Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes disease in humans and equines eliciting both an agricultural and public health threat. In humans, the disease typically presents as a febrile illness with common signs of fever and malaise. Four to fourteen percent of Venezuelan equine encephalitis (VEE) cases are associated with severe neurological complications due to encephalitis caused by VEEV's propensity to infect the brain. Public health concerns are exacerbated by VEEV's aerosolization capabilities, low infectious dose and affordability to mass produce. These qualities drove interest in the pathogen as a bioweapon by the US and the former Soviet Union during the cold war. As a precautionary response to VEEV's notoriety as a biothreat, the National Institute of Allergies and Infectious Diseases has classified VEEV as a category B priority pathogen, and the Human Health Services and United States Department of Agriculture list live virulent strains of VEEV as a select agent and require the pathogen to be manipulated in highly regulated biosafety level 3 (BSL3) facilities. There are currently no FDA approved vaccines or antivirals to target VEEV or other closely related alphaviruses associated with clinical disease in humans. The research performed in this dissertation aimed to elucidate new antiviral targets and treatments to help bridge gaps in current understanding of alphaviruses. The current market lacks available antibodies for E1 specific isolation. In response, a recombinant VEEV TC-83 was produced with a V5 tag at the C-terminal of the E1 sequence to enable VEEV E1 detection. Sequencing was used to verify V5 insertion in the plasmid and immunoprecipitation was used to verify V5 insertion within the E1 glycoprotein. Replication kinetics experiments verified the virus replicated similarly to the parental VEEV TC-83 strain, while passaging experiments verified the tag was highly stable for up to 10 passages. This research produced a cost-effective and highly efficient means to probe and isolate the E1 glycoprotein without modifying the viability of the virus. Knowledge of host protein interactions with VEEV E1 glycoprotein has been limited, with most E1 research focusing on its fusion capabilities. Utilizing 293-T cells infected with E1-V5 TC-83, co-immunoprecipitation was performed to isolate E1 and associated interactors. A total of 486 host and 5 viral protein interactors of E1 were discovered after normalization to the negative control. The top peptide spectrum matches (PSMs) revealed a number of chaperone proteins and ubiquitin proteins as top interactors of VEEV E1. These results effectively revealed a number of previously unknown alphavirus interactions that can be targeted by antivirals and explored further for implications in viral replication. LC-MS/MS results showed that protein disulfide isomerase family A member 6 (PDIA6) interacted with E1. High PSMs, presence in all 3 replicates, similar cellular localization to E1 and known associations between other viruses and protein disulfide isomerase (PDI) family members made this protein an optimum target for further analysis. Co-immunoprecipitation and co-localization experiments were used to validate the LC-MS/MS results. Involvement of PDIs in VEEV replication were explored utilizing two known PDI inhibitors, LOC14 and Nitazoxanide. LOC14, a non-FDA approved broad-spectrum PDI inhibitor, showed broad-spectrum alphavirus antiviral potential, decreasing titers of VEEV TC-83, VEEV Trinidad Donkey strain, eastern equine encephalitis virus (EEEV), chikungunya virus (CHIKV) and Sindbis (SINV) virus in a dose dependent manner. Nitazoxanide, an FDA approved drug known to inhibit PDIA3, was shown to have minimal toxicity and effectively reduced VEEV TC-83 and EEEV titers at concentrations with 100% cell viability. Time of addition assays, E1 expression time course studies, and early event assays showed PDI inhibition with these drugs effects early viral production events. RNA quantification, confocal microscopy and biotin switch assay experiments show that the drugs also prevented proper folding of the E1 glycoprotein and decreased expression of E1 on the peripheral membrane. With no current treatments for alphaviruses, these data provide an effective broad-spectrum target that affects viral replication at multiple stages in-vitro. Nitazoxanide also presents as a promising, non-toxic drug that could be repurposed to combat a number of clinically relevant alphaviruses. Valosin containing protein (VCP) was also