Retrovirology. 2012 Jun 22;9:33. doi: 10.1186/1742-4690-9-33.

Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation.

Mukerji J, Olivieri KC, Misra V, Agopian KA, Gabuzda D.

Source

Department of Cancer Immunology and AIDS, Dana Farber Cancer Institute, Boston, MA, USA.

Abstract

BACKGROUND:

HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown.

RESULTS:

To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery.

CONCLUSIONS:

Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses.

PMID:: 22534017; [PubMed - indexed for MEDLINE]
PMCID:: PMC3382630

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Figure 4

Visualization of protein interaction networks of Nef-interacting proteins and exocyst complex components, regulators, and effectors.A) IPA network of Nef-interacting proteins and exocyst complex components, regulators, and effectors. Inputting 16 molecules (depicted as red nodes, described in the text) into Ingenuity Pathways Analysis [Ingenuity® Systems, www.ingenuity.com] yielded a network of 35 molecules, of which 13 [Calmodulin, Dock1-Pak, ERK1/2, Exocyst, Jnk, NFkB (complex), Phosphatidylinositol 4,5 kinase, PI3K (complex), PIP4K2C, Pkc(s) Pld, Ras, and TCR] were omitted for clarity. Components of the exocyst complex are encircled by a gold oval. Manually-curated edges are shown in dark blue, and dashed arrows denote activation via indirect association. IPA assigned this network a score of 42, which is based on the hypergeometric distribution and is the -log(right-tailed Fisher’s exact test result). The score implies a 1 in 10⁴² chance of obtaining a network containing at least the same number of network eligible molecules by chance when randomly picking 16 molecules that are present in networks from the Ingenuity Knowledge Base. B) STRING network of Nef-interacting proteins and exocyst complex components, regulators, and effectors. Inputting 19 molecules (16 molecules input into IPA, plus EXOC5, EXOC7, and EXOC8; depicted as spheres) into STRING yielded a network visualizing linkages between Nef, the exocyst complex, and regulators of the actin cytoskeleton. Components of the exocyst complex are encircled by a gold oval. Manually-curated edges are shown in dark blue, and dashed arrows denote activation via indirect association. Other edge colors reflect the evidence channels (see legend) by which protein nodes are related in the STRING database. Scores STRING assigned to relationships depicted in this network are provided in Additional file 4: Table S3.

Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

Retrovirology. 2012;9:33-33.

Figure 5

EXOC2 depletion abrogates Nef-mediated enhancement of nanotube formation.A) Lysates of Jurkat E6-1 cells transduced with vector virions bearing pLKO.1-shRNA followed by pHAGE-Nef-IRES-ZsGreen encoding 5C (wild-type) or empty pHAGE were harvested 32 h post-pHAGE transduction and immunoblotted to detect endogenous EXOC2, HA-tagged Nef, and β-tubulin. Samples were treated with an shRNA targeting EXOC2 (right lane, EXOC2) or a control shRNA that does not target the human transcriptome (left lane, Control). B -F) Jurkat cells transduced as described above were stimulated with 1 ug/mL PHA-P for 1 h at 24 h post-pHAGE transduction, incubated on fibronectin-coated coverslips for 5 h, fixed at 30 h post-pHAGE transduction and phalloidin-stained prior to visualization by confocal fluorescence microscopy. B) Percentages of ZsGreen-positive Jurkat cells that formed nanotubes and nanotube-like structures in populations treated with shEXOC2 (right) or shControl (left) are shown. Differences betweenNef-expressing samples treated with control versus EXOC2 shRNAs were significant, as were differences between control shRNA-treated cells with versus without Nef expression (“*” denotes p < 0.05, Mann–Whitney U). The percentage of Nef-expressing cells that formed nanotubes and nanotube-like structures was calculated by manually counting the number of connected cells and dividing the result by the total number of ZsGreen-positive cells for a given sample. The total numbers of ZsGreen-positive cells counted in 30 fields at 40X magnification are detailed in the Methods. Data represent percentages of ZsGreen-positive cells connected by nanotubes, and are reported as mean ± SEM. C-F) Selected fields that contain nanotubes (C and D) and representative confocal fluorescence images (E and F) from the samples quantified in B are shown. Actin-containing nanotubes and nanotube-like structures were visualized via Alexa Fluor 555-conjugated phalloidin; pHAGE-transduced cells were detected via ZsGreen fluorescence. Scale bars, 5 μm.

Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

Retrovirology. 2012;9:33-33.

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