Proteome-wide characterization of the RNA-binding protein RALY-interactome using the in vivo-biotinylation-pulldown-quant (iBioPQ) approach

RALY is a member of the heterogeneous nuclear ribonucleoproteins, a family of RNA-binding proteins generally involved in many processes of mRNA metabolism. No quantitative proteomic analysis of RALY-containing ribonucleoparticles (RNPs) has been performed so far, and the biological role of RALY remains elusive. Here, we present a workflow for the characterization of RALY's interaction partners, termed iBioPQ, that involves in vivo biotinylation of biotin acceptor peptide (BAP)-fused protein in the presence of the prokaryotic biotin holoenzyme synthetase of BirA so that it can be purified using streptavidin-coated magnetic beads, circumventing the need for specific antibodies and providing efficient pulldowns. Protein eluates were subjected to tryptic digestion and identified using data-independent acquisition on an ion-mobility enabled high-resolution nanoUPLC-QTOF system. Using label-free quantification, we identified 143 proteins displaying at least 2-fold difference in pulldown compared to controls. Gene Ontology overrepresentation analysis revealed an enrichment of proteins involved in mRNA metabolism and translational control. Among the most abundant interacting proteins, we confirmed RNA-dependent interactions of RALY with MATR3, PABP1 and ELAVL1. Comparative analysis of pulldowns after RNase treatment revealed a protein-protein interaction of RALY with eIF4AIII, FMRP, and hnRNP-C. Our data show that RALY-containing RNPs are much more heterogeneous than previously hypothesized

Live-Cell Imaging of Early Steps of Single HIV-1 Infection

Live-cell imaging of single HIV-1 entry offers a unique opportunity to delineate the spatio-temporal regulation of infection. Novel virus labeling and imaging approaches enable the visualization of key steps of HIV-1 entry leading to nuclear import, integration into the host genome, and viral protein expression. Here, we discuss single virus imaging strategies, focusing on live-cell imaging of single virus fusion and productive uncoating that culminates in HIV-1 infection

Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells.

Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process

Localization and functions of native and eGFP-tagged capsid proteins in HIV-1 particles.

In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components

CypA-DsRed dissociation from permeabilized HIV-1 cores is concurrent with the loss of CA and is inhibited by cytosolic extract.

<p>(A) IN-sfGFP/CypA-DsRed-labeled viruses were adhered to coverslips, permeabilized as above, fixed with PFA after varied times at 37°C and immunostained for CA/p24. Control viruses (leftmost column) were fixed prior to permeabilization. The two rightmost columns show the effects of CsA (5 μM) and the cytosolic extract from TZM-bl cells. Scale bar 5 μm. (B) Spontaneous release of CypA-DsRed from IN-sfGFP labeled permeabilized viral cores incubated at 37°C without fixation. Sum fluorescence profiles were obtained by single particle tracking and plotted as a function of time TX-100 permeabilization. (C) The average ratios of CypA-DsRed, p24 and IN-sfGFP puncta per image field were determined and normalized to that immediately after permeabilization of pseudoviruses containing WT and the mutant E45A and K203A CA. Four fields of view were imaged for each time point. Data are means and SD from 3 independent experiments. (D) Effect of cytosolic extract on the loss of p24 and CypA-DsRed from the cores bearing the mutant and WT capsids. TX-100-permeabilized viruses were incubated for 80 min at 37°C in the presence or absence of 25 ng/μl cytosol prepared from TZM-bl cells. Results are normalized to samples fixed immediately after permeabilization.</p

Loss of CypA-DsRed following virus-cell fusion.

<p>VSV-G pseudotyped HIV-1 particles bearing WT CA or CA mutants and co-labeled with IN-sfGFP and CypA-DsRed were used to infect TZM-bl cells (~10 pg of p24 per 5·10⁴ cells, MOI 0.008). Viruses were pre-bound to cells in the cold and virus entry/fusion was initiated by adding a pre-warmed buffer. Time-lapse images of a single field of view were taken every 30 sec for 80 min, at which point, 10 μM CsA was added and image acquisition continued for 40 min. (A) Kinetics of CypA-DsRed loss from IN-sfGFP puncta was measured for WT CA and each of the CA mutant viruses in 5 independent experiments. <i>Inset</i>: The fraction of double-labeled cores that lose CypA-DsRed in response to 10 μM CsA addition at 80 min (arrow). (B) Same as in panel A, but CsA was added just before starting the image acquisition (0 min) to measure the fusion kinetics. (C, D) Selected images and corresponding CypA-DsRed and IN-sfGFP intensity profiles obtained by single particle tracking. The tracks and images illustrate the relatively quick (C) and the less frequently occurring slow (D) release of CypA-DsRed. Scale bar 0.5 μm. (E) Effects of CA-binding compounds PF74 (10 μM) and BI-2 (20 μM) on the kinetics of CypA-DsRed loss from single post-fusion cores. (F) Reverse transcription accelerates the loss of CypA-DsRed from IN-sfGFP labeled particles. Inhibition of viral DNA synthesis by Nevirapine (10 μM) or by the RT D185N mutation, delayed the kinetics of CypA-DsRed disappearance after fusion. The E478Q mutation, which abolishes RNase H activity, did not exert a significant effect on the CypA-DsRed retention time. <i>Insets to panels E and F</i>: The fraction of double-labeled cores that lose CypA-DsRed in response to 10 μM CsA added at 80 min (arrows). Statistical significance was determined by the Mann-Whitney Rank-Sum test.</p

