Theses and Dissertations from UMD
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Item Characterization of the GBF1-Arf1 axis in enterovirus RNA replication(2024) Gabaglio Velazquez, Samuel Maria; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Enterovirus genus includes many known and emerging pathogens, such as poliovirus, enteroviruses A71 and D68, rhinoviruses, and others. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, called replication organelles. The cellular protein Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) is essential for the replication of enteroviruses, but its molecular role in the replication process is unclear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including the functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and other enteroviruses directly interact with and recruits GBF1 to the replication organelles. Moreover, enterovirus infection induces the massive recruitment of all isoforms of the small cellular Arf GTPases to the replication organelles, but the mechanistic role of these proteins in the replication process is not understood either. Here, we sought to characterize the role of the GBF1-Arf1 axis in enterovirus replication. First, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrated that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results showed that the Arf-activating property, but not the primary structure of the Sec7 catalytic domain is essential for viral replication. They also suggest a redundant mechanism for recruiting GBF1 to the replication sites. This mechanism depends not only on the direct interaction of the protein with the viral protein 3A but also on elements located in the noncatalytic C-terminal domains of GBF1. Next, we investigated the distribution of viral proteins and Arf1 on the replication organelles and their biochemical environment. Pulse-labeling of viral RNA with 5-ethynyl uridine showed that active RNA replication is associated with Arf1-enriched membranes. We observed that Arf1 forms isolated microdomains in the replication organelles and that viral antigens are localized in both Arf1-depleted and Arf1-enriched microdomains. We investigated the viral protein composition of the Arf1-enriched membranes using peroxidase-based proximity biotinylation. Viral protein biotinylation was detected as early as 3 h.p.i., and the non-cleaved fragments of the viral polyprotein were overrepresented in the Arf1-enriched domains. Furthermore, we show that after 4 h.p.i. viral proteins could be efficiently biotinylated only upon digitonin permeabilization of the replication organelle membranes, while such permeabilization inhibited the Arf1 biotinylation signal at the Golgi in non-infected cells. Together, these data support a model that recruitment of GBF1 to the replication organelles generates foci of activated Arfs on the membranes, which further differentiate into specific microdomains through the recruitment of a specific complex of viral proteins and cellular Arf effectors likely needed to establish the lipid and protein composition required for viral replication.Item Identification and Characterization of New Small Molecule Inhibitors of Picornavirus Replication(2016) Siltz, Lauren Ashley Ford; Belov, George A; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Picornaviridae family consists of positive-strand RNA viruses that are the causative agents of a variety of diseases in humans and animals. Few drugs targeting picornaviruses are available, making the discovery of new antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. The antiviral effect of these compounds is not likely related to their known cellular targets because other inhibitors targeting the same pathways did not inhibit viral replication. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited the formation of a functional replication complex, while E5(1) and E7(2) affected replication after the replication complex had formed. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Poliovirus resistant to E7(2) had a single mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target either PI4KIIIβ (major enviroxime-like compounds) or OSBP (minor enviroxime-like compounds), cellular factors involved in lipid metabolism and shown to be important for replication of diverse positive-strand RNA viruses. We classified E7(2) as a minor enviroxime-like compound, because the localization of OSBP changed in the presence of this inhibitor. Interestingly, both E7(2) and major enviroxime-like compound GW5074 interfered with the viral polyprotein processing. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds. Studies with these compounds shed light on pathways shared by diverse picornaviruses that could be potential targets for the development of broad-spectrum antiviral drugs.Item MUTATIONAL ANALYSIS OF POLIOVIRUS PROTEIN 3AB TO IDENTIFY REGIONS CRITICAL TO NUCLEIC ACID CHAPERONE ACTIVITY(2013) Gangaramani, Divya; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Poliovirus 3AB protein was the first picornavirus protein demonstrated to have nucleic acid chaperone activity. Current results demonstrate that chaperone activity requires the C-terminal 22 amino acid (3B region (also referred to as VPg), amino acid 88-109) of the protein as mutations in this region abrogated nucleic acid binding and chaperone activity. Protein 3B alone had no chaperone activity as determined by established assays testing chaperone activity including: the ability to stimulate nucleic acid hybridization in a primer-template annealing assay, or helix-destabilization in a nucleic acid unwinding assay, or aggregation of nucleic acids. In contrast, the putative 3AB C-terminal cytoplasmic domain (N terminal amino acid 81-109, 3B + the last 7 C-terminal amino acids of 3A, termed 3B+7 in this report) possessed strong activity in these assay, albeit at much higher concentrations than 3AB. Results from several mutations in 3B+7 are described and a model proposing that 3B+7 possesses the "intrinsic" chaperone activity of 3AB while the 3A N-terminal region (amino acid 1-58) and/or membrane anchor domain (amino acid 59-80) serve to increase the effective concentration of the 3B+7 region leading to the potent chaperone activity of 3AB. Two mutations with reduced chaperone activity in vitro, K81A and F83A in 3AB were tested in tissue culture. Viruses with these mutations produced near wildtype and minute plaques, respectively. F83A gave rise to revertants with either wildtype 3AB sequences or additional nearby compensatory mutations. Translation and polyprotein processing were not affected by these mutations but RNA synthesis compared to wildtype, was slightly lower for K81A and significantly lower for F83A. This data suggests that mutations that decrease chaperone activity of 3AB may lead to decreased RNA synthesis, although the exact steps that are affected need to be determined.