Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination.

Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.

Regulation of kynurenine biosynthesis during influenza virus infection.

Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control modalities requires better understanding of virus-host interactions. It has previously been shown that IAV induces the production of kynurenine, which suppresses T-cell responses, enhances pain hypersensitivity and disturbs behaviour in infected animals. However, the regulation of kynurenine biosynthesis during IAV infection remains elusive. Here we showed that IAV infection induced expression of interferons (IFNs), which upregulated production of indoleamine-2,3-dioxygenase (IDO1), which catalysed the kynurenine biosynthesis. Furthermore, IAV attenuated the IDO1 expression and the production of kynurenine through its NS1 protein. Interestingly, inhibition of viral replication prior to IFN induction limited IDO1 expression, while inhibition after did not. Finally, we showed that kynurenine biosynthesis was activated in macrophages in response to other stimuli, such as influenza B virus, herpes simplex virus 1 and 2 as well as bacterial lipopolysaccharides. Thus, the tight regulation of the kynurenine biosynthesis by host cell and, perhaps, pathogen might be a basic signature of a wide range of host-pathogen interactions, which should be taken into account during development of novel antiviral and antibacterial drugs.

Multi-Omics Studies towards Novel Modulators of Influenza A Virus-Host Interaction.

Human influenza A viruses (IAVs) cause global pandemics and epidemics. These viruses evolve rapidly, making current treatment options ineffective. To identify novel modulators of IAV-host interactions, we re-analyzed our recent transcriptomics, metabolomics, proteomics, phosphoproteomics, and genomics/virtual ligand screening data. We identified 713 potential modulators targeting 199 cellular and two viral proteins. Anti-influenza activity for 48 of them has been reported previously, whereas the antiviral efficacy of the 665 remains unknown. Studying anti-influenza efficacy and immuno/neuro-modulating properties of these compounds and their combinations as well as potential viral and host resistance to them may lead to the discovery of novel modulators of IAV-host interactions, which might be more effective than the currently available anti-influenza therapeutics.

Influenza virus NS1 protein binds cellular DNA to block transcription of antiviral genes.

Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication.

JNJ872 inhibits influenza A virus replication without altering cellular antiviral responses.

JNJ-63623872 (formally known as VX-787; referred to here as JNJ872) is an orally bioavailable compound, which is in phase II clinical trials for the treatment of influenza A virus (IAV) infections. Here we show that JNJ872 inhibits at nanomolar concentrations the transcription of viral RNA in IAV-infected human macrophages by targeting a highly conserved site on the cap-snatching domain of influenza polymerase basic 2 protein (PB2). Furthermore, even lower concentrations of JNJ872 protected macrophages from IAV-mediated death when given in combination with 100 nM gemcitabine, which also attenuated transcription and replication of viral RNA. Importantly, treating human macrophages with JNJ872 allowed expression of many immune-related and other genes, involved in antiviral responses, such as indoleamine 2,3-dioxygenase 1 (IDO), and cytosolic 5'-nucleotidase 3A (NT5C3A). Moreover, our targeted metabolomics analysis indicate that treatment with JNJ782 did not interfere with metabolic responses to infection, which further supported our transcriptomics results. Thus, VX-737 alone or in combination with other drugs could be beneficial for treating IAV infected patients, because it would allow the development of antiviral responses and, thereby, protect individuals from current and future infections with closely related IAV strains.

Protein profiling of nasopharyngeal aspirates of hospitalized and outpatients revealed cytokines associated with severe influenza A(H1N1)pdm09 virus infections: A pilot study.

Influenza A viruses (IAV) mutate rapidly and cause seasonal epidemics and occasional pandemics, which result in substantial number of patient visits to the doctors and even hospitalizations. We aimed here to identify inflammatory proteins, which levels correlated to clinical severity of the disease. For this we analysed 102 cytokines and growth factors in human nasopharyngeal aspirate (NPA) samples of 27 hospitalized and 27 outpatients diagnosed with influenza A(H1N1)pdm09 virus infection. We found that the relative levels of monocyte differentiation antigen CD14, lipocalin-2 (LCN2), C-C-motif chemokine 20 (CCL20), CD147, urokinase plasminogen activator surface receptor (uPAR), pro-epidermal growth factor (EGF), trefoil factor 3 (TFF3), and macrophage migration inhibitory factor (MIF) were significantly lower (p<0.008), whereas levels of retinol-binding protein 4 (RBP4), C-X-C motif chemokine 5 (CXCL5), interleukin-8 (IL-8), complement factor D (CFD), adiponectin, and chitinase-3-like 1 (CHI3L1) were significantly higher (p<0.008) in NPA samples of hospitalized than non-hospitalized patients. While changes in CD14, LCN2, CCL20, uPAR, EGF, MIF, CXCL5, IL-8, adiponectin and CHI3L1 levels have already been correlated with severity of IAV infection in mice and humans, our study is the first to describe association of CD147, RBP4, TFF3, and CFD with hospitalization of IAV-infected patients. Thus, we identified local innate immune profiles, which were associated with the clinical severity of influenza infections.