scholarly journals βTrCP is Required for HIV-1 Vpu Modulation of CD4, GaLV Env, and BST-2/Tetherin

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 573 ◽  
Author(s):  
Yul Song ◽  
Daniel Cyburt ◽  
Tiffany Lucas ◽  
Devon Gregory ◽  
Terri Lyddon ◽  
...  
Keyword(s):  
Cell Line ◽  
Ubiquitin Ligase ◽  
Leukemia Virus ◽  
Adapter Proteins ◽  
Primary Role ◽  
Dependent Manner ◽  
Gibbon Ape Leukemia Virus ◽  
F Box Protein ◽  
Hiv 1

The Human immunodeficiency virus-1 (HIV-1) accessory protein Vpu modulates numerous proteins, including the host proteins CD4 and BST-2/tetherin. Vpu interacts with the Skp, Cullin, F-Box (SCF) ubiquitin ligase through interactions with the F-Box protein βTrCP (1 and/or 2). This interaction is dependent on phosphorylation of S52,56 in Vpu. Mutation of S52,56, or inhibition of the SCF, abolishes most Vpu activity against CD4 and partly reduces activity against BST-2/tetherin. Recently, Vpu has also been reported to interact with the clathrin adapter proteins AP-1 and AP-2, and these interactions were also found to be required for BST-2/tetherin antagonism in an S52,56 -dependent manner. In assays where HIV-1 is pseudotyped with gibbon ape leukemia virus (GaLV Env), Vpu has also been found to prevent GaLV Env from being incorporated into viral particles, but the mechanism for this antagonism is not fully understood. To clarify the role of the βTrCPs in Vpu function we used CRISPR/Cas9 to generate a clonal cell line lacking both βTrCP-1 and -2. Vpu activity against CD4 and GaLV Env was abolished in this cell line, and activity against BST-2/tetherin reduced significantly. Mutation of the S52,56 residues no longer affected Vpu activity against BST-2/tetherin in this cell line. These data suggest that the primary role of the S52,56 residues in antagonism of CD4, GaLV Env, and BST-2/tetherin is to recruit the SCF/βTrCP ubiquitin ligase.

scholarly journals HTLV-2 Encoded Antisense Protein APH-2 Suppresses HIV-1 Replication

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1432
Author(s):  
Rajkumar Londhe ◽  
Smita Kulkarni
Keyword(s):  
Leukemia Virus ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Cell Leukemia Virus Type ◽  
Human T Cell ◽  
T Cell Leukemia Virus ◽  
Dose Dependent ◽  
Hiv 1

Antisense protein of Human T-cell Leukemia Virus Type 2 (HTLV-2), also called APH-2, negatively regulates the HTLV-2 and helps the virus to maintain latency via scheming the transcription. Despite the remarkable occurrence of HTLV-2/HIV-1 co-infection, the role of APH-2 influencing HIV-1 replication kinetics is poorly understood and needs investigation. In this study, we investigated the plausible role of APH-2 regulating HIV-1 replication. Herein, we report that the overexpression of APH-2 not only hampered the release of HIV-1 pNL4.3 from 293T cells in a dose-dependent manner but also affected the cellular gag expression. A similar and consistent effect of APH-2 overexpression was also observed in case of HIV-1 gag expression vector HXB2 pGag-EGFP. APH-2 overexpression also inhibited the ability of HIV-1 Tat to transactivate the HIV-1 LTR-driven expression of luciferase. Furthermore, the introduction of mutations in the IXXLL motif at the N-terminal domain of APH-2 reverted the inhibitory effect on HIV-1 Tat-mediated transcription, suggesting the possible role of this motif towards the downregulation of Tat-mediated transactivation. Overall, these findings indicate that the HTLV-2 APH-2 may affect the HIV-1 replication at multiple levels by (a) inhibiting the Tat-mediated transactivation and (b) hampering the virus release by affecting the cellular gag expression.

scholarly journals Loss of Cdc20 Causes a Securin-Dependent Metaphase Arrest in Two-Cell Mouse Embryos

2007 ◽  
Vol 27 (9) ◽  
pp. 3481-3488 ◽  
Author(s):  
Min Li ◽  
J. Philippe York ◽  
Pumin Zhang
Keyword(s):  
Ubiquitin Ligase ◽  
Cyclin B1 ◽  
Mitotic Exit ◽  
Adapter Proteins ◽  
Anaphase Promoting Complex ◽  
Metaphase Arrest ◽  
Cell Stage ◽  
Gene Trapping ◽  
Protein Substrates

ABSTRACT The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.

