Benzene with Alkyl Chains Is a Universal Scaffold for Multivalent Virucidal Antivirals.

Most viruses start their invasion by binding to glycoproteins' moieties on the cell surface (heparan sulfate proteoglycans [HSPG] or sialic acid [SA]). Antivirals mimicking these moieties multivalently are known as broad-spectrum multivalent entry inhibitors (MEI). Due to their reversible mechanism, efficacy is lost when concentrations fall below an inhibitory threshold. To overcome this limitation, we modify MEIs with hydrophobic arms rendering the inhibitory mechanism irreversible, i.e., preventing the efficacy loss upon dilution. However, all our HSPG-mimicking MEIs only showed reversible inhibition against HSPG-binding SARS-CoV-2. Here, we present a systematic investigation of a series of small molecules, all containing a core and multiple hydrophobic arms terminated with HSPG-mimicking moieties. We identify the ones that have irreversible inhibition against all viruses including SARS-CoV-2 and discuss their design principles. We show efficacy in vivo against SARS-CoV-2 in a Syrian hamster model through both intranasal instillation and aerosol inhalation in a therapeutic setting (12 h postinfection). We also show the utility of the presented design rules in producing SA-mimicking MEIs with irreversible inhibition against SA-binding influenza viruses.

cART Restores Transient Responsiveness to IFN Type 1 in HIV-Infected Humanized Mice.

The lack of a human immunodeficiency virus (HIV) cure has heightened interest in immunotherapy. As such, type I interferons (IFNs), in particular, IFN alpha (IFN-α), have gained renewed attention. However, HIV pathogenesis is driven by sustained IFN-mediated immune activation, and the use of IFNs is rather controversial. The following questions therein remain: (i) which IFN-α subtype to use, (ii) at which regimen, and (iii) at what time point in HIV infection it might be beneficial. Here, we used IFN-α14 modified by PASylation for its long half-life in vivo to eventually treat HIV infection. We defined the IFN dosing regimen based on the maximum increase in interferon-stimulated gene (ISG) expression 6 h after its administration and a return to baseline of ubiquitin-specific protease 18 (USP18) prior to the next dose. Notably, USP18 is the major negative regulator of type I IFN signaling. HIV infection resulted in increased ISG expression levels in humanized mice. Intriguingly, high baseline ISG levels correlated with lower HIV load. No effect was observed on HIV replication when PASylated IFN-α14 was administered in the chronic phase. However, combined antiretroviral therapy (cART) restored responsiveness to IFN, and PASylated IFN-α14 administered during analytical cART interruption resulted in a transiently lower HIV burden than in the mock-treated mice. In conclusion, cART-mediated HIV suppression restored transient IFN responsiveness and provided a potential window for immunoenhancing therapies in the context of analytical cART interruption. IMPORTANCE cART is highly efficient in suppressing HIV replication in HIV-infected patients and has resulted in a dramatic reduction in morbidity and mortality in HIV-infected people, yet it does not cure HIV infection. In addition, cART has several disadvantages. Thus, the HIV research community is exploring novel ways to control HIV infection for longer periods without cART. Here, we explored novel, long-acting IFN-α14 for its efficacy to control HIV replication in HIV-infected humanized mice. We found that IFN-α14 had no effect on chronic HIV infection. However, when mice were treated first with cART, we observed a transiently restored responsiveness to INF and a transiently lower HIV burden after stopping cART. These data emphasize (i) the value of cART-mediated HIV suppression and immune reconstitution in creating a window of opportunity for exploring novel immunotherapies, (ii) the potential of IFNs for constraining HIV, and (iii) the value of humanized mice for exploring novel immunotherapies.

Immunophenotypic characterization of TCR γδ T cells and MAIT cells in HIV-infected individuals developing Hodgkin's lymphoma.

