HIV-host interactome revealed directly from infected cells

Abstract

Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection. Importantly, interacting proteins were recovered without bias, regardless of their potential for positive, negative or neutral impact on viral replication. We identified specific host associations made with trimerized Env during its biosynthesis, at virological synapses, with innate immune effectors (such as HLA-E) and with certain cellular signalling pathways (for example, Notch1). We also defined Vif associations with host proteins involved in the control of nuclear transcription and nucleoside biosynthesis as well as those interacting stably or transiently with the cytoplasmic protein degradation apparatus. Our approach is broadly applicable to elucidating pathogen-host interactomes, providing high-certainty identification of interactors by their direct access during cycling infection. Understanding the pathophysiological consequences of these associations is likely to provide strategic targets for antiviral intervention.

Figure 1. Selection of replication-competent, tagged derivatives of HIV-1

a, Viral gene segments (yellow) were independently subcloned to focus a step of Tn7-linker scanning-saturation mutagenesis. A proviral plasmid library was then reconstituted that contained ~3 × 10⁵ to 5 × 10⁵ independent clones, each harbouring a random PmeI insertion (red). DNA was pooled to generate an equally diverse library of PmeI-modified viruses by DNA transfection and used to infect a human T cell line. A stringent biological step selected for those viruses that can accommodate a five-amino-acid insertion yet remain fully replication-competent over several cycles of growth. Defective viruses (crossed out, X) are vastly overrepresented at early time points. Some mutant viruses capable of gaining entry into target cells do persist initially. However, the vast majority of these initially persisting viruses are non-competitive and are rapidly lost. Surviving PmeI-inserted HIV-1 derivative proviral clones exhibiting high replication competency at the end of selection are candidates for further recombinant manipulation and can be used as the recipient vectors for the insertion of foreign immunoaffinity tags (that is, 3xFLAG). b, Representative time course (n = 3; 1–12 d.p.i.) detailing the emergence of survivors from the Env-PmeI-modified clonal library (8% TBE PAGE). Lanes: black, uncut PCR product spanning the mutagenic targeted env gene segment; red, PmeI-digested PCR product. An uncleaved product is observed for any virus that has lost its PmeI insertion post day one (star). This species is (on sequence analysis) WT DNA, and indicates the occurrence of either intragenic retroviral recombination during reverse transcription or, less probably, an artefact of PCR recombination. c, Deep sequencing analysis details the mutational complexity of the plasmid DNA PmeI insertional library of the targeted env gene segment (top). The DNA sequences of viral survivors were determined 1, 3 and 5 d.p.i. Triplicate infection experiments were used to reveal the initially stochastic nature of successful selection pathways for viability. d, Amino acid sequence of the 3xFLAG (flanked by glycine/serine linkers) used to epitope tag a subset of replication-competent PmeI inserted viruses. Replication kinetics of Env- and Vif-modified viruses in comparison with the untagged and otherwise isogenic WT virus over several days of growth in their respective T cell line (n = 3; CEM cells for env recombinants; MT-2 cells for vif recombinants).

Figure 2. Permissible sites of insertion within Env and Vif and visualization of each tagged protein during infection

a,b,PmeI (orange) and 3xFLAG tag (red) insertion sites compatible with viral replication competency within the primary sequence and redrawn using MacPyMol for trimerized Env (PDB 4NCO) (a) and a pentameric Vif-containing complex (PDB 4N9F) (b). Five amino acids flanking the tag/linker and originating from transposition are underlined. The lollipop-like symbols denote N-linked Env glycosylation, and disulfide bonds are in yellow (a). Amino acids in the disordered/unresolved portion of the crystal structure are in light grey (a, top), and the dashed line (a, bottom left) shows an unresolved section in the crystal structure; two post-selection Env PmeI insertional survivors map here. The 3xFLAG tag is immediately adjacent to this region, perhaps reflecting a flexible and/or alternative structural configuration(s). The epitope tag in Vif-3xF is between the Vif BC and Cullin boxes, shown within the primary sequence (b, top) and in the context of a pentameric complex where it is appointed away from all other protein–protein interfaces (b, bottom). c, The 3xFLAG-tagged Vif protein exhibits a cytoplasmic punctate staining pattern within Vif-3xF infected HOS-CD4-Fusin cells. d, 3xF-tagged Env protein (red) elaborates a highly reticulated distribution consistent with the endoplasmic reticulum (ER) in an infected CEM cell syncytium (two Z-slices from the same syncytium are shown: a medial and a peripheral slice). e, The 3xFLAG epitope is preferentially immunoreactive early during its maturation pathway within the ER. In contrast, the subcellular localization of 3xFLAG-tagged Env in HOS-CD4-Fusin cells is indistinguishable from WT Env using an antibody directed against a common gp120/gp160 epitope (2G12) as the Env is stained both within the ER and Golgi (the presentation is displayed vertically for all lanes of each panel, and also in Supplementary Fig. 2). Cells were first infected with baculovirus encoding an ER- or Golgi-specific fluorescent green probe and then infected with HIV Env-3xF or WT and incubated for an additional 48 h. Env was stained with mouse anti-FLAG mAb (red) or human anti-Env Ab (2G12; green or red). Counterstaining for nuclear DNA is in blue. For all fluorescent studies shown, the bar denotes 5 μm. All images are representative of three independent experiments with almost all stained cells in the population illustrating the respective phenotype displayed.