Role of fatty acid oxidation in mechanism of action of gastric secretagogues

1980 ◽  
Vol 238 (3) ◽  
pp. G255-G262
Author(s):  
J. Chacin ◽  
G. Martinez ◽  
E. Severin
Keyword(s):  
Fatty Acid ◽  
Gastric Mucosa ◽  
Acid Secretion ◽  
Fatty Acid Oxidation ◽  
Acid Oxidation ◽  
Primary Role ◽  
Dependent Manner ◽  
Beta Oxidation

The role of beta-oxidation in the mechanism of stimulation of acid secretion was examined in toad gastric mucosa in vitro. The incubation with 4-pentenoate selectively inhibited in a dose-dependent manner the rate of 14CO2 formation from [1-14C]octanoate. Pretreatment with 20 mM 4-pentenoate sharply reduced the respiratory and secretory responses to theophylline and histamine. Tracer studies showed a major utilization of exogenous octanoate over glucose and pyruvate by the in vitro toad gastric mucosa. Theophylline and histamine stimulated by 69% the rate of octanoate oxidation. Over 60% of the increments in oxygen uptake produced by theophylline and histamine accounted for the increments in octanoate oxidation, whereas glucose and pyruvate together accounted for less than 25%. Octanoate-dependent respiration was shown to correlate with octanoate oxidation under both inhibition with 4-pentenoate and stimulation with theophylline. Theophylline stimulated by 25% the rate of octanoate oxidation in Cl--free glucuronate-nutrient solutions. The present work provides further evidence for the primary role of fatty acid oxidation in the mechanism of acid secretion in amphibian.

Human Immunodeficiency Virus (HIV)-Resistant CD4+ UT-7 Megakaryocytic Human Cell Line Becomes Highly HIV-1 and HIV-2 Susceptible Upon CXCR4 Transfection: Induction of Cell Differentiation by HIV-1 Infection

Blood ◽  
1997 ◽  
Vol 89 (8) ◽  
pp. 2670-2678 ◽  
Author(s):  
Marta Baiocchi ◽  
Eleonora Olivetta ◽  
Cristiana Chelucci ◽  
Anna Claudia Santarcangelo ◽  
Roberta Bona ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Hiv Infection ◽  
Cell Line ◽  
Viral Entry ◽  
Human Cell Line ◽  
Immunodeficiency Virus ◽  
Hiv Entry ◽  
Stromal Cell Derived Factor ◽  
Hiv 1

Abstract Recent findings have shown that the expression of the seven trans-membrane G-protein–coupled CXCR4 (the receptor for the stromal cell-derived factor [SDF]-1 chemokine) is necessary for the entry of T-lymphotropic human immunodeficiency virus (HIV) strains, acting as a coreceptor of the CD4 molecule. In the human system, the role of CXCR4 in HIV infection has been determined through env-mediated cell fusion assays and confirmed by blocking viral entry in CD4+/CXCR4+ cells by SDF-1 pretreatment. We observed that the human megakaryoblastic CD4+ UT-7 cell line fails to express CXCR4 RNA and is fully resistant to HIV entry. Transfection of an expression vector containing the CXCR4 c-DNA rendered UT-7 cells readily infectable by different T-lymphotropic syncytium-inducing HIV-1 and HIV-2 isolates. Interestingly, HIV-1 infection of CXCR4 expressing UT-7 cells (named UT-7/fus) induces the formation of polynucleated cells through a process highly reminiscent of megakaryocytic differentiation and maturation. On the contrary, no morphologic changes were observed in HIV-2–infected UT-7/fus cells. These findings further strengthen the role of CXCR4 as a molecule necessary for the replication of T-lymphotropic HIV-1 and HIV-2 isolates and provide a useful model to study the functional role of CD4 coreceptors in HIV infection.

scholarly journals FUNDC1 interacts with FBXL2 to govern mitochondrial integrity and cardiac function through an IP3R3-dependent manner in obesity

2020 ◽  
Vol 6 (38) ◽  
pp. eabc8561 ◽  
Author(s):  
Jun Ren ◽  
Mingming Sun ◽  
Hao Zhou ◽  
Amir Ajoolabady ◽  
Yuan Zhou ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Ubiquitin Ligase ◽  
High Fat Diet ◽  
Dependent Manner ◽  
Cardiac Damage ◽  
Ubiquitin Ligase Complex ◽  
F Box Protein ◽  
Trisphosphate Receptor ◽  
Type 3 ◽  
Mitochondrial Anomalies