BACKGROUND: Despite successful combined antiretroviral therapy (cART), the risk of non-AIDS defining cancers (NADCs) remains higher for HIV-infected individuals than the general population. The reason for this increase is highly disputed. Here, we hypothesized that T-cell receptor (TCR) γδ cells and/or mucosal-associated invariant T (MAIT) cells might be associated with the increased risk of NADCs. γδ T cells and MAIT cells both serve as a link between the adaptive and the innate immune system, and also to exert direct anti-viral and anti-tumor activity. METHODS: We performed a longitudinal phenotypic characterization of TCR γδ cells and MAIT cells in HIV-infected individuals developing Hodgkin's lymphoma (HL), the most common type of NADCs. Cryopreserved PBMCs of HIV-infected individuals developing HL, matched HIV-infected controls without (w/o) HL and healthy controls were used for immunophenotyping by polychromatic flow cytometry, including markers for activation, exhaustion and chemokine receptors. RESULTS: We identified significant differences in the CD4+ T cell count between HIV-infected individuals developing HL and HIV-infected matched controls within 1 year before cancer diagnosis. We observed substantial differences in the cellular phenotype mainly between healthy controls and HIV infection irrespective of HL. A number of markers tended to be different in Vδ1 and MAIT cells in HIV+HL+ patients vs. HIV+ w/o HL patients; notably, we observed significant differences for the expression of CCR5, CCR6 and CD16 between these two groups of HIV+ patients. CONCLUSION: TCR Vδ1 and MAIT cells in HIV-infected individuals developing HL show subtle phenotypical differences as compared to the ones in HIV-infected controls, which may go along with functional impairment and thereby may be less efficient in detecting and eliminating malignant cells. Further, our results support the potential of longitudinal CD4+ T cell count analysis for the identification of patients at higher risk to develop HL.

Systems-based analysis of Arabidopsis leaf growth reveals adaptation to water deficit.

Leaves have a central role in plant energy capture and carbon conversion and therefore must continuously adapt their development to prevailing environmental conditions. To reveal the dynamic systems behaviour of leaf development, we profiled Arabidopsis leaf number six in depth at four different growth stages, at both the end-of-day and end-of-night, in plants growing in two controlled experimental conditions: short-day conditions with optimal soil water content and constant reduced soil water conditions. We found that the lower soil water potential led to reduced, but prolonged, growth and an adaptation at the molecular level without a drought stress response. Clustering of the protein and transcript data using a decision tree revealed different patterns in abundance changes across the growth stages and between end-of-day and end-of-night that are linked to specific biological functions. Correlations between protein and transcript levels depend on the time-of-day and also on protein localisation and function. Surprisingly, only very few of >1700 quantified proteins showed diurnal abundance fluctuations, despite strong fluctuations at the transcript level.

pep2pro: a new tool for comprehensive proteome data analysis to reveal information about organ-specific proteomes in Arabidopsis thaliana.

pep2pro is a comprehensive proteome analysis database specifically suitable for flexible proteome data analysis. The pep2pro database schema offers solutions to the various challenges of developing a proteome data analysis database and because data integrated in pep2pro are in relational format, it enables flexible and detailed data analysis. The information provided here will facilitate building proteome data analysis databases for other organisms or applications. The capacity of the pep2pro database for the integration and analysis of large proteome datasets was demonstrated by creating the pep2pro dataset, which is an organ-specific characterisation of the Arabidopsis thaliana proteome containing 14 522 identified proteins based on 2.6 million peptide spectrum assignments. This dataset provides evidence of protein expression and reveals organ-specific processes. The high coverage and density of the dataset are essential for protein quantification by normalised spectral counting and allowed us to extract information that is usually not accessible in low-coverage datasets. With this quantitative protein information we analysed organ- and organelle-specific sub-proteomes. In addition we matched spectra to regions in the genome that were not predicted to have protein coding capacity and provide PCR validation for selected revised gene models. Furthermore, we analysed the peptide features that distinguish detected from non-detected peptides and found substantial disagreement between predicted and detected proteotypic peptides, suggesting that large-scale proteomics data are essential for efficient selection of proteotypic peptides in targeted proteomics surveys. The pep2pro dataset is available as a resource for plant systems biology at www.pep2pro.ethz.ch.

Proteome dynamics during plastid differentiation in rice.

