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All organisms sense and respond to pathogenic challenge. Tissue-specific responses are required to combat pathogens infecting distinct cell types. Cyclic dinucleotides (CDNs) are produced endogenously downstream of pathogen recognition or by pathogens themselves which bind to STING to activate NF-kB-dependent antimicrobial gene expression programs. It remains unknown whether there are distinct immune responses to CDNs in Drosophila tissues. Here, we investigated tissue specific CDN-STING responses and uncovered differences in gene-induction patterns across tissues that play important roles in viral infections. Using tissue-and cell-specific genetic studies we found that dSTING in the fat body controls CDN-induced expression of dSTING-regulated gene 1 ( Srg1 ) but not dSTING-regulated gene 2 ( Srg2 ) or 3 ( Srg3 ). In contrast, the gastrointestinal tract largely controls expression of Srg2 and Srg3 . We found that Srg3 is antiviral against the natural fly pathogen Drosophila C virus and the human arthropod-borne Rift Valley Fever virus (RVFV), but not other arthropod-borne viruses including Sindbis virus and dengue virus. Furthermore, we found that Srg3 has an important role in controlling RVFV infection of the ovary which has important implications in understanding vertical transmission of viruses and RVFV in mosquitoes. Overall, our study underscores the importance of tissue-specific responses in antiviral immunity and highlights the complex tissue regulation of the CDN-STING pathway.

Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cell-intrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi) pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV), a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts.

A unique facet of arthropod-borne virus (arbovirus) infection is that the pathogens are orally acquired by an insect vector during the taking of a blood meal, which directly links nutrient acquisition and pathogen challenge. We show that the nutrient responsive ERK pathway is both induced by and restricts disparate arboviruses in Drosophila intestines, providing insight into the molecular determinants of the antiviral “midgut barrier.” Wild-type flies are refractory to oral infection by arboviruses, including Sindbis virus and vesicular stomatitis virus, but this innate restriction can be overcome chemically by oral administration of an ERK pathway inhibitor or genetically via the specific loss of ERK in Drosophila intestinal epithelial cells. In addition, we found that vertebrate insulin, which activates ERK in the mosquito gut during a blood meal, restricts viral infection in Drosophila cells and against viral invasion of the insect gut epithelium. We find that ERK’s antiviral signaling activity is likely conserved in Aedes mosquitoes, because genetic or pharmacologic manipulation of the ERK pathway affects viral infection of mosquito cells. These studies demonstrate that ERK signaling has a broadly antiviral role in insects and suggest that insects take advantage of cross-species signals in the meal to trigger antiviral immunity.

Dietary self-monitoring is considered the core of behavioral weight control programs. As software for personal digital assistants (PDA) has become more available, this study investigated whether the use of a PDA would improve dietary self-monitoring frequency and subsequent weight loss over the use of traditional paper diaries. One-hundred-seventy-six adults (BMI 25-39.9) participated in a 6-month behavioral weight control program. Treatment subjects (n = 61) were provided with a PalmZire 21 with Calorie King's Diet Diary software installed. Their self-monitoring habits and weight loss were compared with the results from a previous program (n = 115) which followed the same protocol using paper diaries for self-monitoring. No significant differences in weight loss or dietary self-monitoring were found. More frequent self-monitoring correlated with weight loss in both groups (p<.001). People seeking to lose weight should be encouraged to self-monitor and be matched with a mode of self-monitoring that is fitting to their lifestyle and skills.

