Neutral Bystander, Intrusive Micromanager, or Useful Catalyst?

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73380/1/j.1541-0072.1995.tb01746.x.pd

HIV-1 Vpu Protein Mediates the Transport of Potassium in Saccharomyces cerevisiae

Human immunodeficiency virus type 1 (HIV-1) Vpu is an integral membrane protein that belongs to the viroporin family. Viroporins interact with cell membranes, triggering membrane permeabilization and promoting release of viral particles. In vitro electrophysiological methods have revealed changes in membrane ion currents when Vpu is present; however, in vivo the molecular mechanism of Vpu at the plasma membrane is still uncertain. We used the yeast Saccharomyces cerevisiae as a genetic model system to analyze how Vpu ion channel impacts cellular homeostasis. Inducible expression of Vpu impaired cell growth, suggesting that this viral protein is toxic to yeast cultures. This toxicity decreased with extracellular acidic pH. Also, Vpu toxicity diminished as the extracellular K(+) concentration was increased. However, expression of the Vpu protein suppresses the growth defect of K(+) uptake-deficient yeast (Δtrk1,2). The phenotype rescue of these highly hyperpolarized cells was almost total when they were grown in medium supplemented with high concentrations of KCl (100 mM) at pH 7.0 but was significantly reduced when the extracellular K(+) concentration or pH was decreased. These results indicate that Vpu has the ability to modify K(+) transport in both yeast strains. Here, we show also that Vpu confers tolerance to the aminoglycoside antibiotic hygromycin B in Δtrk1,2 yeast. Our results suggest that Vpu interferes with cell growth of wild-type yeast but improves proliferation of the hyperpolarized trk1,2 mutant by inducing plasma membrane depolarization. Furthermore, evaluation of the ion channel activity of the Vpu protein in Δtrk1,2 yeast could aid in the development of a high-throughput screening assay for molecules that target the retroviral protein.This study was supported by Grants PI PI05/00013 and PI08/0912 from Fondo de Investigación Sanitaria. L.H. and N.M. were holders of Predoctoral Fellowships from Instituto de Salud Carlos III.S

HIV-1 NEF and VPU interfere with L-selectin (CD62L) cell surface expression to inhibit adhesion and signaling in infected CD4⁺ t lymphocytes.

Leukocytes recirculation between blood and lymphoid tissues is required for the generation and maintenance of immune responses against pathogens and is crucially controlled by the L-selectin (CD62L) leukocyte homing receptor. CD62L has adhesion and signaling functions and initiates the capture and rolling on the vascular endothelium of cells entering peripheral lymph nodes. This study reveals that CD62L is strongly down-regulated on primary CD4(+) T lymphocytes upon infection with human immunodeficiency virus type 1 (HIV-1). Reduced cell-surface CD62L expression was attributable to the Nef and Vpu viral proteins and not due to increased shedding via matrix metalloproteases. Both Nef and Vpu associated with and sequestered CD62L in perinuclear compartments thereby impeding CD62L transport to the plasma membrane. Besides, Nef decreased total CD62L protein levels. Importantly, infection with wild type but not Nef- and Vpu-deficient HIV-1 inhibited the capacity of primary CD4(+) T lymphocytes to adhere to immobilized fibronectin in response to CD62L ligation. Moreover, HIV-1 infection impaired the signaling pathways and co-stimulatory signals triggered in primary CD4(+) T cells by CD62L ligation. We propose that HIV-1 dysregulates CD62L expression to interfere with the trafficking and activation of infected T cells. Altogether, this novel HIV-1 function could contribute to virus dissemination and evasion of host immune responses. IMPORTANCE: L-selectin (CD62L) is an adhesion molecule that mediates the first steps of leukocytes homing to peripheral lymph nodes, thus crucially controlling the initiation and maintenance of immune responses against pathogens. Here we report that CD62L is down-modulated on the surface of HIV-1-infected T cells through the activity of two viral proteins, Nef and Vpu, that prevent newly synthesized CD62L molecules to reach the plasma membrane. We provide evidence that CD62L down-regulation on HIV-1-infected primary T cells results in impaired adhesion and signaling functions upon CD62L triggering. Removal of cell-surface CD62L may predictably keep HIV-1-infected cells away from lymph nodes, the privileged sites of both viral replication and immune response activation, with important consequences such as systemic viral spread and evasion of the host immune surveillance. Altogether, we propose that Nef- and Vpu-mediated subversion of CD62L function could represent a novel determinant of HIV-1 pathogenesis

A novel pVHL-independent but NEMO-driven pathway in renal cancer promotes HIF stabilization

Activation of hypoxia-inducible factor (HIF) is due to loss of von Hippel-Lindau protein (pVHL) function in most clear cell renal cell carcinomas (ccRCCs). Here we describe a novel pVHL-independent mechanism of HIF regulation and identify nuclear factor (NF)-κB essential modulator (NEMO) as a hitherto unknown oncogenic factor influencing human ccRCC progression. Over 60% of human ccRCCs (n=157) have negative or weak NEMO protein expression by immunohistochemistry. Moderate/strong NEMO protein expression is more frequent in VHL wild-type ccRCCs. We show that NEMO stabilizes HIFα via direct interaction and independently of NF-κB signaling in vitro. NEMO prolongs tumor cell survival via regulation of apoptosis and activation of epithelial-to-mesenchymal transition, facilitating tumor metastasis. Our findings suggest that NEMO-driven HIF activation is involved in progression of ccRCC. Therefore, NEMO may represent a clinically relevant link between NF-κB and the VHL/HIF pathways. Targeting NEMO with specific inhibitors in patients with metastatic ccRCC could be a novel treatment approach in patients with ccRCC expressing functional pVHL.Oncogene advance online publication, 26 October 2015; doi:10.1038/onc.2015.400