We have analyzed proteome dynamics during light-induced development of rice (Oryza sativa) chloroplasts from etioplasts using quantitative two-dimensional gel electrophoresis and tandem mass spectrometry protein identification. In the dark, the etioplast allocates the main proportion of total protein mass to carbohydrate and amino acid metabolism and a surprisingly high number of proteins to the regulation and expression of plastid genes. Chaperones, proteins for photosynthetic energy metabolism, and enzymes of the tetrapyrrole pathway were identified among the most abundant etioplast proteins. The detection of 13 N-terminal acetylated peptides allowed us to map the exact localization of the transit peptide cleavage site, demonstrating good agreement with the prediction for most proteins. Based on the quantitative etioplast proteome map, we examined early light-induced changes during chloroplast development. The transition from heterotrophic metabolism to photosynthesis-supported autotrophic metabolism was already detectable 2 h after illumination and affected most essential metabolic modules. Enzymes in carbohydrate metabolism, photosynthesis, and gene expression were up-regulated, whereas enzymes in amino acid and fatty acid metabolism were significantly decreased in relative abundance. Enzymes involved in nucleotide metabolism, tetrapyrrole biosynthesis, and redox regulation remained unchanged. Phosphoprotein-specific staining at different time points during chloroplast development revealed light-induced phosphorylation of a nuclear-encoded plastid RNA-binding protein, consistent with changes in plastid RNA metabolism. Quantitative information about all identified proteins and their regulation by light is available in plprot, the plastid proteome database (http://www.plprot.ethz.ch).

The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions.

BACKGROUND: Chloroplasts are plant cell organelles of cyanobacterial origin. They perform essential metabolic and biosynthetic functions of global significance, including photosynthesis and amino acid biosynthesis. Most of the proteins that constitute the functional chloroplast are encoded in the nuclear genome and imported into the chloroplast after translation in the cytosol. Since protein targeting is difficult to predict, many nuclear-encoded plastid proteins are still to be discovered. RESULTS: By tandem mass spectrometry, we identified 690 different proteins from purified Arabidopsis chloroplasts. Most proteins could be assigned to known protein complexes and metabolic pathways, but more than 30% of the proteins have unknown functions, and many are not predicted to localize to the chloroplast. Novel structure and function prediction methods provided more informative annotations for proteins of unknown functions. While near-complete protein coverage was accomplished for key chloroplast pathways such as carbon fixation and photosynthesis, fewer proteins were identified from pathways that are downregulated in the light. Parallel RNA profiling revealed a pathway-dependent correlation between transcript and relative protein abundance, suggesting gene regulation at different levels. CONCLUSIONS: The chloroplast proteome contains many proteins that are of unknown function and not predicted to localize to the chloroplast. Expression of nuclear-encoded chloroplast genes is regulated at multiple levels in a pathway-dependent context. The combined shotgun proteomics and RNA profiling approach is of high potential value to predict metabolic pathway prevalence and to define regulatory levels of gene expression on a pathway scale.

Residual cell-associated unspliced HIV-1 RNA in peripheral blood of patients on potent antiretroviral therapy represents intracellular transcripts.

Unspliced HIV-RNA (HIV-UsRNA) associated with peripheral blood mononuclear cells (PBMCs) persists in patients on potent antiretroviral therapy even in the absence of detectable plasma HIV-RNA. To further characterize such residual HIV-RNA, cell-associated virion-encapsidated HIV and intracellular unspliced HIV-RNA were differentiated and monitored using a novel highly sensitive method. In addition, expression of HIV-mRNA encoding tat and rev was assessed. PBMCs of patients with unsuppressed plasma viraemia harboured an extracellular fraction of HIV-UsRNA, which correlated highly with intracellular HIV-RNA levels. Thus, extracellular PBMC-associated HIV-RNA may, to a significant extent, reflect nascent virions attached to productively infected cells. Upon treatment with potent antiretroviral therapy resulting in plasma viraemia <50 copies/ml, expression of cell-associated viral particles was hardly discernible in PBMCs but transcription of unspliced HIV-RNA persisted. Given the virtual absence of rev-mRNA, translation of residual HIV-UsRNA was probably precluded by retention of these transcripts in the nucleus. As shown by limiting dilution analysis, HIV-1 infected cells with such a repressed viral transcription pattern were observed at high frequencies in PBMC from untreated patients.