Limits...
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

HIV-Tat protein induces PKR expression in infected macrophages.(A) RAW 264.7 cells were transiently transfected with a PKR-promoter-luciferase reporter plasmid, then treated with Tat (100 ng/mL) and/or infected with L. amazonensis 24 h after transfection. Whole-cell lysates were analyzed for luciferase activity 24 hours later. (B) THP-1 cells were infected with L. amazonensis for one hour, non-internalized promastigotes were washed out, fresh medium was added and then the cells were treated for an additional three hours with Tat (100 ng/mL). Total RNA was extracted and a quantitative real-time RT-PCR was performed. Western blot was performed using anti-PKR (C), anti-phospho-PKR (D) or anti-phospho-eIF2α (E) antibodies in infected and/or treated THP-1 cells as indicated. The results are representative of three independent experiments. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4660360&req=5

f1: HIV-Tat protein induces PKR expression in infected macrophages.(A) RAW 264.7 cells were transiently transfected with a PKR-promoter-luciferase reporter plasmid, then treated with Tat (100 ng/mL) and/or infected with L. amazonensis 24 h after transfection. Whole-cell lysates were analyzed for luciferase activity 24 hours later. (B) THP-1 cells were infected with L. amazonensis for one hour, non-internalized promastigotes were washed out, fresh medium was added and then the cells were treated for an additional three hours with Tat (100 ng/mL). Total RNA was extracted and a quantitative real-time RT-PCR was performed. Western blot was performed using anti-PKR (C), anti-phospho-PKR (D) or anti-phospho-eIF2α (E) antibodies in infected and/or treated THP-1 cells as indicated. The results are representative of three independent experiments. *P < 0.05.

Mentions: L. amazonensis induces the expression of PKR and triggers PKR signaling in macrophages610. Because PKR signaling plays a pivotal role in the host macrophage-Leishmania interaction, we investigated whether the HIV-1 Tat protein would enhance the expression and the levels of activated PKR in Leishmania-infected macrophages. We used a luciferase gene reporter construct containing inducible elements of the PKR promoter to test the role of Tat in Leishmania-infected macrophages. As observed in Fig. 1A, the addition of Tat increased the activity of the PKR promoter and enhanced the luciferase levels induced by parasite infection. Accordingly, PKR expression was highly increased in infected macrophages treated with Tat (Fig. 1B,C). The pre-incubation of HIV-1 Tat with Leishmania did not affect the parasite association with macrophages or the parasite load 48 hours post-infection, thereby ruling out a direct effect of Tat on the parasite (Supplementary Figure 1). Importantly, PKR underwent an increase in phosphorylation in Leishmania-infected cells treated with Tat (Fig. 1D), as revealed by the use of anti-pPKR (Thr451). Therefore, we investigated whether the main PKR substrate, the initiation factor eIF2α, was phosphorylated. Figure 1E shows that eIF2α was phosphorylated in infected macrophages. However, the addition of Tat remarkably reduced eIF2α phosphorylation, suggesting that Tat may be a PKR substrate and compete with eIF2α, as described elsewhere14. Taken together, these results indicate that HIV-1Tat is able to induce an increase in PKR levels and phosphorylation upon Leishmania infection.


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)

HIV-Tat protein induces PKR expression in infected macrophages.(A) RAW 264.7 cells were transiently transfected with a PKR-promoter-luciferase reporter plasmid, then treated with Tat (100 ng/mL) and/or infected with L. amazonensis 24 h after transfection. Whole-cell lysates were analyzed for luciferase activity 24 hours later. (B) THP-1 cells were infected with L. amazonensis for one hour, non-internalized promastigotes were washed out, fresh medium was added and then the cells were treated for an additional three hours with Tat (100 ng/mL). Total RNA was extracted and a quantitative real-time RT-PCR was performed. Western blot was performed using anti-PKR (C), anti-phospho-PKR (D) or anti-phospho-eIF2α (E) antibodies in infected and/or treated THP-1 cells as indicated. The results are representative of three independent experiments. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660360&req=5

f1: HIV-Tat protein induces PKR expression in infected macrophages.(A) RAW 264.7 cells were transiently transfected with a PKR-promoter-luciferase reporter plasmid, then treated with Tat (100 ng/mL) and/or infected with L. amazonensis 24 h after transfection. Whole-cell lysates were analyzed for luciferase activity 24 hours later. (B) THP-1 cells were infected with L. amazonensis for one hour, non-internalized promastigotes were washed out, fresh medium was added and then the cells were treated for an additional three hours with Tat (100 ng/mL). Total RNA was extracted and a quantitative real-time RT-PCR was performed. Western blot was performed using anti-PKR (C), anti-phospho-PKR (D) or anti-phospho-eIF2α (E) antibodies in infected and/or treated THP-1 cells as indicated. The results are representative of three independent experiments. *P < 0.05.
Mentions: L. amazonensis induces the expression of PKR and triggers PKR signaling in macrophages610. Because PKR signaling plays a pivotal role in the host macrophage-Leishmania interaction, we investigated whether the HIV-1 Tat protein would enhance the expression and the levels of activated PKR in Leishmania-infected macrophages. We used a luciferase gene reporter construct containing inducible elements of the PKR promoter to test the role of Tat in Leishmania-infected macrophages. As observed in Fig. 1A, the addition of Tat increased the activity of the PKR promoter and enhanced the luciferase levels induced by parasite infection. Accordingly, PKR expression was highly increased in infected macrophages treated with Tat (Fig. 1B,C). The pre-incubation of HIV-1 Tat with Leishmania did not affect the parasite association with macrophages or the parasite load 48 hours post-infection, thereby ruling out a direct effect of Tat on the parasite (Supplementary Figure 1). Importantly, PKR underwent an increase in phosphorylation in Leishmania-infected cells treated with Tat (Fig. 1D), as revealed by the use of anti-pPKR (Thr451). Therefore, we investigated whether the main PKR substrate, the initiation factor eIF2α, was phosphorylated. Figure 1E shows that eIF2α was phosphorylated in infected macrophages. However, the addition of Tat remarkably reduced eIF2α phosphorylation, suggesting that Tat may be a PKR substrate and compete with eIF2α, as described elsewhere14. Taken together, these results indicate that HIV-1Tat is able to induce an increase in PKR levels and phosphorylation upon Leishmania infection.

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