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Endocytic activity of HIV-1 Vpu: Phosphoserine-dependent interactions with clathrin adaptors.

ABSTRACT: HIV-1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune-evasion, triggering ubiquitin-mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP-1 and AP-2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking signals encoded by Vpu, we fused the cytoplasmic domain (CD) of Vpu to the extracellular and transmembrane domains of the CD8 ?-chain. CD8-VpuCD was rapidly endocytosed in a clathrin- and AP-2-dependent manner. Multiple determinants within the Vpu CD contributed to endocytic activity, including phosphoserines of the ?-TrCP binding site and a leucine-based ExxxLV motif. Using recombinant proteins, we confirmed ExxxLV-dependent binding of the Vpu CD to the ?/?2 subunit hemicomplex of AP-2 and showed that this is enhanced by serine-phosphorylation. Remarkably, the Vpu CD also bound directly to the medium (?) subunits of AP-2 and AP-1; this interaction was dependent on serine-phosphorylation of Vpu and on basic residues in the ? subunits. We propose that the flexibility with which Vpu binds AP complexes broadens the range of cellular targets that it can misdirect to the virus' advantage.

SUBMITTER: Stoneham CA

PROVIDER: S-EPMC5600163 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Endocytic activity of HIV-1 Vpu: Phosphoserine-dependent interactions with clathrin adaptors.

Stoneham Charlotte A CA Singh Rajendra R Jia Xiaofei X Xiong Yong Y Guatelli John J

Traffic (Copenhagen, Denmark) 20170627 8

HIV-1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune-evasion, triggering ubiquitin-mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP-1 and AP-2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking ...[more]

PMID: 28504462

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Project description:UnlabelledBacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world's population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake.ImportanceBacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake of Helicobacter pylori vesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs the H. pylori vesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

Dataset Information

Endocytic activity of HIV-1 Vpu: Phosphoserine-dependent interactions with clathrin adaptors.

Publications

Endocytic activity of HIV-1 Vpu: Phosphoserine-dependent interactions with clathrin adaptors.

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