shown to interact with the E1 glycoprotein. Exploration of VCP's interaction with alphavirus E1 has never been explored, yet it was previously shown to be involved in alphavirus replication. Co-localization and co-immunoprecipitation experiments were performed validating the interaction between VCP and E1. siRNA knockdown of VCP in 293-T cells and U87-MG cells showed a significant reduction in VEEV TC-83 titers. The allosteric VCP inhibitor, NMS-873, also reduced VEEV TC-83 titers, but was shown to be less effective against CHIKV, SINV and EEEV, suggesting the NMS-873 mechanism is more selective for VEEV. Mechanism experiments showed that reduction of VCP with NMS-873 inhibits early events of VEEV replication. These results elucidate VCP's association with E1 and show that VCP can be targeted to decrease VEEV viral replication. / Doctor of Philosophy / Venezuelan equine encephalitis virus (VEEV) causes disease in humans, as well as horses, donkeys and other closely related animals. In humans, the virus causes a flu-like disease and sometimes swelling of the brain. This can be associated with symptoms such as light sensitivity, confusion and sometimes coma. Prior to the Cold War, VEEV was researched by the US and previous Soviet Union's militaries in hopes to deploy the virus as a bioweapon. Current treaties prevent active production of such weapons, yet allows for defensive research to continue in preparation for a worst-case scenario. Currently no FDA approved medications or vaccines exist to combat the virus further exacerbating concerns. In order to protect laboratorians and prevent unintentional or intentional introduction of the virus into the community, the virus is only manipulated in highly secure facilities with barriers that separate the virus from personnel and the outside environment. A component of the virus called E1, allows for the virus to be released from a structure, called an endosome, that transports the virus into the cell. Currently, E1 is mostly known for this function, yet our research found that E1 interacts with 486 protein components of the host cell, suggesting a more elaborate role of E1 than previously understood. This list of interactors provides numerous new targets for potential medications to combat VEEV and other closely related viruses. Discovered E1 interactors, protein disulfide isomerase family A member 6 (PDIA6) and valosin containing protein (VCP), were validated through extensive experimentation and their function in viral replication was further explored. Protein disulfide isomerases (PDI), such as PDIA6, play an important role in folding proteins, which are cellular components made of organic building blocks called amino acids. PDIs do so by creating organic pillars, called disulfide bonds, between two cysteine amino acid residues. These disulfide bonds contribute to the 3D shape of the proteins they fold which are essential for the protein's function. E1 of VEEV has a total of eight disulfide bonds within its structure, highlighting that disulfide bonds are likely essential for the protein's structure, and therefore, function. We verified that E1 could not properly fold without PDI function by using two compounds that prevented PDI from forming or breaking disulfide bonds, specifically LOC14 and FDA approved drug nitazoxanide. Cells treated with one of either compound before and after infection with VEEV, were found to produce E1 protein with significantly less disulfide bonds therefore producing less viable virus. Further experiments also showed that the compounds also affected early stages in the virus production cycle. These two mechanisms explain the significant reduction in production of VEEV and related viruses when PDI is inhibited. These results provide a new VEEV drug target, PDIs, as well as two compounds that can potentially be used to combat VEEV and other related viruses that have no current treatment options. Another host interactor, VCP, functions throughout the cell and is known for unfolding of numerous substrates, including proteins. It is involved in numerous cellular functions thus making this interactor a promising target for drug treatment. Cells with reduced VCP function were shown to produce less progeny VEEV. Cells treated with NMS-873, a compound that reduces VCP function was also shown to reduce VEEV production. NMS-863 inhibition of VCP was shown to effect early events in VEEV replication. These results further emphasize the E1 interactors discovered are invaluable novel targets for VEEV drug treatment.

