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Some findings of FADD knockdown in inhibition of HIV-1 replication in Jurkat cells and PBMCs

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Abstract

Fas-associated protein with death domain (FADD) is a key adaptor molecule transmitting the death signal mediated by death receptors, and it is also required for T cell proliferation. A recent study indicated that FADD is able to affect HIV-1 production, but the mechanism is not known. Using the susceptible Jurkat cell line and peripheral blood mononuclear cells, we studied the effects of FADD on HIV-1 production. TaqMan RT-PCR was used to quantify HIV-1 viral RNA copies, and Western blot analysis was used to detect protein expression. FADD knockdown decreased HIV-1 replication and inactivated caspase-3 activity in the cells and blocked CD4 translocation to the lipid rafts of the plasma membrane. Reduced expression of FADD suppressed TCR signaling through downregulation of TCR, CD3, and Zap-70 in response to HIV-1 infection and blocked the trafficking of TCR, CD3, CD28, and Zap-70 to lipid rafts, leading to reduced activation of NF-κB and NFAT, which are required for HIV-1 replication. FADD knockdown diminished caspase-8 migration to lipid rafts and its expression in response to HIV-1 infection. These results indicate that FADD, as a host pro-apoptotic protein, plays important roles in regulating HIV-1 replication and production in several ways, and apoptotic pathway inhibition is able to decrease HIV-1 replication and production.

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Acknowledgments

The authors wish to acknowledge Dr. Mingjie Zhang, Dr Cheng Sun, and Ewan Plant for critical review of this manuscript. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

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Authors and Affiliations

  1. Lab of Molecular Virology, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Building 29B, Rm 4NN22 8800 Rockville Pike, Bethesda, MD, 20892, USA

    Xue Wang, Jiying Tan, Jiangqin Zhao, Viswannath Ragupathy, Mohan Haleyurgirisetty & Indira Hewlett

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  1. Xue Wang
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  2. Jiying Tan
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  3. Jiangqin Zhao
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Corresponding authors

Correspondence to Xue Wang or Indira Hewlett.

Additional information

Xue Wang and Jiying Tan contributed equally to this work.

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11010_2014_2058_MOESM1_ESM.ppt

A. No significant effect of Adenovirus containing LacZ on HIV-1 production in Jurkat cells. To investigate whether adenovirus containing LacZ affects HIV-1 production, Jurkat cells were infected with HIV-1 (MN) for 2 hours, and then incubated with/without adenovirus inserted a gene, LacZ, and cultured for 3 days. One hundred forty µl of the culture supernatants containing HIV-1 particles were used to isolate viral RNA. Five µl in 50 µl of the RNA was used as template to perform real-time PCR. Statistically, as shown in Fig. A, there was no significant difference of HIV-1 production between the treatments of HIV-1 infection and HIV-1 + adenovirus containing LacZ. B. Lipid rafts and caveolae are plasma membrane domains that are enriched in cholesterol and sphingolipid. Both lipid rafts and caveolae are docking sites for specific proteins involved in signal transduction. Caveolae are a specialised form of lipid raft, defined by the presence of a specific cellular protein marker, caveolin. The term ‘lipid rafts’ reflects the ability of these microdomains to serve as relay stations in intracellular signaling pathways or as vehicles for the transport of selected lipid membrane molecules.Jurkat cells infected HIV-1 for 2 hours, and then incubated with adenovirus inserted a gene LacZ or anti-sense FADD for 3 days. Total cell lysates were loaded on the discontinuous sucrose gradients for 18 h (39,000 rpm, SW 41 rotor). Twelve fractions of each were obtained and subjected to Western blot analysis for Flotillin-1. Flotillins are lipid raft components, and belongs to stomatin protein family, whose members are characterized by the presence of a hydrophobic N-terminal region that is predicted to form a single, outside to inside, transmembrane domain. (PPT 67 kb)

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Wang, X., Tan, J., Zhao, J. et al. Some findings of FADD knockdown in inhibition of HIV-1 replication in Jurkat cells and PBMCs. Mol Cell Biochem 393, 181–190 (2014). https://doi.org/10.1007/s11010-014-2058-7

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  • DOI: https://doi.org/10.1007/s11010-014-2058-7

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