Figure 3. Reciprocal mass spectrometric isotopic differentiation of interactions as random or targeted (MS I-DIRT) (ref. 19) plot for Env (including inset of region from 0.97 to 1.0) and histogram of the number of proteins versus average I-DIRT ratios for Env

a, I-DIRT ratios for forward and reverse I-DIRT affinity isolations for the 3xFLAG-tagged HIV-1 Env protein. The blue rectangular region delimits the cutoff for statistical significance (<0.01% chance of being nonspecific). Selected proteins of interest are labelled. The average protein abundance within the two-affinity isolation is indicated by darkness of shading. Proteins registering greater than 0.97 on both I-DIRT and reverse I-DIRT are circled in red. b, Histogram showing the number of identified proteins in the Env affinity isolation plotted versus their average (forward and reverse) I-DIRT ratios. The calculated nonspecific distribution is indicated in blue, and putative specific interactors (>0.97) are boxed in red.

Figure 4. Vif interactors identified by reciprocal MS I-DIRT analysis and documentation of differential binding kinetics of its interactors

a, Plot of I-DIRT ratios for forward and reverse I-DIRT affinity isolations for the 3xFLAG-tagged HIV-1 Vif protein. The blue region delimits the cutoff for statistical significance (<0.01% chance of being nonspecific). Selected proteins of interest are labelled. Protein abundance is indicated by the darkness of shading. b, Histogram showing the number of identified proteins in the Vif affinity isolation plotted versus their average (forward and reverse) I-DIRT ratios. The calculated nonspecific distribution is indicated in blue. c, Left: Results of the isotopic exchange experiment in which immunoaffinity-isolated, heavy isotope-labelled tagged Vif and heavy isotope-labelled associated proteins were exposed to protein extract of normal (light) peptide isotopic content for the indicated times. Per cent exchange between the heavy labelled proteins on the beads and their light counterparts in the extract was plotted for tagged Vif itself and the known interactors CBFβ, Cul2, Cul5, APOBEC3F and APOBEC3G (left), showing that although CBFβ and Cul2 exhibit very little exchange, Cul5, APOBEC3F and APOBEC3G exchange rapidly. c, Right: Percentage exchange curves of Vif interactors TCP1, CCT2, CCT3, CCT3, CCT5, CCT6A, CCT7 and CCT8, which together form the T-complex (inset: End-on view of a cryogenic-electron microscopy (cryo-EM) density map Protein Data Bank (PDB) accession code 3IYG (ref. 20) as redrawn using MacPyMol), were almost superimposable, indicating that these proteins dissociate from the Vif affinity pull down as a complex, rather than as individual factors.

Figure 5. Experimental documentation for select interactors

a, Co-immunoprecipitation of selected Env interactors confirms I-DIRT results. 293T cells were co-transfected with gp140 Env and the indicated plasmids. Extracts were immunoprecipitated, electrophoresed and silver stained (top) or western blotted against Env or GAPDH control. b, Tor3A occupies the same intracellular compartments as Env, and when expressed in producer cells, inhibits the next round of infection. Immunofluorescence: 293T cells co-transfected with Env-3xFLAG proviral DNA and an expression vector for human Tor3A were co-stained for FLAG (red) and Tor3A (green). Infection: 293T cells were co-transfected individually with WT env⁻ proviral DNA, a pseudotype expression vector for the envelope proteins of HIV-1 (WT), HIV-1 ΔCT, SIV or VSV and with either pCMV-empty or pCMV-TOR3A. TZM-bl (HeLa*; n = 4) or MT4-GFP (MT4*; n = 2) indicator cells were infected with p24-normalized viral stocks. c, WT HIV-1 infectivity is increased when produced in TOR1AIP2 (LULL1) knockout cells. Virus produced in LULL1⁺ or LULL1⁻ cells was normalized for reverse transcriptase activity and scored for infectivity on MT4* indicator cells (Supplementary Fig. 11a). d, HIV-1 infection alters Notch1 proteolytic processing while Env increases Notch1 levels. GFP-positive cells infected with either Env⁻ or Env⁺ virus were purified by FACS (Supplementary Fig. 11b) and compared with uninfected cells. Western blots were probed against the Notch1 C-terminal domain (anti-CTD) or against a neo-epitope created upon γ-secretase cleavage (anti-NICD). Gamma (γ)-tubulin is a loading control, while Gag-and Env-specific antibodies confirm lane identities. e, HIV-1 infection leads to hypophosphorylation of the NICD independently of Env. Precalibrated lysates used in d were either untreated, treated with calf intestinal alkaline phosphatase (CIP) or treated with CIP plus phosphatase inhibitors and the lysates were electrophoresed, blotted and probed with anti-NICD. Upper bands (asterisk), hyperphosphorylated NICD; lower bands, hypophosphorylated NICD. f, Vif expression reduces DDB1 and AMBRA1 levels in 293T cells. For the DDB1 Vif titration (top right), indicated amounts of Vif construct were co-transfected with 5 μg of DDB1 plasmid (total DNA, 10 μg). For other transfections, Vif and the interactor or control plasmid were present at 5 μg each. Extracts were immunoprecipitated, electrophoresed and silver stained or probed by western blot (DDB1 and GAPDH). Residual antibody light chain was used as a loading control. n ≥ 2 for all experiments. Error bars represent standard deviation.

Figure 6. Summary of biological subclasses of cellular interactors engaged with Vif and Env during active viral growth

An abbreviated schematic of the HIV life cycle starting at the upper left with infection of a susceptible target cell and at the bottom right showing perpetuation of the infectious cycle by viral egress and transmission via cell-free infection of both nearby and distant cells or by cell-to-cell transmission of the virus to an immediately adjacent cell. The members of each class of cellular interactor are colour-coded based on their predicted intracellular location and/or function. The dashed horizontal arrow intersecting the figure indicates the progression of the viral life cycle and separates the members of the Vif interactome from those interacting with Env during the series of steps that occur during the Env biosynthetic and maturation pathways.