Stable Signal Peptide Requirement for Lymphocytic Choriomeningitis Virus Glycoprotein Synthesis and Maturation

Arenaviruses persist in distinct rodent hosts in regions defined by the host rodent's geographic distribution. The prototype arenavirus, the lymphocytic choriomeningitis virus (LCMV) uses the house mouse, Mus musculus, as its reservoir host, and as a result, LCMV has a near-global geographic distribution. In general, LCMV cause non-cytolytic infections and the resulting tissue pathology is caused by immune cell-mediated mechanisms rather than direct viral cytopathology. LCMV has also been implicated in the deaths of organ recipients upon transplantation of infected donor tissue. Several arenaviruses, namely the Lassa Virus and the Junín Virus, are causative agents of viral hemorrhagic fever. Arenavirus infections are initiated by the binding of the viral surface glycoprotein complex to receptors on the target host cell. A series of protein interactions ensues with the ultimate release of the viral core into the cytoplasm of the newly infected cell. The work presented here targets two aspects of the arenavirus life cycle that, due to their conservation across the Arenaviridae family, are attractive targets for therapeutic developments: the genome promoter element and the glycoprotein stable signal peptide (SSP). The 5' and 3' termini of each genome segment share sequence complementarity, as each drive expression of the viral open reading frames in an ambisense manner. This highly conserved genome element was the target for antisense peptide-conjugated phosphorodiamidate morpholino oligonucleotide inhibitor development (P-PMO). Cells treated with P-PMO compounds, followed by infection with LCMV resulted in a decrease in viral protein and RNA production. Total viral protein and titer inhibition was observed with 2 uM P-PMO treatment. In addition to my contribution to the development of antisense oligonucleotide compounds with antiviral activity, I created a panel of glycoprotein tools that directly target SSP with the goal of elucidating this glycoprotein subunit's role in the synthesis and maturation of the entire glycoprotein complex. SSP is a fascinating component of arenaviruses. This highly conserved 58 amino acid long polypeptide is retained as a critical factor for several steps in the production of a mature glycoprotein complex, which studs the surface of nascent virions originating at the infected cell plasma membrane. Further, I created a glycoprotein plasmid containing an epitope-tagged SSP for experimental manipulation and detection of SSP. Using a series of biochemical assays, confocal microscopy, and flow cytometry, I present compelling evidence that SSP is the driving force for the synthesis of a properly organized and functional glycoprotein complex. The tools I have developed will aide future studies to further elucidate the mechanism and cell biology behind how this viral protein borrows cellular machinery for virus propagation. Since SSP is a highly conserved arenavirus component, a better understanding of how this subunit functions has the potential to become a target for antiviral therapeutic development