Mouse adenoviruses (MAdVs) are non-enveloped double stranded DNA viruses. There are two different types of MAdVs with different tropisms or ability to infect particular cells or tissues. MAdV-1 infects macrophages and endothelial cells and causes encephalitis. MAdV-2 infects intestinal cells but causes no disease. Although, the MAdV fiber capsid proteins are important for attachment of the virus to host cells, it is not known if they are the major determinant of tissue tropism in the mouse. To address this question, I use recombination-mediated genetic engineering to make chimeric MAdVs, wherein I keep most of the genome of one strain but replace the fiber protein with that of the other strain. I then use transfection to introduce the DNA of the chimeric virus into a mouse cell line to allow the virus to replicate. I am currently designing and testing the proper chimeric fiber construct that will result in a replication-competent virus. Ultimately, I compare infection of the chimeric virus to that of the parent viruses in intestinal organoids, a tissue culture model that allows us to faithfully test tropism without the need for mouse studies. These experiments may reveal general principles of AdV tropism that will allow us to understand why different human AdVs cause disease in specific organs.

Mouse adenoviruses (MAdV), like human adenoviruses (HAdVs), have specific tissue tropisms. MAdV-1 infects macrophages and vascular endothelial cells, which can result in encephalitis, while MAdV-2 infects epithelial cells of the intestine but does not cause overt disease. The viral protein that determines MAdV tropism is unknown; however, for many viral families it is the viral attachment protein that is critical. For MAdVs, fiber is the viral attachment protein, and the receptors used by MAdV-1 and MAdV-2, although unknown, are distinct. To test whether MAdV receptor usage dictates tissue tropism, I constructed a MAdV-2 chimeric virus, replacing its fiber protein with that of MAdV-1 using a gene-editing recombination system. The chimera was used to infect a 3D culture model of the intestinal epithelium called “enteroids.” As expected, MAdV-1 does not replicate in enteroids and MAdV-2 does, consistent with their in vivo tropisms. Remarkably, the chimera replicated efficiently, indicating that the fiber protein is not the sole determinant of MAdV-2 intestinal tropism. Although fiber is not the main contributor to tropism, its interactions with host factors are still likely important for productive infection. A recent study identified N-acetylglucosamine (GlcNAc) as a specific ligand for MAdV-2 fiber. We have shown that GlcNAc is not the primary receptor for MAdV-2; however, binding to GlcNAc may aid in adhesion of MAdV-2 and penetration through the mouse intestinal mucus layer. To test this hypothesis, I mutated the GlcNAc interacting residues in MAdV-2 fiber to prevent GlcNAc binding. I am currently comparing the infectivity of this mutant virus to wild type MAdV-2 in both epithelial tumor cells and enteroids. Unlike tumor cell cultures, enteroids contain mucus-secreting goblet cells which will recreate the in vivo context more accurately. Together, these studies of MAdV may help us to understand why different HAdVs infect specific tissues.

For many years, concerted efforts to combat malaria through the use of antimalarial drugs, bed nets, and other public health measures led to marked reductions in morbidity and mortality. Unfortunately, progress has stalled. Reductions in malaria have leveled off and even reversed in certain areas (WHO, 2017). As of 2016, there were 216 million cases and 445,000 deaths annually due to Plasmodium infections (WHO, 2017). To regain momentum and accelerate malaria eradication efforts, an effective and durable vaccine is needed. The Murphy Laboratory focuses on developing novel pre-erythrocytic (PE) malaria vaccines that can effectively stop the Plasmodium sporozoite (spz) before the clinically symptomatic blood stage begins. Identification and inclusion of multiple different protective Plasmodium antigens is thought to be crucial to developing a broad immune response and durable protection against this intracellular parasite. To test and define protective antigens, the Laboratory developed an “Acute Challenge” (AC) model in order to sensitively measure T-cell responses that are completely or partially protective. In this model, DNA vaccines encoding Plasmodium yoelii proteins are delivered by gene gun to induce CD8+ T-cell responses in BALB/c mice. At the peak of the immune response, we challenge the mice with luciferase-expressing P. yoelli sporozoites and measure the parasite burden and protection using IVIS imaging. A known protective epitope derived from P. yoelii circumsporozoite protein (CSP) induces a potent and protective response in this system. My project is to utilize the AC model to assess P. yoelli candidate antigens, of unknown protective potential, that are putatively exported or secreted from the parasite-containing vacuole into the host cell cytoplasm. Confirmed protective antigens will then be assessed for their localization and defined T-cell epitopes. The results will be used to create vaccines designed to maximize such responses and target the responding T-cells to the liver.

The dynamic nature of chromatin, the complex of DNA and histones, is not fully understood and continues to be studied. During infection, viruses utilize host cell machinery, including chromatin, to promote viral proliferation. Adenovirus protein VII is a small histone-like core protein that tightly binds DNA and disrupts host chromatin in the infected cell . By studying protein VII, we aim to uncover how chromatin manipulation affects the health of the cell. To this end, our lab is using the model eukaryote, Saccharomyces cerevisiae or budding yeast, to understand the interaction between protein VII and chromatin. Yeast is an excellent model for studying chromatin due to the high conservation of histone proteins across all eukaryotes. By inducibly expressing protein VII in yeast, we tested how the expression of protein VII affects yeast growth and viability. Our preliminary findings show that protein VII expression is toxic to yeast—cell growth is slowed resulting in the formation of fewer and smaller colonies. This result suggests that protein VII perturbation of host chromatin is sufficient to inhibit growth in our model organism. Currently, we are generating yeast mutants and expressing different viral protein variants to understand the mechanism of these phenotypes . Through studying the chromatin perturbation caused by adenovirus protein VII, we will further our understanding of fundamental chromatin structure.

Human alpha defensins, a component of the innate immune system, are small cationic peptides that possess antiviral activity against non-enveloped viruses. The effect of defensins on human adenoviruses (HAdV) is serotype-dependent, infection by some serotypes is enhanced while for others it is neutralized. Enhanced infection correlates with increased cell binding; however, the mechanism of increased binding is unclear. One hypothesis is that defensins mediate receptor-independent binding. Inhibitor studies support this hypothesis, although formal proof is still needed. To test this hypothesis, we used CRISPR/Cas9 lentivirus to knockout the primary receptor, coxsackie adenovirus receptor(CAR), in A549 lung cells. In order to vet these cell lines, they were infected with different HAdV serotypes that use either CAR or an unrelated molecule, sialic acid, as their primary receptors. As expected, the sialic acid-utilizing but not the CAR-utilizing serotype was able to infect the CAR KO A549 cells. We have used these cell lines in combination with integrin co-receptor inhibitors to measure binding and infection of wildtype and mutant adenoviruses in the presence and absence of defensins. These experiments allowed us to determine the extent to which defensin-mediated attachment and entry is receptor-independent.