| Summary: | 博士 === 國立中興大學 === 微生物暨公共衛生學研究所 === 100 === Human immunodeficiency virus-1 (HIV-1) is a member of lentiviruses in the Retroviridae family. HIV-1 infection may lead to the depletion of CD4+ T cells, deficiency of immune system, and the subsequent fatal consequences. Viral protein R (Vpr), an HIV-1 accessory protein, induces apoptosis of CD4+ T cells via a mitochondria-associated pathway; however, the molecular mechanism of it is yet unclear. In this thesis, our results show that Vpr is integrated into the endoplasmic reticulum (ER) membrane and mitochondria outer membrane via its C-terminal transmembrane domain. In addition, Vpr can also be detected at the mitochondria-associated membrane (MAM), suggesting that Vpr is transported from the ER to the mitochondria via MAM. This transport pathway is mediated by mitofusin 2 (Mfn2), the ATPase AAA domain-containing 3A (ATAD3A), and dynamin-related protein 1 (DRP1). Vpr reduces Mfn2 expression and increases nuclear level of DRP1, leading to the accumulation of Vpr in MAM, reduction of mitochondrial proteins, and loss of mitochondrial membrane potential (MMP) that finally results in cell death. Overexpression of Mfn2 and DRP1 significantly resurrects MMP loss and alleviates Vpr-related cell death. Taken together, our results suggest that Vpr-mediated cellular damage occurs mainly by influencing protein transport from the ER, via MAM to the mitochondria, which are mediated by Mfn2, ATAD3A and DRP1.
Moreover, mitochondria play important roles in the host defense mechanism against viral infections. Certain viral proteins are known to interact with mitochondrial proteins or target directly to mitochondria to induce cell death. Hepatocyte growth factor (HGF) is a potent pleiotropic factor that can promote cell growth and mobility, and inhibit apoptosis. HGF can prevent cell death by activating Bcl-2 expression and inhibiting mitochondrial translocation of Bax protein. Our results show that HGF suppressed apoptosis by reducing apoptosis-inducing factor (AIF) expression, not by inhibiting nuclear translocation of AIF. We further identifies that HGF reduces AIF expression via c-Met receptor transduced downstream effector, focal adhesion kinase (FAK). Knockout of FAK gene or interfering FAK function by introducing dominant-negative mutants can increase AIF expression. These results suggest that HGF may suppress apoptosis by reducing mitochondrial AIF expression via c-Met and its downstream protein FAK.
In conclusion, this study elucidates the protein transport pathway of HIV-1 Vpr to mitochondria and the mechanism of HGF alleviated mitochondria-dependent apoptosis. Our results indicate that Vpr induces mitochondrial apoptosis by influencing protein transport between the ER and the mitochondria; and HGF, on the other hand, reduces mitochondria-dependent apoptosis by inhibiting AIF expression. Taken together, our study leads to a better understanding of HIV-1 Vpr-induced mitochondrial apoptosis, which could be regulated by growth factor. We expect that these informations would be useful to the future development of effective therapeutic regimens for HIV infection.
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