CypA-DsRed enables visualization of HIV-1 CA shedding.

<p>CypA-DsRed remains associated with post-fusion cores until CA is shed from IN-sfGFP-labeled cores. Loss of CA/CypA-DsRed occurs either within the first hour after initiating virus entry (pathway 1) or over a period of several hours (pathway 2). An alternative uncoating at the nuclear pore (pathway 3) is also illustrated. Dashed arrows indicate putative nuclear import of pre-integration complexes; these are not meant to relate the sites of uncoating to productive infection.</p

CypA-DsRed specifically incorporates into HIV-1 cores through tight binding to capsid and obviates the need for CypA in target Jurkat cells.

<p>(A) VSV-G pseudotyped particles containing WT or G89V CA were co-labeled with IN-sfGFP and CypA-DsRed. Bottom panel: viruses bearing WT-CA and the CypA-DsRed H126Q mutant. (B) TZM-bl cells were inoculated with equal p24 amounts of VSV-G pseudotyped particles containing BlaM-Vpr and containing or lacking CypA-DsRed. Virus fusion activity was measured in the presence or absence of 5 μM CsA. Data are means and SEM from 3 triplicate experiments. (C) Infectivity of unlabeled and CypA-DsRed-containing pseudoviruses. TZM-bl cells were inoculated with equal RT units of viruses in the presence or absence of CsA, and the resulting luciferase activity was measured at 48 h.p.i. Data are means and SD from 5 triplicate experiments. (D) Jurkat CypA+/+ or CypA-/- cells were infected with VSV-G pseudotyped viruses containing or lacking CypA-DsRed. The resulting luciferase activity was measured after 48 h. Results are means and SD from 3 duplicate experiments. (E) Pseudotyped viruses co-labeled with IN-sfGFP and CypA-DsRed were bound to coverslips, permeabilized with TX-100 and fixed with PFA. In parallel samples, the initial colocalization of IN-sfGFP, CypA-DsRed and p24 was preserved by PFA fixation prior to permeabilization (left column). Particles were then treated with CsA (5 μM) or DMSO for 2 min, fixed and immunostained for p24/CA. Scale bars in all panels are 5 μm.</p

Analyses of spontaneous and CsA-induced release of CypA-DsRed from post-fusion cores.

<p>(A) CsA-induces loss of CypA-DsRed from the IN-sfGFP labeled particle 1 in a TZM-bl cell at 4 h.p.i. Particle 2 retained CypA-DsRed. The cell nucleus is colored blue. The lower image panels show blow up views of the two particles at indicated times after CsA addition. Scale bar 10 μm. (B) Kinetics of spontaneous, apparently complete loss of CypA-DsRed from IN-sfGFP labeled cores in TZM-bl cells within 80 min of imaging, starting at 2, 4 or 6 h after infection. CsA (10 μM) was added at 80 min after beginning of image acquisition. (C-G) Single particle tracking analysis of CypA-DsRed loss from IN-sfGFP puncta in TZM-bl cells at 6 h post-infection. Cells were imaged for 80 min, at which point 10 μM CsA was added and imaging continued for 40 min. Examples of a steady level of the CypA marker (C), as well as partial (D) and nearly complete (E) loss of CypADsRed are shown. (F) Typical stable CypA-DsRed and IN-sfGFP signals in the presence of Nevirapine (10 μM). Dashed lines in panels C-F mark the time of CsA addition. (G) Analysis of the single particle tracking results exemplified in panels C-F. The changes in CypA-DsRed intensity within the 80 min imaging window were measured for 142 randomly chosen post-fusion cores in control samples and 122 cores in Nevirapine treated samples. The CypA-DsRed loss was categorized as no release (less than 40% loss of the initial signal), slow (40–70% of the signal) and quick (>70% loss) during the 80 min imaging interval. The fraction of particles that did not release CypA-DsRed within 80 min of imaging increased from 43.0% in control to 65.6% in Nevirapine-treated cells, whereas the slow and quick release events dropped from 42.4% to 32.8% and from 14.6% to 1.6%, respectively.</p