Dr Marine J. Petit

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.

Eukaryotic translation initiation factor SA (eIF5A) is essential for cell proliferation, becoming functionally active only after post-translational conversion of a specific Lys to hypusine [N-epsilon-(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 alpha-difluoromethylornithine (inhibitor of polyamine biosynthesis) and (R)-3-MeSpd did not cause depletion of hypusinated eIFSA, and the cells were still able to grow, whereas the combination of alpha-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 [C-14]-labeled spermidine for cellular uptake less efficiently than the (S)-3-MeSpd (K-i = 141 mu M vs 19 mu M, 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.

Author summaryViruses rely on cellular metabolism for productive infection. How viruses interact with and manipulate metabolism impacts viral replication and pathogenesis. Metabolic pathways are frequently interconnected, and perturbation of one pathway can have wide-ranging impacts on other metabolites within the cell. Here, we show that polyamines, small positively-charged metabolites critical to virus infection, regulate cholesterol synthesis, which ultimately impacts viral attachment. We find that polyamines' role in translation is critical to their regulation of cholesterol synthesis. These data have important implications for the connections between polyamine and sterol synthesis as well as how these molecules impact virus replication. 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.

Ticks are vectors of arboviruses in many parts of the world. The rising incidence and emergence of tick-borne arboviral infections across human populations indicates that further transmission control strategies including those based on vectors, will be required to reduce the burden of disease. However, arbovirus-tick interactions at the cellular level remain poorly understood in general, and particularly neglected for negative strand RNA arboviruses.  In this study we developed a proteomics informed by transcriptomics approach to characterize the cellular response of Rhipicephalus microplus-derived cell cultures to infection with the tick-borne pathogen severe fever with thrombocytopenia syndrome virus (SFTSV, Phenuiviridae). For this, we generated the first de novo transcriptomes and confirmed proteomes of SFTSV- or mock-infected tick cell cultures derived from a vector species that transmits the virus in nature. Through comprehensive annotation of genes, proteins and pathway analysis, we identified core host responses and regulatory processes mediated in response to SFTSV infection. Moreover, examining the interactome of the virally encoded nucleoprotein (N) allowed us to integrate host responses with the analysis of cellular factors required for viral replication. The influence of specific host genes on SFTSV replication was systematically assessed through dsRNA-mediated gene silencing. This functional genomics approach pinpointed two tick-derived RNA helicases as critical antiviral factors capable of restricting SFTSV infection: the DexD/box helicase (DHX9) and the Up-Frameshift Protein 1 (UPF1). Collectively, our findings enrich the repository of resources available for understanding the antiviral response to SFTSV infection in Rh. microplus vector cells and support the identification of SFTSV-antiviral restrictions factors. 

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.

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.

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.

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.