Polyamines and eIF5A hypusination facilitate SREBP2 synthesis and cholesterol production leading to enhanced enterovirus attachment and infection.

Metabolism is key to cellular processes that underlie the ability of a virus to productively infect. Polyamines are small metabolites vital for many host cell processes including proliferation, transcription, and translation. Polyamine depletion also inhibits virus infection via diverse mechanisms, including inhibiting polymerase activity and viral translation. We showed that Coxsackievirus B3 (CVB3) attachment requires polyamines; however, the mechanism was unknown. Here, we report polyamines' involvement in translation, through a process called hypusination, promotes expression of cholesterol synthesis genes by supporting SREBP2 synthesis, the master transcriptional regulator of cholesterol synthesis genes. Measuring bulk transcription, we find polyamines support expression of cholesterol synthesis genes, regulated by SREBP2. Thus, polyamine depletion inhibits CVB3 by depleting cellular cholesterol. Exogenous cholesterol rescues CVB3 attachment, and mutant CVB3 resistant to polyamine depletion exhibits resistance to cholesterol perturbation. This study provides a novel link between polyamine and cholesterol homeostasis, a mechanism through which polyamines impact CVB3 infection.

Transcriptomic profiling implicates PAF1 in both active and repressive immune regulatory networks.

BACKGROUND: Sitting at the interface of gene expression and host-pathogen interaction, polymerase associated factor 1 complex (PAF1C) is a rising player in the innate immune response. The complex localizes to the nucleus and associates with chromatin to modulate RNA polymerase II (RNAPII) elongation of gene transcripts. Performing this function at both proximal and distal regulatory elements, PAF1C interacts with many host factors across such sites, along with several microbial proteins during infection. Therefore, translating the ubiquity of PAF1C into specific impacts on immune gene expression remains especially relevant. RESULTS: Advancing past work, we treat PAF1 knockout cells with a slate of immune stimuli to identify key trends in PAF1-dependent gene expression with broad analytical depth. From our transcriptomic data, we confirm PAF1 is an activator of traditional immune response pathways as well as other cellular pathways correlated with pathogen defense. With this model, we employ computational approaches to refine how PAF1 may contribute to both gene activation and suppression. Specifically focusing on transcriptional motifs and regulons, we predict gene regulatory elements strongly associated with PAF1, including those implicated in an immune response. Overall, our results suggest PAF1 is involved in innate immunity at several distinct axes of regulation. CONCLUSIONS: By identifying PAF1-dependent gene expression across several pathogenic contexts, we confirm PAF1C to be a key mediator of innate immunity. Combining these transcriptomic profiles with potential regulatory networks corroborates the previously identified functions of PAF1C. With this, we foster new avenues for its study as a regulator of innate immunity, and our results will serve as a basis for targeted study of PAF1C in future validation studies.

Functional connectivity of the medial prefrontal cortex related to mindreading abilities.

The medial prefrontal cortex is a key region of mindreading belonging to the mentalizing system, a set of brain areas underlying mental state inference based on reasoning on social concepts. The aim of this study was to characterize the functional connectivity between regions involved in mindreading and to highlight the processes it underpins, focusing on the dorsal and ventral parts of the medial prefrontal cortex. We analyzed resting-state functional magnetic resonance imaging of 56 healthy volunteers, to study the relationship between mindreading abilities and functional connectivity of the medial prefrontal cortex. Cognitive mindreading performances were correlated with connectivity between the medial prefrontal cortex and frontal regions involved in the regulation of the salience of one's own mental contents, with a distinction between the dorsal part connected to regions subtending inhibition processes and the ventral part to emotional regions. Affective mindreading performances were negatively correlated with negative connectivity of the ventro- and dorsomedial prefrontal cortex with sensorimotor regions belonging to the mirror neuron system subtending the simulation of mental states. These findings suggested a role of the medial prefrontal cortex to decrease the salience of one's own mental content and in the antisynchronous interaction between the mentalizing and mirror neurons systems.

Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes.

Dengue virus (DENV) disruption of the innate immune response is critical to establish infection. DENV non-structural protein 5 (NS5) plays a central role in this disruption, such as antagonism of STAT2. We recently found that DENV serotype 2 (DENV2) NS5 interacts with Polymerase associated factor 1 complex (PAF1C). The primary members of PAF1C are PAF1, LEO1, CTR9, and CDC73. This nuclear complex is an emerging player in the immune response. It promotes the expression of many genes, including genes related to the antiviral, antimicrobial and inflammatory responses, through close association with the chromatin of these genes. Our previous work demonstrated that NS5 antagonizes PAF1C recruitment to immune response genes. However, it remains unknown if NS5 antagonism of PAF1C is complementary to its antagonism of STAT2. Here, we show that knockout of PAF1 enhances DENV2 infectious virion production. By comparing gene expression profiles in PAF1 and STAT2 knockout cells, we find that PAF1 is necessary to express immune response genes that are STAT2-independent. Finally, we mapped the viral determinants for the NS5-PAF1C protein interaction. We found that NS5 nuclear localization and the C-terminal region of the methyltransferase domain are required for its interaction with PAF1C. Mutation of these regions rescued the expression of PAF1-dependent immune response genes that are antagonized by NS5. In sum, our results support a role for PAF1C in restricting DENV2 replication that NS5 antagonizes through its protein interaction with PAF1C.

Age-Related Differences in Functional and Structural Connectivity in the Spatial Navigation Brain Network.

