Virus-host interactions are a delicate interplay of opposing forces: the virus attempts to subvert cellular machinery to aid in its replication while the host mounts an immune response to eliminate the infection. The goal of this thesis is to take advantage of the powerful genetic tools in Drosophila to identify novel cell-intrinsic innate immune pathways that restrict arboviral infections, thereby contributing to our knowledge of virus- host interactions for pathogens that cause emerging infectious illnesses.
By performing a cell-based RNAi screen against a panel of disparate arboviruses, we identified broadly antiviral candidates for further investigation. In
Chapter 3, we explore the role of two of these genes belong to the NELF complex, called NELF-B and NELF-D, which led us to discover a novel antiviral transcriptional gene program that is activated during early infection. Innate immune responses are characterized by precise gene expression, whereby gene subsets are temporally induced to limit infection. The current paradigm for orchestrating such innate immune responses is at the step of transcription initiation. Controlling innate response genes at alternate steps in the transcription cycle has not been well explored. We found that antiviral immunity in Drosophila requires an alternate gene regulatory mechanism, called transcriptional pausing. Depletion of components of this pathway, including negative elongation factor (NELF) that pauses RNA polymerase II (Pol II) and positive elongation factor b (P-TEFb), which releases paused Pol II to produce full-length transcripts, resulted in increased viral infection in Drosophila cells. This led us to identify a set of genes that is rapidly transcribed upon arbovirus infection, including components of antiviral pathways (RNA silencing, autophagy, JAK/STAT, Toll, and IMD) and various Toll receptors. Many of these genes require P-TEFb for expression and exhibit pausing- associated chromatin features. Furthermore, transcriptional pausing is critical for antiviral immunity in insects, since NELF and P-TEFb are required to restrict viral replication in adult flies and vector mosquito cells. Thus, transcriptional pausing primes virally induced genes to facilitate rapid gene induction and robust antiviral immunity.
In Chapter 4, we explored a subset of genes that are transcriptionally induced during early viral infection, which led us to discover that the ERK pathway is involved in antiviral defenses of disparate insects. A unique facet of arthropod-borne virus
taking of a blood meal. Hence, there is a direct link between nutrient acquisition and pathogen challenge. A coupling of this nutrient rich signal with host defense would likely benefit the organism. We found that the nutrient responsive ERK pathway is both induced by and restricts disparate viral infections, including human arboviruses, in
Drosophila and mosquito cells. Furthermore, ERK signaling is essential for antiviral defense in the insect intestinal epithelium. While wild type flies are refractory to oral infection by arboviruses including Sindbis virus (SINV) and Vesicular Stomatitis virus (VSV), this innate restriction can be overcome chemically by oral administration of an ERK pathway inhibitor or genetically via the specific loss of ERK in the intestinal epithelial cells. Either treatment results in robust viral infection of the gut. Furthermore, vertebrate insulin that activates ERK signaling in the mosquito gut during a blood meal [150] can both restrict viral infection in insect cells and protect against viral invasion of the gut epithelium. These studies collectively demonstrate that ERK signaling in the insect intestines potently restricts viral infection, suggesting that insects take advantage of signals in the meal to preemptively activate antiviral immunity.
Figure 1. Probable temporal sequence and dispersal routes of WNV from its proposed center of origin in sub-Saharan Africa [1,151]. WNV is distributed
circumglobally, with two main genetic lineages: Lineage 1 is widely distributed and highly invasive, whereas Lineage 2 remains in Africa. Sub clades of Lineage 1 are widespread throughout Africa and the Mediterranean: Lineage 1b (aka Kunjin virus) is restricted to Australia and Lineage 1c is found in Central Asia through the central highlands of India.
Figure 2. Global distribution of Dengue Virus infection in 2012. Blue regions represent areas of ongoing transmission risk as defined by the Centers for Disease Control and Prevention (CDC). Red markers indicate recent reports of local and regional Dengue or imported cases of Dengue.
Figure 3. Generalized Arboviral Transmission Cycle. Arboviral infection alternates between vertebrate reservoirs and arthropod vectors. The insect will bite the vertebrate host and infect it, and then later, another insect will bite that same host, contract the infection, and continue to spread it to other vertebrate hosts. This type of life cycle comes to a dead end if an insect carries the infection to a human or animal; once there, the infection stops reproducing and does not re-transmit to a new host.
Figure 4. General workflow for arrayed cell-based RNAi screening. In brief,
Drosophila cells are plated in a 384-well format with spotted dsRNA. The cells are incubated for 3 days to allow for efficient depletion of the genes-of-interest. Then, cells are infected with virus. Immunofluorescence and automated microscopy are performed to measure the percentage of infected cells. Statistical analysis is used to identify viral permissivity and restriction factors.
Figure 5. The heat shock-inducible UAS/Gal4 system for In vivo RNAi in
Drosophila. In F1 progeny, heat shock at 37°C will induce the expression of Gal4, which
binds to the UAS element to activate the synthesis of the inverted repeat sequence. The inverted repeat forms a snapback that is processed by the RNAi machinery to deplete the gene-of-interest.
Figure 6. Antiviral pathways in Drosophila.2This figure highlights the major antiviral
pathways in insects: Toll, IMD, JAK/STAT, autophagy, and RNA interference pathways.
2 Adapted from Sabin LR, Hanna SL, Cherry S. Innate Immunity in Drosophila. Curr Opin Immunol. 2010 Feb;22(1):4-9.