PKR K296R (RAW-DN-PKR cells) were transiently transfected with the 6kB-Luciferase consensus vector. 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, whole-cell lysates were analyzed for luciferase activity. (C) Western blot was performed for THP-1 cell nuclear extracts. The cells were treated with Tat and/or PKR two hours post-infection. Nuclear proteins were extracted and incubated with the anti-p65 antibody in PVDF membranes. The results are representative of three independent experiments. *P < 0.05.

Mentions: The transcription factor NF-κB is activated in viral and parasitic infections25. Briefly, the heterodimer RelA/p50 is translocated to the nuclei of infected cells and regulates the expression of a number of cytokines26. RelA/p50 is also important for HIV-1 LTR expression and may be induced by bacterial co-infections27 or by HIV-1-encoded proteins such as Tat28. We investigated the activation of NF-κB in L. amazonensis-infected macrophages treated with Tat and the PKR inhibitor through gene reporter assays (Fig. 3A). The NF-κB reporter construct was induced by Tat, and the inhibition of PKR partially prevented this effect. Importantly, Tat treatment increased NF-κB activation in uninfected cells, while Leishmania infection diminished this activation. These results were corroborated by employing the DN-PKR-RAW 264.7 cell line, thereby strengthening the hypothesis that Tat-mediated NF-κB activation was dependent on PKR signaling and that Leishmania infection reduced this effect (Fig. 3B). Next, we investigated the nuclear levels of RelA (p65) induced by Tat and the dependence of this phenomenon on PKR. Figure 3C shows that Tat increased the nuclear levels of RelA and that PKR inhibition prevented this effect. Taken together, these observations corroborate previous results on the role of Tat in NF-κB activation29 and demonstrate the importance of PKR signaling in this event. Importantly, L. amazonensis infection per se did not induce the canonical NF-κB p65/50 dimer, as previously described30.


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 and Tat induced NF-kB activation downstream of PKR signaling.(A) THP-1 and (B) RAW 264.7 cells stably transfected with either the empty vector (RAW-WT-Bla PKR cells) or the dominant-negative PKR K296R (RAW-DN-PKR cells) were transiently transfected with the 6kB-Luciferase consensus vector. 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, whole-cell lysates were analyzed for luciferase activity. (C) Western blot was performed for THP-1 cell nuclear extracts. The cells were treated with Tat and/or PKR two hours post-infection. Nuclear proteins were extracted and incubated with the anti-p65 antibody in PVDF membranes. The results are representative of three independent experiments. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4660360&req=5

f3: L. amazonensis and Tat induced NF-kB activation downstream of PKR signaling.(A) THP-1 and (B) RAW 264.7 cells stably transfected with either the empty vector (RAW-WT-Bla PKR cells) or the dominant-negative PKR K296R (RAW-DN-PKR cells) were transiently transfected with the 6kB-Luciferase consensus vector. 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, whole-cell lysates were analyzed for luciferase activity. (C) Western blot was performed for THP-1 cell nuclear extracts. The cells were treated with Tat and/or PKR two hours post-infection. Nuclear proteins were extracted and incubated with the anti-p65 antibody in PVDF membranes. The results are representative of three independent experiments. *P < 0.05.
Mentions: The transcription factor NF-κB is activated in viral and parasitic infections25. Briefly, the heterodimer RelA/p50 is translocated to the nuclei of infected cells and regulates the expression of a number of cytokines26. RelA/p50 is also important for HIV-1 LTR expression and may be induced by bacterial co-infections27 or by HIV-1-encoded proteins such as Tat28. We investigated the activation of NF-κB in L. amazonensis-infected macrophages treated with Tat and the PKR inhibitor through gene reporter assays (Fig. 3A). The NF-κB reporter construct was induced by Tat, and the inhibition of PKR partially prevented this effect. Importantly, Tat treatment increased NF-κB activation in uninfected cells, while Leishmania infection diminished this activation. These results were corroborated by employing the DN-PKR-RAW 264.7 cell line, thereby strengthening the hypothesis that Tat-mediated NF-κB activation was dependent on PKR signaling and that Leishmania infection reduced this effect (Fig. 3B). Next, we investigated the nuclear levels of RelA (p65) induced by Tat and the dependence of this phenomenon on PKR. Figure 3C shows that Tat increased the nuclear levels of RelA and that PKR inhibition prevented this effect. Taken together, these observations corroborate previous results on the role of Tat in NF-κB activation29 and demonstrate the importance of PKR signaling in this event. Importantly, L. amazonensis infection per se did not induce the canonical NF-κB p65/50 dimer, as previously described30.

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