Upon infection, pathogen recognition leads to a rapidly activated gene expression program that induces antimicrobial effectors to clear the invader. We recently found that Nup98 regulates the expression of a subset of rapidly activated antiviral genes to restrict disparate RNA virus infections in Drosophila by promoting RNA polymerase occupancy at the promoters of these antiviral genes. How Nup98 specifically targets these loci was unclear; however, it is known that Nup98 participates with transcription factors to regulate developmental-gene activation. We reasoned that additional transcription factors may facilitate the Nup98-dependent expression of antiviral genes. In a genome-wide RNA interference (RNAi) screen, we identified a relatively understudied forkhead transcription factor, FoxK, as active against Sindbis virus (SINV) in Drosophila . Here we find that FoxK is active against the panel of viruses that are restricted by Nup98, including SINV and vesicular stomatitis virus (VSV). Mechanistically, we show that FoxK coordinately regulates the Nup98-dependent expression of antiviral genes. Depletion of FoxK significantly reduces Nup98-dependent induction of antiviral genes and reduces the expression of a forkhead response element-containing luciferase reporter. Together, these data show that FoxK-mediated activation of gene expression is Nup98 dependent. We extended our studies to mammalian cells and found that the mammalian ortholog FOXK1 is antiviral against two disparate RNA viruses, SINV and VSV, in human cells. Interestingly, FOXK1 also plays a role in the expression of antiviral genes in mammals: depletion of FOXK1 attenuates virus-inducible interferon-stimulated response element (ISRE) reporter expression. Overall, our results demonstrate a novel role for FOXK1 in regulating the expression of antiviral genes, from insects to humans. IMPORTANCE Innate immunity is characterized by rapid gene expression programs, from insects to mammals. Furthermore, we find that Nup98, known for its roles in the nuclear pore, plays a noncanonical role in binding the promoters and poising a subset of loci for rapid antiviral gene induction. It was unclear how Nup98 accesses these specific genes, and we here demonstrate that Nup98 cooperates with the transcription factor FoxK to regulate this gene expression program. Depletion of FoxK specifically reduces the induction of Nup98-dependent genes. Further, we find that the antiviral function of FoxK is conserved, as the human ortholog FOXK1 is also antiviral and regulates gene expression from virus-induced promoters. Although other forkhead transcription factors have been implicated in immunity, a role for FoxK in antiviral defense was previously unappreciated. Our findings reveal a conserved and novel role for FoxK in coordinating with Nup98 to promote a robust and complex antiviral transcriptional response. Innate immunity is characterized by rapid gene expression programs, from insects to mammals. Furthermore, we find that Nup98, known for its roles in the nuclear pore, plays a noncanonical role in binding the promoters and poising a subset of loci for rapid antiviral gene induction. It was unclear how Nup98 accesses these specific genes, and we here demonstrate that Nup98 cooperates with the transcription factor FoxK to regulate this gene expression program. Depletion of FoxK specifically reduces the induction of Nup98-dependent genes. Further, we find that the antiviral function of FoxK is conserved, as the human ortholog FOXK1 is also antiviral and regulates gene expression from virus-induced promoters. Although other forkhead transcription factors have been implicated in immunity, a role for FoxK in antiviral defense was previously unappreciated. Our findings reveal a conserved and novel role for FoxK in coordinating with Nup98 to promote a robust and complex antiviral transcriptional response.

Arthropod-borne viruses are diverse pathogens and are associated with human disease. Through high-throughput drug screening, we found that sirtuin inhibitors are potently antiviral against diverse arboviruses, including flaviviruses such as West Nile virus, bunyaviruses such as Rift Valley fever virus, and alphaviruses such as chikungunya virus. Sirtuin inhibitors block infection of these viruses in multiple human cell types. Moreover, we found that sirtuin inhibitors arrest infection downstream of entry but that they do so at an early step, preventing the accumulation of viral RNA and protein. Since these viruses infect vector insects, we also tested whether sirtuin inhibitors impacted infection of adult flies and found that these inhibitors blocked infection; therefore, they target highly conserved facets of replication. Taken together, these results suggest that sirtuin inhibitors represent a new class of potent host-targeting antivirals. Arthropod-borne viruses are diverse pathogens and are often associated with human disease. These viruses span multiple genera, including flaviviruses, alphaviruses, and bunyaviruses. In a high-throughput drug screen, we found that tenovin-1 was antiviral against the flaviviruses Zika virus and dengue virus. Tenovin-1 is a sirtuin inhibitor, and here we found that inhibition of sirtuins, but not inhibition of the related histone deacetylases, is potently antiviral against diverse arboviruses. Sirtuin inhibitors block infection of arboviruses in multiple human cell types. We found that sirtuin inhibitors arrest infection downstream of entry but that they do so at an early step, preventing the accumulation of viral RNA and protein. However, sirtuin inhibitors had no impact on the replication of flaviviral replicons, suggesting a defect in the establishment of replication. Consistent with this, we found that sirtuin inhibitors impacted double-stranded RNA (dsRNA) accumulation during flaviviral infection. Since these viruses infect vector insects, we also tested whether sirtuin inhibitors impacted infection of adult flies and found that these inhibitors blocked infection; therefore, they target highly conserved facets of replication. Taken together, these results suggest that sirtuin inhibitors represent a new class of potent host-targeting antivirals. IMPORTANCE Arthropod-borne viruses are diverse pathogens and are associated with human disease. Through high-throughput drug screening, we found that sirtuin inhibitors are potently antiviral against diverse arboviruses, including flaviviruses such as West Nile virus, bunyaviruses such as Rift Valley fever virus, and alphaviruses such as chikungunya virus. Sirtuin inhibitors block infection of these viruses in multiple human cell types. Moreover, we found that sirtuin inhibitors arrest infection downstream of entry but that they do so at an early step, preventing the accumulation of viral RNA and protein. Since these viruses infect vector insects, we also tested whether sirtuin inhibitors impacted infection of adult flies and found that these inhibitors blocked infection; therefore, they target highly conserved facets of replication. Taken together, these results suggest that sirtuin inhibitors represent a new class of potent host-targeting antivirals.

