Browsing by Subject "HIV-1 Nef"
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Item HIV-1 Impairment via UBE3A and HIV-1 Nef Interactions Utilizing the Ubiquitin Proteasome System(MDPI, 2019-11-27) Pyeon, Dohun; Rojas, Vivian K.; Price, Lenore; Kim, Seongcheol; Singh, Meharvan; Park, In-WooMolecular basis of HIV-1 life cycle regulation has thus far focused on viral gene stage-specificity, despite the quintessence of post-function protein elimination processes in the virus life cycle and consequent pathogenesis. Our studies demonstrated that a key pathogenic HIV-1 viral protein, Nef, interacted with ubiquitin (Ub)-protein ligase E3A (UBE3A/E6AP), suggesting that interaction between Nef and UBE3A is integral to regulation of viral and cellular protein decay and thereby the competing HIV-1 and host cell survivals. In fact, Nef and UBE3A degraded reciprocally, and UBE3A-mediated degradation of Nef was significantly more potent than Nef-triggered degradation of UBE3A. Further, UBE3A degraded not only Nef but also HIV-1 structural proteins, Gag, thus significantly inhibiting HIV-1 replication in Jurkat T cells only in the presence of Nef, indicating that interaction between Nef and UBE3Awas pivotal for UBE3A-mediated degradation of the viral proteins. Mechanistic study showed that Nef and UBE3A were specific and antagonistic to each other in regulating proteasome activity and ubiquitination of cellular proteins in general, wherein specific domains of Nef overlapping with the long terminal repeat (LTR) were essential for the observed actions. Further, Nef itself reduced the level of intracellular Gag by degrading a cardinal transcription regulator, Tat, demonstrating a broad role for Nef in the regulation of the HIV-1 life cycle. Taken together, these data demonstrated that the Nef and UBE3A complex plays a crucial role in coordinating viral protein degradation and hence HIV-1 replication, providing insights as to the nature of pathobiologic and defense strategies of HIV-1 and HIV-infected host cells.Item Novel role of HIV-1 Nef in regulating the ubiquitination of cellular proteins(Frontiers Media S.A., 2023-03-28) Ghaly, Maria; Proulx, Jessica; Borgmann, Kathleen; Park, In-WooOur recent data established that HIV-1 Nef is pivotal in determining the fate of cellular proteins by modulating ubiquitination. However, it is unknown which proteins are ubiquitinated in the presence of Nef, a question critical for understanding the proliferation/restriction strategies of HIV-1 in infected cells. To identify cellular proteins ubiquitinated by Nef, we conducted a proteomic analysis of cellular proteins in the presence and absence of Nef. Proteomic analysis in HEK293T cells indicated that 93 proteins were upregulated and 232 were downregulated in their ubiquitination status by Nef. Computational analysis classified these proteins based on molecular function, biological process, subcellular localization, and biological pathway. Of those proteins, we found a majority of molecular functions to be involved in binding and catalytic activity. With respect to biological processes, a significant portion of the proteins identified were related to cellular and metabolic processes. Subcellular localization analysis showed the bulk of proteins to be localized to the cytosol and cytosolic compartments, which is consistent with the known function and location of Nef during HIV-1 infection. As for biological pathways, the wide range of affected proteins was denoted by the multiple modes to fulfill function, as distinguished from a strictly singular means, which was not detected. Among these ubiquitinated proteins, six were found to directly interact with Nef, wherein two were upregulated and four downregulated. We also identified 14 proteins involved in protein stability through directly participating in the Ubiquitin Proteasome System (UPS)-mediated proteasomal degradation pathway. Of those proteins, we found six upregulated and eight downregulated. Taken together, these analyses indicate that HIV-1 Nef is integral to regulating the stability of various cellular proteins via modulating ubiquitination. The molecular mechanisms directing Nef-triggered regulation of cellular protein ubiquitination are currently under investigation.Item ROLE OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 NEF IN ACCELERATION OF HEPATITIS C VIRUS-MEDIATED LIVER DISEASE.(2013-04-12) Park, In-WooPurpose: HIV-1 infection has profound, adverse consequences for every stage of the natural history of HCV infection via significantly elevating HCV viral load and expediting HCV-mediated liver disease progression in the co-infected host. However, molecular details for how HIV-1 accelerates this pathogenesis are largely unknown. According to recent publications, HIV-1 Nef can be transferred from HIV-1 susceptible cells to other uninfected susceptible cells and even to non-susceptible target cells by formation of conduits or by exosomes, suggesting that Nef is a leading candidate molecule for explaining the occurrence of HIV-1-mediated opportunistic diseases in non-susceptible target tissues. Accordingly, we have investigated the role of HIV-1 and viral protein Nef in HCV-infected hepatocytes to better understand the pathobiology of HIV-1 and HCV co-infection. Methods: Infectivity of HIV-1 in human hepatocytes was monitored at the indicated time point by measuring reverse transcriptase (RT) activity in the clarified culture supernatants, and effect of Nef on the expression of HCV replicon was examined by measuring reporter gene, Luciferase (Luc), expression in the replicon cells. Transfer of Nef to target hepatocytes and subcellular distribution of lipid droplets (LD) were studied by immunofluoscence and confocal microscopic analyses as well as by flow cytometric analyses. Results: Infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the hepatocytes transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was found to be transferred from expressing T cells to hepatocytes through conduits, wherein up to 16% (average 10%) of hepatocytes harbored the transferred Nef, when the hepatocytes were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of LD, and consistently up-regulated HCV replication by 1.5~2.5 fold in the target hepatocytes, which is remarkable in relation to the initially indolent viral replication. Conclusions: HIV-1 Nef is a critical element in accelerating HCV-mediated liver pathogenesis through modulation of lipid molecules and changes in HCV replication.