Protein-correlation-profiling (PCP), in combination with quantitative proteomics, has emerged as a high-throughput method for the rapid identification of dynamic protein complexes in native conditions. While PCP has been successfully applied to soluble proteomes, characterization of the membrane interactome has lagged, partly due to the necessary use of detergents to maintain protein solubility. Here, we apply the peptidisc, a ‘one-size fits all’ membrane mimetic, for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Analysis of the SILAC-labeled peptidisc library via PCP allows generation of over 4900 possible binary interactions out of >700,000 random associations. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we demonstrate that our dataset is a useful resource for identifying transient and surprisingly novel protein interactions. For example, we discover a trans-periplasmic supercomplex comprising subunits of the Bam and Sec machineries, including membrane-bound chaperones YfgM and PpiD. We identify RcsF and OmpA as bone fide interactors of BamA, and we show that MetQ association with the ABC transporter MetNI depends on its N-terminal lipid anchor. We also discover NlpA as a novel interactor of MetNI complex. Most of these interactions are largely undetected by standard detergent-based purification. Together, the peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions and without genetic manipulation.

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Summary The dicistrovirus, Cricket paralysis virus (CrPV) encodes an RNA interference (RNAi) suppressor, 1A, which modulates viral virulence. Using the Drosophila model, we combined structural, biochemical, and virological approaches to elucidate the strategies by which CrPV-1A restricts RNAi immunity. The atomic resolution structure of CrPV-1A uncovered a flexible loop that interacts with Argonaute 2 (Ago-2), thereby inhibiting Ago-2 endonuclease-dependent immunity. Mutations disrupting Ago-2-binding attenuates viral pathogenesis in wild-type but not Ago-2-deficient flies. CrPV-1A also contains a BC-box motif that enables the virus to hijack a host Cul2-Rbx1-EloBC ubiquitin ligase complex, which promotes Ago-2 degradation and virus replication. Our study uncovers a viral-based dual regulatory program that restricts antiviral immunity by direct interaction with and modulation of host proteins. While the direct inhibition of Ago-2 activity provides an efficient mechanism to establish infection, the recruitment of a ubiquitin ligase complex, enables CrPV-1A to amplify Ago-2 inactivation to restrict further antiviral RNAi immunity.

Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K ؉ glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high-and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.

Background: The type I interferon (IFN) response is an ancient pathway that protects cells against viral pathogens by inducing the transcription of hundreds of IFNstimulated genes. Comprehensive catalogs of IFN-stimulated genes have been established across species and cell types by transcriptomic and biochemical approaches, but their antiviral mechanisms remain incompletely characterized. Here, we apply a combination of quantitative proteomic approaches to describe the effects of IFN signaling on the human proteome, and apply protein correlation profiling to map IFN-induced rearrangements in the human protein-protein interaction network. Results: We identify > 26,000 protein interactions in IFN-stimulated and unstimulated cells, many of which involve proteins associated with human disease and are observed exclusively within the IFN-stimulated network. Differential network analysis reveals interaction rewiring across a surprisingly broad spectrum of cellular pathways in the antiviral response. We identify IFN-dependent protein-protein interactions mediating novel regulatory mechanisms at the transcriptional and translational levels, with one such interaction modulating the transcriptional activity of STAT1. Moreover, we reveal IFN-dependent changes in ribosomal composition that act to buffer IFN-stimulated gene protein synthesis. Conclusions: Our map of the IFN interactome provides a global view of the complex cellular networks activated during the antiviral response, placing IFN-stimulated genes in a functional context, and serves as a framework to understand how these networks are dysregulated in autoimmune or inflammatory disease.

Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication.IMPORTANCE RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.

