Large-scale mapping of human protein–protein interactions by mass spectrometry

Mapping protein–protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein–protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein–protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations

Exocytosis of Alphaherpesvirus Virions, Light Particles, and Glycoproteins Uses Constitutive Secretory Mechanisms

Many molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based on total internal reflection fluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway

Exocytosis of Alphaherpesvirus Virions, Light Particles, and Glycoproteins Uses Constitutive Secretory Mechanisms

Many molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based on total internal reflection fluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding▿ †

The human immunodeficiency virus type 1 structural polyprotein Pr55Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other

Nucleocapsid promotes localization of HIV-1 gag to uropods that participate in virological synapses between T cells.

T cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear. Using multiple uropod markers, we observed that HIV-1 Gag localizes to the uropod in polarized T cells. Infected T cells formed contacts with uninfected target T cells preferentially via HIV-1 Gag-containing uropods compared to leading edges that lack plasma-membrane-associated Gag. Cell contacts enriched in Gag and CD4, which define the virological synapse (VS), are also enriched in uropod markers. These results indicate that Gag-laden uropods participate in the formation and/or structure of the VS, which likely plays a key role in cell-to-cell transmission of HIV-1. Consistent with this notion, a myosin light chain kinase inhibitor, which disrupts uropods, reduced virus particle transfer from infected T cells to target T cells. Mechanistically, we observed that Gag copatches with antibody-crosslinked uropod markers even in non-polarized cells, suggesting an association of Gag with uropod-specific microdomains that carry Gag to uropods. Finally, we determined that localization of Gag to the uropod depends on higher-order clustering driven by its NC domain. Taken together, these results support a model in which NC-dependent Gag accumulation to uropods establishes a preformed platform that later constitutes T-cell-T-cell contacts at which HIV-1 virus transfer occurs

Compartmented neuronal cultures reveal two distinct mechanisms for alpha herpesvirus escape from genome silencing.

Alpha herpesvirus genomes encode the capacity to establish quiescent infections (i.e. latency) in the peripheral nervous system for the life of their hosts. Multiple times during latency, viral genomes can reactivate to start a productive infection, enabling spread of progeny virions to other hosts. Replication of alpha herpesviruses is well studied in cultured cells and many aspects of productive replication have been identified. However, many questions remain concerning how a productive or a quiescent infection is established. While infections in vivo often result in latency, infections of dissociated neuronal cultures in vitro result in a productive infection unless lytic viral replication is suppressed by DNA polymerase inhibitors or interferon. Using primary peripheral nervous system neurons cultured in modified Campenot tri-chambers, we previously reported that reactivateable, quiescent infections by pseudorabies virus (PRV) can be established in the absence of any inhibitor. Such infections were established in cell bodies only when physically isolated axons were infected at a very low multiplicity of infection (MOI). In this report, we developed a complementation assay in compartmented neuronal cultures to investigate host and viral factors in cell bodies that prevent establishment of quiescent infection and promote productive replication of axonally delivered genomes (i.e. escape from silencing). Stimulating protein kinase A (PKA) signaling pathways in isolated cell bodies, or superinfecting cell bodies with either UV-inactivated PRV or viral light particles (LP) promoted escape from genome silencing and prevented establishment of quiescent infection but with different molecular mechanisms. Activation of PKA in cell bodies triggers a slow escape from silencing in a cJun N-terminal kinase (JNK) dependent manner. However, escape from silencing is induced rapidly by infection with UVPRV or LP in a PKA- and JNK-independent manner. We suggest that viral tegument proteins delivered to cell bodies engage multiple signaling pathways that block silencing of viral genomes delivered by low MOI axonal infection

Open LED Illuminator: A Simple and Inexpensive LED Illuminator for Fast Multicolor Particle Tracking in Neurons

<div><p>Dual-color live cell fluorescence microscopy of fast intracellular trafficking processes, such as axonal transport, requires rapid switching of illumination channels. Typical broad-spectrum sources necessitate the use of mechanical filter switching, which introduces delays between acquisition of different fluorescence channels, impeding the interpretation and quantification of highly dynamic processes. Light Emitting Diodes (LEDs), however, allow modulation of excitation light in microseconds. Here we provide a step-by-step protocol to enable any scientist to build a research-grade LED illuminator for live cell microscopy, even without prior experience with electronics or optics. We quantify and compare components, discuss our design considerations, and demonstrate the performance of our LED illuminator by imaging axonal transport of herpes virus particles with high temporal resolution.</p></div

Characterization of the neuroinvasive profile of a pseudorabies virus recombinant expressing the mTurquoise2 reporter in single and multiple injection experiments.