Les virus ont besoin d’interagir avec des facteurs cellulaires pour se répliquer et se propager dans les cellules d’hôtes. Une étude de l'interactome des protéines du virus d'hépatite C (VHC) par Germain et al. (2014) a permis d'élucider de nouvelles interactions virus-hôte. L'étude a également démontré que la majorité des facteurs de l'hôte n'avaient pas d'effet sur la réplication du virus. Ces travaux suggèrent que la majorité des protéines ont un rôle dans d'autres processus cellulaires tel que la réponse innée antivirale et ciblées pas le virus dans des mécanismes d'évasion immune. Pour tester cette hypothèse, 132 interactant virus-hôtes ont été sélectionnés et évalués par silençage génique dans un criblage d'ARNi sur la production interferon-beta (IFNB1). Nous avons ainsi observé que les réductions de l'expression de 53 interactants virus-hôte modulent la réponse antivirale innée. Une étude dans les termes de gène d'ontologie (GO) démontre un enrichissement de ces protéines au transport nucléocytoplasmique et au complexe du pore nucléaire. De plus, les gènes associés avec ces termes (CSE1L, KPNB1, RAN, TNPO1 et XPO1) ont été caractérisé comme des interactant de la protéine NS3/4A par Germain et al. (2014), et comme des régulateurs positives de la réponse innée antivirale. Comme le VHC se réplique dans le cytoplasme, nous proposons que ces interactions à des protéines associées avec le noyau confèrent un avantage de réplication et bénéficient au virus en interférant avec des processus cellulaire tel que la réponse innée. Cette réponse innée antivirale requiert la translocation nucléaire des facteurs transcriptionnelles IRF3 et NF-κB p65 pour la production des IFNs de type I. Un essai de microscopie a été développé afin d'évaluer l’effet du silençage de 60 gènes exprimant des protéines associés au complexe du pore nucléaire et au transport nucléocytoplasmique sur la translocation d’IRF3 et NF-κB p65 par un criblage ARNi lors d’une cinétique d'infection virale. En conclusion, l’étude démontre qu’il y a plusieurs protéines qui sont impliqués dans le transport de ces facteurs transcriptionnelles pendant une infection virale et peut affecter la production IFNB1 à différents niveaux de la réponse d'immunité antivirale. L'étude aussi suggère que l'effet de ces facteurs de transport sur la réponse innée est peut être un mécanisme d'évasion par des virus comme VHC. / Viruses interact with cellular factors in order to successfully replicate and propagate in host cells. Germain et al. (2014) performed a proteomics analysis to elucidate viral-host interactors of hepatitis C virus (HCV). They found that the majority of host factors did not have an effect on viral replication, suggesting that these host proteins may be beneficial to the virus by affecting other cellular processes such as evading the innate antiviral immunity. To test that hypothesis, 132 virus-host interactors were selected and silenced by RNAi for their effect on inteferon-beta (IFNB1) production as a readout of the innate antiviral response. 53 were found to modulate the response with enrichment in the gene ontology (GO) terms related to nucleocytoplasmic transport and the nuclear pore complex. An interesting point is that the genes associated with these terms (CSE1L, KPNB1, RAN, TNPO1, and XPO1) were previously elucidated as HCV NS3/4A interactors by Germain et al. (2014), as well as positive regulators of the innate antiviral response. Although it is surprising that a cytoplasmic-replicating virus like HCV would interact with proteins associated with the nucleus, we proposed that viruses interact with these proteins for their benefit to interfere with the innate immune response. The innate antiviral response requires the nuclear translocation of IRF3 and NF-κB p65 for the production of type I interferons. As it is unclear which transporters or nucleoporins are involved, 60 genes associated with the nuclear pore complex and nucleocytoplasmic transport were studied for their effect on the nuclear translocation of IRF3 and NF-κB p65 via a microscopy-based RNAi screen during a 10-hour viral infection time course. Overall, the study revealed that many of these proteins are involved in the trafficking of these transcription factors during a viral infection, and can affect the production of IFNB1 at different levels of the innate antiviral response. The study also suggests that the effect of these transport factors on the immune response may be an evasion mechanism for viruses such as HCV.