Fig. 1

Identification and validation of HIV-1 and SIV Nef interactors. a Schematic representation of FTS-tagged Nef constructs used for TAP-MS. Nef is myristoylated (Myr) at the N-terminus and comprises three folded domains (represented in blue) (residues 55–65, 84–148 and 178–203 in HIV-1 Nef; residues 87–97, 116–180 and 212–235 in SIVsmm Nef) flanked by flexible segments, comprising an N-terminal flexible anchor (residues 1–54 in HIV-1 Nef; residues 1–86 in SIVsmm Nef) and a C-terminal flexible loop (residues 149–177 in HIV-1 Nef; residues 181–211 in SIVsmm Nef). SIVcpz Nef is similar to HIV-1 Nef and SIVmac to SIVsmm. The FTS tag is composed of one FLAG tag (DYKDDDK) followed by two strep tags (WSHPQFEK). b Flowchart of the TAP-MS protocol conducted to identify Nef-interacting proteins. Detergent extracts of HEK-293T cells stably expressing FTS-tagged Nef from the HIV-1 NL4-3 strain or from three SIV strains (SIVcpz, SIVmac and SIVsmm) were incubated with Strep-Tactin beads and bound proteins eluted with desthiobiotin. The eluate was further purified by binding to anti-FLAG M2 beads and elution with FLAG peptide. Eluted proteins were identified by MS. Raw data were filtered against the CRAPome database, and interaction maps were generated using BioGRID. c Interaction map of HIV-1 Nef with host proteins identified by TAP-MS and grouped according to their cellular functions. d Classification of HIV-1 Nef interactors according to their cellular compartments. e Validation of TAP-MS hits by pulldown (PD) and immunoblotting (IB). HeLa cells were transfected with plasmids encoding Nef from HIV-1 or the three SIV strains, as well as myrlysin or lyspersin as specificity controls, all tagged with FTS. Cells were cross-linked, lysed and the FTS-tagged proteins isolated on Strep-Tactin beads. The isolated proteins were analyzed by SDS-PAGE and immunoblotting with antibodies to endogenous PAK3, SPTLC2 and ATG9A, as well as antibody to the FLAG epitope. The positions of molecular mass markers (in kDa) are indicated on the left. The > 70-kDa species observed in the lyspersin-FTS pulldown is likely a non-specific protein that is recognized by the antibody to SPTLC2