Drosophila use small‐interfering RNA mechanisms to limit the amplification of viral genomes. However, it is unclear how small RNA interference components recognize and separate viral from cellular RNA. Dnmt2 enzymes are highly conserved RNA methyltransferases with substrate specificity towards cellular tRNAs. We report here that Dnmt2 is required for efficient innate immune responses in Drosophila . Dnmt2 mutant flies accumulate increasing levels of Drosophila C virus and show activated innate immune responses. Binding of Dnmt2 to DCV RNA suggests that Dnmt2 contributes to virus control directly, possibly by RNA methylation. These observations demonstrate a role for Dnmt2 in antiviral defence. Adult Dnmt2 mutants accumulate increasing levels of Drosophila C virus and activate stress and immune responses. This work describes a role of this highly conserved tRNA methyltransferases Dnmt2 for efficient innate immunity in Drosophila .

Significance The innate immune system is a highly conserved mode of defense that induces gene expression programs to restrict microbial infections. However, much remains unknown about how the target genes are poised for their rapid induction. Using a Drosophila model, we demonstrate that Nup98 plays an essential antiviral role in insects against human insect-borne viruses. Although Nup98 is known for its role in nuclear-cytoplasmic transport, our data suggest that this antiviral function is not at the nuclear pore, rather at promoters controlling expression of a subset of virus-induced genes. Our findings suggest that the Nup98 primes virus-stimulated genes by regulating the occupancy of active RNA polymerase at these promoters poising them for rapid induction, thereby coordinating a robust and complex antiviral response.

Lipid-derived prodrugs offer a strategy to improve the oral bioavailability of nucleos(t)ide antiretrovirals by enabling their absorption into the lymphatic system, providing access to HIV reservoirs such as lymph nodes, lung, and gut-associated lymphoid tissue. We hypothesized that chylomicrons (CMs), lipoproteins responsible for the transport of dietary lipids into the lymphatics, may facilitate the distribution of these lipidic drugs through this pathway.To test this hypothesis, this dissertation presents the development of a novel in vitro assay that models CM-mediated drug transport, enabling direct measurement of prodrug incorporation into Caco-2 cell-derived CMs. Using this platform, a series of lipid prodrugs of tenofovir (TFV)— a widely used antiretroviral agent—was evaluated. These prodrugs featured a benzyloxyglycerol (BOG) linker in the lipid chain and/or an ω-trifluoromethyl (CF3) motif to enhance lipophilicity, thereby promoting incorporation into CMs, and metabolic stability, ensuring maximal systemic delivery of these agents. In vitro, these modifications improved antiviral potency and resistance to metabolism compared to tenofovir exalidex (TXL), an exploratory unfunctionalized lipid prodrug of TFV. Moreover, we observed that prodrug incorporation into CMs in vitro correlated strongly with lipophilicity, with BOG and ω-CF3 derivatives exhibiting the highest levels of CM-association relative to TXL.To assess the translatability of this assay in vivo, we carried out a pharmacokinetic (PK) study in mice. This experiment revealed that the more lipophilic prodrugs achieved significantly higher systemic exposure levels and enhanced lung delivery when compared with TXL. Given that lymph drains into the venous system upstream of pulmonary circulation, lung concentrations were used as a surrogate marker for lymphatic transport. These data offer the first direct evidence of drug packaging into cell-derived CMs and demonstrate that CM uptake in vitro correlates with lymphatic distribution in vivo. Together, this work establishes a new framework to study lymphatic drug delivery. Moreover, it supports the development of our lipid prodrugs of TFV as potentially affordable, orally available therapies that target HIV-enriched tissues and improve adherence, particularly in resource-limited settings.

Internet-based weight-loss programs appear promising in the short-term but, to date, have not been able to produce the level of weight loss seen in traditional in-person treatment; thus, novel approaches are necessary. Using a combination of interactive technology and in-person support has been beneficial in other areas of medicine. The aim of this study is to compare 12-month weight-loss outcomes of an Internet-only behavioral weight-loss treatment with the same program supplemented with monthly in-person meetings. One hundred and twenty-three participants were randomized to an Internet-only (n = 62) or an Internet + in-person treatment (I+IPS; n = 61). All participants then participated in a 12-month behavioral weight-loss program conducted over the Internet. The groups met online weekly for the first 6 months and biweekly for the second half of the intervention. The I+IPS group had access to the same Web site as the Internet-only group but, once a month, attended an in-person meeting in place of an online chat. Assessments included body weight, program adherence, and social support measures. An intent-to-treat analysis (n = 123) revealed there were no significant Group x Time differences (p = .15) in weight loss at either 6 (-6.8 +/- 7.8 vs. -5.1 +/- 4.8, p = .15) or 12 months (-5.1 +/- 7.1 kg vs. -3.5 +/- 5.1 kg, p = .17, for Internet-only and I+IPS, respectively). Differences between groups for those completing all measures (n = 77) also revealed no significant differences at 6 months (-9.2 +/- 7.0 kg vs. -6.9 +/- 4.2 kg, p = .08) or 12 months (-8.0 +/- 7.5 kg vs. -5.6 +/- 5.5 kg, p = .10 for the Internet-only and I+IPS conditions, respectively). Supplementation of an Internet weight-loss treatment with monthly in-person meetings did not result in greater weight losses over 12 months. Dynamic, socially supportive, and interactive elements of the Web site may have obviated the need for further interpersonal behavioral counseling.