Defective mitophagy is causally linked to obesity complications. Here, we identified an interaction between mitophagy protein FUNDC1 (FUN14 domain containing 1) and receptor subunit of human SCF (SKP1/cullin/F-box protein) ubiquitin ligase complex FBXL2 as a gatekeeper for mitochondrial Ca2+ homeostasis through degradation of IP3R3 (inositol 1,4,5-trisphosphate receptor type 3). Loss of FUNDC1 in FUNDC1−/− mice accentuated high-fat diet–induced cardiac remodeling, functional and mitochondrial anomalies, cell death, rise in IP3R3, and Ca2+ overload. Mass spectrometry and co-immunoprecipitation analyses revealed an interaction between FUNDC1 and FBXL2. Truncated mutants of Fbox (Delta-F-box) disengaged FBXL2 interaction with FUNDC1. Activation or transfection of FBXL2, inhibition of IP3R3 alleviated, whereas disruption of FBXL2 localization sensitized lipotoxicity-induced cardiac damage. FUNDC1 deficiency accelerated and decelerated palmitic acid–induced degradation of FBXL2 and IP3R3, respectively. Our data suggest an essential role for interaction between FUNDC1 and FBXL2 in preserving mitochondrial Ca2+ homeostasis and cardiac function in obese hearts.

scholarly journals Evidence that Ecotropic Murine Leukemia Virus Contamination in TZM-bl Cells Does Not Affect the Outcome of Neutralizing Antibody Assays with Human Immunodeficiency Virus Type 1

2009 ◽  
Vol 83 (16) ◽  
pp. 8289-8292 ◽  
Author(s):  
Emily J. Platt ◽  
Miroslawa Bilska ◽  
Susan L. Kozak ◽  
David Kabat ◽  
David C. Montefiori
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Line ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT The TZM-bl cell line that is commonly used to assess neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) was recently reported to be contaminated with an ecotropic murine leukemia virus (MLV) (Y. Takeuchi, M. O. McClure, and M. Pizzato, J. Virol. 82:12585-12588, 2008), raising questions about the validity of results obtained with this cell line. Here we confirm this observation and show that HIV-1 neutralization assays performed with a variety of serologic reagents in a similar cell line that does not harbor MLV yield results that are equivalent to those obtained in TZM-bl cells. We conclude that MLV contamination has no measurable effect on HIV-1 neutralization when TZM-bl cells are used as targets for infection.

scholarly journals Overproduction of Polypeptides Corresponding to the Amino Terminus of the F-Box Proteins Cdc4p and Met30p Inhibits Ubiquitin Ligase Activities of Their SCF Complexes

2003 ◽  
Vol 2 (1) ◽  
pp. 123-133 ◽  
Author(s):  
Cheryl Dixon ◽  
Lee Ellen Brunson ◽  
Mary Margaret Roy ◽  
Dechelle Smothers ◽  
Michael G. Sehorn ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Cellular Responses ◽  
Amino Terminus ◽  
Variable Component ◽  
Amino Terminal ◽  
F Box Protein ◽  
Substrate Proteins

ABSTRACT Ubiquitin ligases direct the transfer of ubiquitin onto substrate proteins and thus target the substrate for proteasome-dependent degradation. SCF complexes are a family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. Distinct SCF complexes, defined by a particular F-box protein, target different substrate proteins for degradation. Although a few have been identified to be involved in important biological pathways, such as the cell division cycle and coordinating cellular responses to changes in environmental conditions, the role of the overwhelming majority of F-box proteins is not clear. Creating inhibitors that will block the in vivo activities of specific SCF ubiquitin ligases may provide identification of substrates of these uncharacterized F-box proteins. Using Saccharomyces cerevisiae as a model system, we demonstrate that overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p results in specific inhibition of their SCF complexes. Analyses of mutant amino-terminal alleles demonstrate that the interaction of these polypeptides with their full-length counterparts is an important step in the inhibitory process. These results suggest a common means to inhibit specific SCF complexes in vivo.

Role of CCL5 and Interleukin-6 in the Biology of Waldenström Macroglobulinemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 688-688
Author(s):  
Sherine F. Elsawa ◽  
Anne J. Novak ◽  
Steven C. Ziesmer ◽  
Thomas E. Witzig ◽  
Steven P. Treon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cells ◽  
Gene Array ◽  
Serum Levels ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Malignant Cells ◽  
Monoclonal Protein