Osmosensing by transporter ProP is modulated by its cardiolipin (CL)-dependent concentration at the poles of Escherichia coli cells. Other contributors to this phenomenon were sought with the BACterial Two-Hybrid System (BACTH). The BACTH-tagged variants T18-ProP and T25-ProP retained ProP function and localization. Their interaction confirmed the ProP homo-dimerization previously established by protein crosslinking. YdhP, YjbJ and ClsA were prominent among the putative ProP interactors identified by the BACTH system. The functions of YdhP and YjbJ are unknown, although YjbJ is an abundant, osmotically induced, soluble protein. ClsA (CL Synthase A) had been shown to determine ProP localization by mediating CL synthesis. Unlike a deletion of clsA, deletion of ydhP or yjbJ had no effect on ProP localization or function. All three proteins were concentrated at the cell poles, but only ClsA localization was CL-dependent. ClsA was shown to be N-terminally processed and membrane-anchored, with dual, cytoplasmic, catalytic domains. Active site amino acid replacements (H224A plus H404A) inactivated ClsA and compromised ProP localization. YdhP and YjbJ may be ClsA effectors, and interactions of YdhP, YjbJ and ClsA with ProP may reflect their colocalization at the cell poles. Targeted CL synthesis may contribute to the polar localization of CL, ClsA and ProP.

ProQ is a cytoplasmic protein with RNA chaperone activities that reside in FinO-and Hfq-like domains. Lesions at proQ decrease the level of the osmoregulatory glycine betaine transporter ProP. Lesions at proQ eliminated ProQ and Prc, the periplasmic protease encoded by the downstream gene prc. They dramatically slowed the growth of Escherichia coli populations and altered the morphologies of E. coli cells in high-salinity medium. ProQ and Prc deficiencies were associated with different phenotypes. ProQ-deficient bacteria were elongated unless glycine betaine was provided. High-salinity cultures of Prcdeficient bacteria included spherical cells with an enlarged periplasm and an eccentric nucleoid. The nucleoid-containing compartment was bounded by the cytoplasmic membrane and peptidoglycan. This phenotype was not evident in bacteria cultivated at low or moderate salinity, nor was it associated with murein lipoprotein (Lpp) deficiency, and it differed from those elicited by the MreB inhibitor A-22 or the FtsI inhibitor aztreonam at low or high salinity. It was suppressed by deletion of spr, which encodes one of three murein hydrolases that are redundantly essential for enlargement of the murein sacculus. Prc deficiency may alter bacterial morphology by impairing control of Spr activity at high salinity. ProQ and Prc deficiencies lowered the ProP activity of bacteria cultivated at moderate salinity by approximately 70% and 30%, respectively, but did not affect other osmoregulatory functions. The effects of ProQ and Prc deficiencies on ProP activity are indirect, reflecting their roles in the maintenance of cell structure.O smotic stress perturbs cell structure, composition, and function (1). Despite retaining their rod-like shape, Escherichia coli cells cultivated in high-salinity minimal medium maintain lower hydration, turgor pressure, and growth rate than those cultivated at a lower salinity that is optimal for growth (2). The elastic murein sacculus is believed to buffer effects of osmotically induced water fluxes on cell structure (3). E. coli can attenuate osmotically induced dehydration by accumulating small, uncharged, or zwitterionic organic solutes called osmolytes (1, 4, 5). For example, transporter ProP mediates the accumulation of diverse solutes, including proline and glycine betaine, thereby restoring cellular hydration and stimulating bacterial growth in high-salinity media (6, 7).ProQ is a cytoplasmic protein that binds RNA, facilitating RNA duplexing and strand exchange (8). Previous work showed that proQ lesions decreased ProP levels and attenuated ProP activity (the proQ transport phenotype). These effects occurred when bacteria expressed proP from the chromosome or a plasmid-based P BAD promoter during growth in low-to moderate-salinity media and were reversed by plasmid-based proQ expression (8, 9). Here, we show that proQ lesions dramatically slow the growth of E. coli populations in high-salinity medium and alter the morphologies of E. coli cells (the proQ growth and morphological phenotypes...