p97 is a member of the AAA family of proteins (ATPase Associated with various cellular Activities). It is a highly conserved and abundant protein and functions in numerous ubiquitin-mediated processes including ERAD. Endoplasmic Reticulum Associated Degradation is the process by which misfolded/ubiquitinated proteins translocate out of the ER and migrate to the proteasome for degradation. p97 maintains substrate misfolding and mediates its exit from the ER and trafficking to the 26S proteasome. It also plays important roles in protein trafficking, the cell-cycle, apoptosis and homeotypic Golgi Apparatus and Endoplasmic Reticulum membrane fusion after mitosis. In addition, p97 plays a role in the aggresome-autophagy degradation pathway, which handles the ubiquitin-mediated destruction of aggregate-prone, misfolded, cytosolic proteins. p97 mutation is the causative alteration in the disorder, IBMPFD, which is marked by defects in autophagy. This broad diversity of function is mediated through p97's interaction with a large group of adaptor proteins. Many of these adaptors harbor both p97 interaction motifs and ubiquitin association domains. However, more than half of known p97 adaptors do not. Their function is largely unknown. UBXD1 is one known adaptor for p97 that does not have a ubiquitin association domain (UBA), and has been shown to have decreased interaction with IBMPFD mutant p97R155H and p97A232E. Recently, it has been suggested to perform a role in protein trafficking, specifically in monoubiquitinated caveolin-1 internalization and trafficking to the endosome. A novel high abundance UBXD1 interacting partner has been identified via solution-based mass spectrometric analyses. ERGIC-53, the namesake of the ER-Golgi Intermediate Compartment, has been shown to be involved in bi-directional trafficking between the ER and Golgi. The association between UBXD1 and ERGIC-53 is unique among UBX family members. Deletional analysis has shown that unlike p97, the ERGIC-53-UBXD1 interaction takes place in the extreme amino terminus of UBXD1, (within the first 10 amino acids) which is predicted by computer modeling to form a hydrophobic binding pocket. Further site-directed mutagenesis work has clearly shown four amino acids (3 highly hydrophobic) are crucial for maintaining this interaction. They have been modeled to form a conserved alpha-helix. ßCOPI, a primary member of the COPI coatomer complex which is involved in protectively coating ERGIC-53 positive vesicles, is also thought to be involved with the ERGIC-53-UBXD1-p97 pathway. ßCOPI has been identified as a UBXD1-independent interactor with p97. Modest UBXD1 over- expression using a ponasterone inducible system has shown that UBXD1 modulates ERGIC-53 localization. Additionally, a functional link between UBXD1, p97 and ERGIC-53 in autophagy has been discovered through the use of a highly efficient, miR30-based, inducible knockdown system. Upon individual knockdown of UBXD1, p97 and ERGIC-53, autophagic markers p62 and LC3-II accumulate at relatively high levels in normal culture conditions, strongly suggesting a role in mediating basal autophagy. However, when placed under starvation conditions, autophagy progresses and p62 is degraded. It is speculated from these studies that a p97/UBXD1 complex plays a role in regulating the trafficking of ERGIC-53 positive vesicles and this activity plays an important role in autophagy. / Molecular Biology and Genetics

Arenaviruses and hantaviruses are human pathogens that cause significant morbidity and mortality. The current lack of vaccines and treatment options for these viruses is a global concern. Despite producing only 4 proteins, these viruses are able to maintain a persistent and asymptomatic infection in wild rodents while being continuously shed into the environment. In humans, these viruses cause a spectrum of diseases ranging from aseptic meningitis to severe hemorrhagic fever syndromes. Little is known about how arenavirus and hantavirus proteins engage and interact with the human proteome during the complex process of viral biogenesis, or how the interactions with human proteins contribute to viral propagation as well as the onset and progression of disease. This dissertation provides a road map of the protein interactions formed between a prototypic envelope glycoprotein encoded by either an arenavirus or hantavirus, and the human proteome. The viral envelope glycoprotein (GP) decorates the surface of the virion. The primary function of the GP is to mediate attachment of the virus to specific cellular receptors, and after internalization of the virion, fuse the viral membrane with an internal endosomal membrane. In order to carry out these specific tasks, the viral GPs must first co-opt the extensive machinery found within the cellular secretory pathway to coordinate the proper glycosylation, folding, proteolytic maturation, and targeting of the GP during its biosynthesis. We identified a human protein with a conserved interaction amongst these two groups of viral GPs termed the Endoplasmic Reticulum (ER)-Golgi Intermediate Compartment Protein of 53 kiloDaltons (ERGIC-53). ERGIC-53 is an intracellular cargo receptor that normally cycles within the early secretory pathway of cells, where it is responsible for ferrying a small subset of cellular glycoproteins, most notably the coagulation factors FV and FVIII, from the ER to the Golgi apparatus. Herein we describe a novel role for ERGIC-53 in the propagation of not only arenaviruses, but also coronaviruses and filoviruses. Following infection with an arenavirus, ERGIC-53 leaves the early secretory pathway and becomes incorporated into the virus as it pinches off from the cell surface. Newly formed viruses lacking ERGIC-53 are no longer infectious due, in part, to a defect in their ability to attach to host cells. We suggest that ERGIC-53 represents a promising broad-spectrum antiviral target because of its association with the GPs from many families of pathogenic viruses, as well as its ability to exert control over their infectivity; and finally, because ERGIC-53 itself is not required for human health. The discovery of ERGIC-53 outside of its normal location inside of cells suggests that it may have additional unknown functions. Lastly, by revealing the importance of the cellular protein in controlling viral infectivity, we provide insight into the ongoing co-evolution of virus and host.