BACKGROUND:Viral transneuronal tracing has become a well established technology used to define the synaptic architecture of polysynaptic neural networks. NEW METHOD:In this report we define the neuroinvasive profile and reporter expression of a new recombinant of the Bartha strain of pseudorabies virus (PRV). The new recombinant, PRV-290, expresses the mTurquoise2 fluorophor and is designed to complement other isogenic recombinants of Bartha that express different reporters of infection. Results & Comparison with Existing Methods: PRV-290 was injected either alone or in combination with isogenic recombinants of PRV that express enhanced green fluorescent protein (EGFP; PRV-152) or monomeric red fluorescent protein (mRFP; PRV-614). Circuits previously defined using PRV-152 and PRV-614 were used for the analysis. The data demonstrate that PRV-290 is a retrograde transneuronal tracer with temporal kinetics similar to those of its isogenic recombinants. Stable expression of the diffusible mTurquoise2 reporter filled infected neurons, with the extent and intensity of labeling increasing with advancing post inoculation survival. In multiple injection experiments, PRV-290 established productive infections in neurons also replicating PRV-152 and/or PRV-614. This novel demonstration of three recombinants infecting individual neurons represents an important advance in the technology. CONCLUSION:Collectively, these data demonstrate that PRV-290 is a valuable addition to the viral tracer toolbox for transneuronal tracing of neural circuitry

Triggering allows high-speed multi-channel live-imaging of axonal transport.

<p>Embryonic superior cervical ganglion (SCG) neurons were cultured in tri-chambers [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.ref004" target="_blank">4</a>] and neurons in the soma-compartment were infected with PRV 137 expressing gM-EGFP (gM, green) and mRFP-VP26 (VP26, red). 10 hours post infection viral particle trafficking was imaged in axons penetrating into the middle and neuron compartment using non-triggered Arc lamp illumination (Arc lamp–no trigger) or triggered LED illumination (LED–trigger) (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#sec002" target="_blank">Materials and Methods</a>), a) Three consecutive stills of a representative track for each mode with roughly the same length (≈14 sec) and average particle speed of ≈1.5μm/sec are shown. The time between consecutive dual-color images is ~106 ms for LED illumination and 625 ms for Arc lamp illumination. b) The same tracks as in a) shown as a Kymograph. As triggered LED illumination allows much higher frame rates, particle positions with LED illumination almost overlap in both channels while they do not for non-triggered Arc lamp illumination. c) Signal-to-noise ratio (SNR) calculated for each imaging condition. Scale bar indicates 5 μm.</p

Koehler illumination results in superior illumination evenness.

<p>a) Schematic of critical (top) and Koehler modes. Images were inspired by [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.ref015" target="_blank">15</a>]. In critical illumination, the LED light (green) is collimated using a collimation lens (blue) and projected onto the objective. The objective focuses the light, resulting in an image of the LED at the sample plane. In Koehler illumination mode, the collimated light gets focused onto the back focal plane (BFP) of the objective using a second lens (right most lens, blue). This way, the objective projects a collimated cone of light onto the sample plane, resulting in better illumination flatness. A set of two more lenses can be used to generate conjugate planes that than can be regulated by diaphragms to adjust the brightness, contrast, resolution and depth of field of the image (Aperture diaphragm) and the illuminated field of view (Field diaphragm) (grey box). In critical illumination with infinite conjugate plate, an Aperture diaphragm can be used to regulate brightness, contrast, resolution and depth of field. b) Brightness and illumination flatness in critical and Koehler illumination modes were measured on a custom-built epifluorescence microscope (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#sec002" target="_blank">Materials and Methods</a> for details). Representative images are shown and intensities are coded as colors as indicated in the legend and can be compared between critical and Koehler modes but not between LEDs as different exposure times were used. Koehler illumination showed a better field flatness and slightly increase in intensity (≈ 1.3–29.9% depending on image position for the 470 nm LED and about 2.8–12.96% for the 595 nm LED (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.s005" target="_blank">S5 Table</a>)). c) Comparison of fluorescence intensity distributions. At least three images were taken and the fluorescent plastic slide was moved between images. Images were analyzed in Fiji [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.ref003" target="_blank">3</a>] by drawing a diagonal line ROI and intensities along that line were measured using the “plot profile” tool (black arrow). Resulting intensity values were imported into Matlab (Mathworks), the mean was calculated for three images each and means were normalized to their individual maxima and plotted as a line plot depicting the degree of illumination differences over the measured diagonal. Critical illumination with directly emitting LEDs (470 nm LED) lead to uneven illumination, while the evenness with the phosphor-converted LED (595 nm LED) was only slightly worse than with Koehler illumination.</p