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Summary: Accessory proteins of lentiviruses, such as HIV-1, target cellular restriction factors to enhance viral replication. Systematic analyses of proteins that are targeted for degradation by HIV-1 accessory proteins may provide a better understanding of viral immune evasion strategies. Here, we describe a high-throughput platform developed to study cellular protein stability in a highly parallelized matrix format. We used this approach to identify cellular targets of the HIV-1 accessory protein Vpu through arrayed coexpression with 433 interferon-stimulated genes, followed by differential fluorescent labeling and automated image analysis. Among the previously unreported Vpu targets identified by this approach, we find that the E2 ligase mediating ISG15 conjugation, UBE2L6, and the transmembrane protein PLP2 are targeted by Vpu during HIV-1 infection to facilitate late-stage replication. This study provides a framework for the systematic and high-throughput evaluation of protein stability and establishes a more comprehensive portrait of cellular Vpu targets. : Retroviruses use their accessory proteins to evade immune detection and enhance viral replication. Jain et al. developed a high-throughput–high-content imaging platform to study protein stability and degradation. This method was then applied to reveal cellular targets of the HIV-1 accessory factor Vpu. Keywords: Vpu, HIV-1, high-content imaging, protein degradation, interferon-stimulated genes, antiviral, ISGylation, immune evasion

Treebanks are important resources for researchers in natural language processing. They provide training and testing materials so that different algorithms can be compared. However, it is not a trivial task to construct high-quality treebanks. We have not yet had a proper treebank for such a low-resource language as Vietnamese, which has probably lowered the performance of Vietnamese language processing. We have been building a consistent and accurate Vietnamese treebank to alleviate such situations. Our treebank is annotated with three layers: word segmentation, part-of-speech tagging, and bracketing. We developed detailed annotation guidelines for each layer by presenting Vietnamese linguistic issues as well as methods of addressing them. Here, we also describe approaches to controlling annotation quality while ensuring a reasonable annotation speed. We specifically designed an appropriate annotation process and an effective process to train annotators. In addition, we implemented several support tools to improve annotation speed and to control the consistency of the treebank. The results from experiments revealed that both inter-annotator agreement and accuracy were higher than 90%, which indicated that the treebank is reliable.

Several systems-level datasets designed to dissect host-pathogen interactions during influenza A infection have been reported. However, apparent discordance among these data has hampered their full utility toward advancing mechanistic and therapeutic knowledge. To collectively reconcile these datasets, we performed a meta-analysis of data from eight published RNAi screens and integrated these data with three protein interaction datasets, including one generated within the context of this study. Further integration of these data with global virus-host interaction analyses revealed a functionally validated biochemical landscape of the influenza-host interface, which can be queried through a simplified and customizable web portal (http://www.metascape.org/IAV). Follow-up studies revealed that the putative ubiquitin ligase UBR4 associates with the viral M2 protein and promotes apical transport of viral proteins. Taken together, the integrative analysis of influenza OMICs datasets illuminates a viral-host network of high-confidence human proteins that are essential for influenza A virus replication.

Background. Accurate timing of antenatal corticosteroids (ACS) has resulted in improved neonatal outcomes.Objectives. Our primary objective was to determine predictors for optimal timing of ACS in women presenting with spontaneous preterm labor.Study Design. A retrospective cohort study of women receiving ACS for spontaneous preterm birth was conducted. Women were included if they presented with preterm labor or preterm premature rupture of membranes. Accurate timing of ACS was defined as administration within 7 days of delivery. Maternal demographic and obstetrics characteristics were compared between the groups receiving ACS ≤7 days and >7 days from delivery. Statistical analyses were performed using parametric and nonparametric tests.P<0.05was considered significant.Results. The study included 215 subjects. Median latency from ACS administration to delivery was 6 days (IQR 32). Accurate timing of ACS occurred in 113 (53%) women and was associated with rupture of membranes (OR 13.8, 95% CI 5.9–32.6), cervical change (OR 7.1, 95% CI 3.0–17.1), and cervical dilation ≥ 2 cm (OR 3.9, 95% CI 1.5–10.3).Conclusions. Rupture of membranes, cervical change, and cervical dilation ≥ 2 cm were strong predictors of optimal timing. 53% of women with preterm labor received ACS optimally.

SummaryPathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent proinflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis, we identify 190 cofactors required for TLR7- and TLR9-directed signaling responses. A set of cofactors were crossprofiled for their activities downstream of several immunoreceptors and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection.

Retinoic acid-inducible gene I (RIG-I) receptor recognizes 5'-triphosphorylated RNA and triggers a signalling cascade that results in the induction of type-I interferon (IFN)-dependent responses. Its precise regulation represents a pivotal balance between antiviral defences and autoimmunity. To elucidate the cellular cofactors that regulate RIG-I signalling, we performed two global RNA interference analyses to identify both positive and negative regulatory nodes operating on the signalling pathway during virus infection. These factors were integrated with experimentally and computationally derived interactome data to build a RIG-I protein interaction network. Our analysis revealed diverse cellular processes, including the unfolded protein response, Wnt signalling and RNA metabolism, as critical cellular components governing innate responses to non-self RNA species. Importantly, we identified K-Homology Splicing Regulatory Protein (KHSRP) as a negative regulator of this pathway. We find that KHSRP associates with the regulatory domain of RIG-I to maintain the receptor in an inactive state and attenuate its sensing of viral RNA (vRNA). Consistent with increased RIG-I antiviral signalling in the absence of KHSRP, viral replication is reduced when KHSRP expression is knocked down both in vitro and in vivo. Taken together, these data indicate that KHSRP functions as a checkpoint regulator of the innate immune response to pathogen challenge.

An ultrahigh-throughput screen was performed to identify novel small molecule inhibitors of influenza virus replication. The screen employed a recombinant influenza A/WSN/33 virus expressing Renilla luciferase and yielded a hit rate of 0.5%, of which the vast majority showed little cytotoxicity at the inhibitory concentration. One of the top hits from this screen, designated S20, inhibits HA-mediated membrane fusion. S20 shows potent antiviral activity (IC50 = 80 nM) and low toxicity (CC50 = 40 μM), yielding a selectivity index of 500 and functionality against all of the group 1 influenza A viruses tested in this study, including the pandemic H1N1 and avian H5N1 viruses. Mechanism of action studies proved a direct S20-HA interaction and showed that S20 inhibits fusion by stabilizing the prefusion conformation of HA. In silico docking studies were performed, and the predicted binding site in HA2 corresponds with the area where resistance mutations occurred and correlates with the known role of this region in fusion. This high-throughput screen has yielded many promising new lead compounds, including S20, which will potentially shed light on the molecular mechanisms of viral infection and serve as research tools or be developed for clinical use as antivirals.

Innate immune sensors such as Toll-like receptors (TLRs) differentially utilize adaptor proteins and additional molecular mediators to ensure robust and precise immune responses to pathogen challenge. Through a gain-of-function genetic screen, we identified the gamma catalytic subunit of protein phosphatase 1 (PP1-γ) as a positive regulator of MyD88-dependent proinflammatory innate immune activation. PP1-γ physically interacts with the E3 ubiquitin ligase TRAF6, and enhances the activity of TRAF6 towards itself and substrates such as IKKγ, whereas enzymatically inactive PP1-γ represses these events. Importantly, these activities were found to be critical for cellular innate responses to pathogen challenge and microbial clearance in both mouse macrophages and human monocyte lines. These data indicate that PP1-γ phosphatase activity regulates overall TRAF6 E3 ubiquitin ligase function and promotes NF-κB-mediated innate signaling responses.