Spatial navigation involves multiple cognitive processes including multisensory integration, visuospatial coding, memory, and decision-making. These functions are mediated by the interplay of cerebral structures that can be broadly separated into a posterior network (subserving visual and spatial processing) and an anterior network (dedicated to memory and navigation planning). Within these networks, areas such as the hippocampus (HC) are known to be affected by aging and to be associated with cognitive decline and navigation impairments. However, age-related changes in brain connectivity within the spatial navigation network remain to be investigated. For this purpose, we performed a neuroimaging study combining functional and structural connectivity analyses between cerebral regions involved in spatial navigation. Nineteen young (µ = 27 years, σ = 4.3; 10 F) and 22 older (µ = 73 years, σ = 4.1; 10 F) participants were examined in this study. Our analyses focused on the parahippocampal place area (PPA), the retrosplenial cortex (RSC), the occipital place area (OPA), and the projections into the visual cortex of central and peripheral visual fields, delineated from independent functional localizers. In addition, we segmented the HC and the medial prefrontal cortex (mPFC) from anatomical images. Our results show an age-related decrease in functional connectivity between low-visual areas and the HC, associated with an increase in functional connectivity between OPA and PPA in older participants compared to young subjects. Concerning the structural connectivity, we found age-related differences in white matter integrity within the navigation brain network, with the exception of the OPA. The OPA is known to be involved in egocentric navigation, as opposed to allocentric strategies which are more related to the hippocampal region. The increase in functional connectivity between the OPA and PPA may thus reflect a compensatory mechanism for the age-related alterations around the HC, favoring the use of the preserved structural network mediating egocentric navigation. Overall, these findings on age-related differences of functional and structural connectivity may help to elucidate the cerebral bases of spatial navigation deficits in healthy and pathological aging.

Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis.

Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms.

Mapping Arbovirus-Vector Interactions Using Systems Biology Techniques.

Studying how arthropod-borne viruses interact with their arthropod vectors is critical to understanding how these viruses replicate and are transmitted. Until recently, these types of studies were limited in scale because of the lack of classical tools available to study virus-host interaction for non-model viruses and non-model organisms. Advances in systems biology "-omics"-based techniques such as next-generation sequencing (NGS) and mass spectrometry can rapidly provide an unbiased view of arbovirus-vector interaction landscapes. In this mini-review, we discuss how arbovirus-vector interaction studies have been advanced by systems biology. We review studies of arbovirus-vector interactions that occur at multiple time and length scales, including intracellular interactions, interactions at the level of the organism, viral and vector populations, and how new techniques can integrate systems-level data across these different scales.

piRNA pathway is not required for antiviral defense in Drosophila melanogaster.

Since its discovery, RNA interference has been identified as involved in many different cellular processes, and as a natural antiviral response in plants, nematodes, and insects. In insects, the small interfering RNA (siRNA) pathway is the major antiviral response. In recent years, the Piwi-interacting RNA (piRNA) pathway also has been implicated in antiviral defense in mosquitoes infected with arboviruses. Using Drosophila melanogaster and an array of viruses that infect the fruit fly acutely or persistently or are vertically transmitted through the germ line, we investigated in detail the extent to which the piRNA pathway contributes to antiviral defense in adult flies. Following virus infection, the survival and viral titers of Piwi, Aubergine, Argonaute-3, and Zucchini mutant flies were similar to those of wild type flies. Using next-generation sequencing of small RNAs from wild type and siRNA mutant flies, we showed that no viral-derived piRNAs were produced in fruit flies during different types of viral infection. Our study provides the first evidence, to our knowledge, that the piRNA pathway does not play a major role in antiviral defense in adult Drosophila and demonstrates that viral-derived piRNA production depends on the biology of the host-virus combination rather than being part of a general antiviral process in insects.

Enantiomers of 3-methylspermidine selectively modulate deoxyhypusine synthesis and reveal important determinants for spermidine transport.

Eukaryotic translation initiation factor 5A (eIF5A) is essential for cell proliferation, becoming functionally active only after post-translational conversion of a specific Lys to hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Deoxyhypusine synthase (DHS) is the rate-limiting enzyme of this two-step process, and the polyamine spermidine is the only natural donor of the butylamine group for this reaction, which is very conserved-hypusine biosynthesis suffers last when the intracellular spermidine pool is depleted. DHS has a very strict substrate specificity, and only a few spermidine analogs are substrates of the enzyme and can support long-term growth of spermidine-depleted cells. Herein, we compared the biological properties of earlier unknown enantiomers of 3-methylspermidine (3-MeSpd) in deoxyhypusine synthesis, in supporting cell growth and in polyamine transport. Long-term treatment of DU145 cells with α-difluoromethylornithine (inhibitor of polyamine biosynthesis) and (R)-3-MeSpd did not cause depletion of hypusinated eIF5A, and the cells were still able to grow, whereas the combination of α-difluoromethylornithine with a racemate or (S)-3-MeSpd caused cessation of cell growth. Noticeably, DHS preferred the (R)- over the (S)-enantiomer as a substrate. (R)-3-MeSpd competed with [(14)C]-labeled spermidine for cellular uptake less efficiently than the (S)-3-MeSpd (Ki = 141 µM vs 19 µM, respectively). The cells treated with racemic 3-MeSpd accumulated intracellularly mainly (S)-3-MeSpd, but not DHS substrate (R)-3-MeSpd, explaining the inability of the racemate to support long-term growth. The distinct properties of 3-MeSpd enantiomers can be exploited in designing polyamine uptake inhibitors, facilitating drug delivery and modulating deoxyhypusine synthesis.