Dissertation > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Acquired Immune Deficiency Syndrome ( AIDS AIDS)

Molecular Mechanisms of HIV-1Vpr-mediated NF-κB Pathway Activation

Author LiuRuiKang
Tutor GengYunZuo
School Nankai University
Course Microbiology
Keywords Vpr NF-κB pathway IKK TAK1 phosphorylation
CLC R512.91
Type PhD thesis
Year 2013
Downloads 33
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Viral protein R (Vpr) is an accessory protein of human immunodeficiency virus type I (HIV-1). Vpr is expressed at the late stage of HIV-1infection and incorporated into virus particles through a direct interaction with the p6domain of Gag. Vpr plays an important role in HIV-1replication and pathogenesis, especially by conferring a favorable cellular environment for efficient replication of HIV-1. Deletion of vpr reduces viral replication. Multiple functions have been discovered for Vpr, including the regulation of cellular NF-κB signaling pathway. But the exact role of Vpr on NF-κB signaling pathway remains unclear. And the detailed mechanisms still need to be illustrated. This study focused on the relationship between Vpr and NF-κB and the main results are listed below.1. We confirmed that Vpr can activate canonical and non-canonical NF-κB signaling pathway. We found that at the early stage of HIV-1infection, Vpr, in the context of HIV-1virions, increased the phosphorylation of IκBα. In the meantime, Vpr elevated the phosphorylation and the nuclear transportation of p65, and stimulated the expression of NF-κB reporter gene in a dose-dependent manner. Furthermore, both ectopic expressed and virion-associated Vpr induced the phosphorylation of p100, and the processing of p100into p52, which resulted in the activation of non-canonical NF-κB pathway.2. Immunoprecipitation assay gave the clues that Vpr interacted with IKKa and IKKP which are the upstream kinases of the canonical and non-canonical NF-κB pathway. And Vpr regulated the phosphorylation of IKKa and IKKβ.In addition, the activation of NF-κB reporter by Vpr is severely reduced in p65, RelB, IKKa or IKKβ knockdown cell lines.3. With the benefit of immunoprecipitation between Vpr and the upstream signaling factors of IKK complex, we found that Vpr interacted with TRAF6and TAK1, but not TRAF2or TAB1. But Vpr didn’t affect the auto-ubiquitination of TRAF6. Over-expressed and virion-associated Vpr enhanced the auto-phosphorylation-of TAK1on Thr187in various cell lines, which brought about the phosphorylation of IKKα, IKKβ, and MKK7, and the activation of downstream signal cascade. The phosphorylation of TAK1by Vpr relied on TAB1and TRAF6. Moreover, Vpr could increase the ubiquitination of TAK1by recruiting TAB3.4. Finally, we envisioned that NF-κB activation might affect the reported functions of Vpr. The HIV-1LTR reporter assay in NF-κB knockdown cell lines indicated that Vpr-mediated NF-κB activation promoted HIV-1LTR transcription. Additionally, preliminary results of Flow cytometry shown that NF-κB activation promoted Vpr-induced cell cycle G2/M arrest.Collectively, in this study, we demonstrate that Vpr activates both canonical and non-canonical NF-κB pathway at the early stage of HIV-1infection. The molecular mechanisms underlying this activation reveal that Vpr employs two patterns to hijack host NF-κB signaling pathway.(1) Vpr increases the phosphorylation of IKKa and IKKβ,(2) Vpr interacts with TAK1and elevated its activation. This results in the promotion of HIV-1LTR transcription, the cell cycle G2/M phase arrest and finally the virus replication.

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