Abstract Waldenström macroglobulinemia (WM) is a B-cell malignancy that is characterized by the production of a monoclonal IgM protein, a lymphoplasmacytic infiltrate in the bone marrow, and associated symptoms including anemia, lymphadenopathy and hyperviscosity. The aberrant production of a monoclonal IgM in the serum is a major factor causing significant morbidity in patients with this disease, yet little is known about the mechanisms that regulate monoclonal protein synthesis. While recent gene array studies and serum analysis have shown that IL-6 is elevated in WM patients suggesting an important role for this cytokine in this disease, the precise role played by IL-6 in WM is unknown. Using a multiplex ELISA approach to screen sera from WM patients, we confirmed that IL-6 was significantly elevated (p<0.0019) in patients (n=20) compared to controls (n=20). Serum levels of IL-6 in WM patients correlated with elevated levels of β2-microglobulin (p<0.0019). Additionally, we also found that serum levels of CCL5 (Rantes) were significantly elevated in WM patients (p<0.0001). CCL5 has been shown to regulate IL-6 secretion, and we therefore wanted to determine if CCL5 influenced IL-6 expression in WM and what the subsequent consequence of IL-6 stimulation was on WM cells. To define the source of IL-6 in the tumor microenvironment, we used stromal cells from the bone marrow of healthy donors, malignant cells from patients with WM, and the BCWM.1 WM cell line, and tested their ability to secrete IL-6 by ELISA. All cell types secreted IL-6, with stromal cells secreting the most. We then tested the ability of CCL5 to induce IL-6 secretion by WM and stromal cells. CCL5 significantly increased IL-6 secretion by stromal cells (p<0.03) and also increased IL-6 secretion by fresh CD19+ CD138+ cells from WM patients (p<0.02). Using fresh patient WM cells and the BCWM.1 WM cell line as a model, we then determined the effect of IL-6 on growth of WM cells. We found that IL-6 had a modest effect (mean=20% increase, range=5–41%) on cell proliferation (p<0.0039) but had no effect on cell viability. In contrast, when we addressed the role of IL-6 on IgM secretion, we found that IL-6 increased IgM secretion by BCWM.1 cells in a dose-dependent manner. The IL-6 mediated increase in IgM secretion was abolished in the presence of neutralizing antibodies to IL-6. When we analyzed the downstream signaling events activated by IL-6 in WM cells we found that stimulation of BCWM.1 cells, which express the IL-6R, resulted in phosphorylation of Stat1, Stat3 and Erk1/2, but not Akt. Using a mitogen activated protein kinase (MAPK) inhibitor, we could inhibit the IL-6-mediated phosphorylation of Erk1/2. Similarly, using a JAK1 Inhibitor, we could inhibit IL-6 mediated signaling through Stat1 and Stat3. In summary, we have clearly shown that IL-6 significantly upregulates IgM secretion by WM cells and increases their proliferation. We have also demonstrated the ability of both the malignant cells and the stromal cells to secrete IL-6, and that this secretion is regulated in part by CCL5. We have found that WM cells express IL-6R, and that IL-6 induced signaling is through both the MAPK and Jak/Stat pathways. Therapies targeting IL-6 secretion or the IL-6 signaling pathways may therefore provide clinical benefit to patients with WM; not just by inhibiting the malignant cells but by down regulating the production of the monoclonal protein.

Multiple components are required for sequence recognition of the AP1 site in the gibbon ape leukemia virus enhancer

1989 ◽  
Vol 9 (11) ◽  
pp. 4713-4721
Author(s):  
J P Quinn ◽  
A R Farina ◽  
K Gardner ◽  
H Krutzsch ◽  
D Levens
Keyword(s):  
Cell Line ◽  
Core Protein ◽  
Leukemia Virus ◽  
Consensus Sequence ◽  
Affinity Column ◽  
The Core ◽  
Sequence Recognition ◽  
Human T Cell ◽  
Specific Complex ◽  
Gibbon Ape Leukemia Virus

At least two subunits contributed to the formation in vitro of a specific complex binding to the AP1 consensus sequence (TGAGTCA) in the gibbon ape leukemia virus (GALV) enhancer in MLA144 cells. This complex can be dissociated on a monomeric GALV oligonucleotide affinity column. One protein, termed the core protein, was retained on the oligonucleotide affinity column. The second protein flowed through the oligonucleotide affinity column and, when alone, did not bind to DNA; however, when present with the core protein, it bound strongly and very specifically to the GALV sequence. MLA144 cells contained only trace amounts of c-fos and c-jun by immunoblot analysis, suggesting that the proteins specifically binding to the GALV AP1 site were distinct from c-fos and c-jun. In addition to the major complex that recognized the GALV element, MLA144 cells contained a minor complex that is chromatographically different from and antigenically related to c-fos. The factor in the flowthrough complemented a human T-cell nuclear extract (Jurkat cell line), which, when alone, had no assayable complex that specifically bound to the GALV enhancer; this complementation gave rise to a specific complex similar to that seen in MLA144 cells. Together, these results suggest that the GALV enhancer can interact with multicomponent protein complexes in a cell-line-specific manner.

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