The Arenaviridae are enveloped, negative-sense RNA viruses with several family members that cause hemorrhagic fevers. These viruses encode only four open reading frames and each protein expressed performs multiple functions in the viral lifecycle. This thesis focuses on elucidating the regulation and functions of Lymphocytic Choriomeningitis Virus (LCMV) nucleoprotein (NP). Phosphorylation is a common and reversible posttranslational modification. This work identifies two residues in the Lymphocytic Choriomeningitis Virus (LCMV) nucleoprotein (NP) that are as conserved in every mammalian arenavirus and are required for recombinant LCMV recovery. One of these sites, NP Y125, was confirmed as a phosphorylation site by LC-MS/MS. Y125 resides in the N-terminal region of NP that becomes disordered when RNA is bound. The other site, NP T206, was predicted to be a phosphorylation site. Immunofluorescence demonstrated that NP T206 is required for the formation of the punctate replication-transcription complexes (RTC) typically found in LCMV infection. A minigenome reporter assay in combination with NP mutants and Northern blot analysis, demonstrated that, while NP T206A remains diffuse in the cytoplasm and does not form RTC, it can facilitate the transcription and replication of a minigenome. However, in the presence of Z and GP, translation of minigenome message with NP T206A was inhibited, suggesting that RTC formation is required to regulate viral replication. Additionally, we provide immunofluorescence evidence that, unlike Old World arenaviruses, LCMV RTC colocalize with eIF4E, and that eIF4E may participate in the translation of LCMV mRNA.

In addition to nucleating RTC formation and participating in replication and transcription, NP also inhibits stimulation of the innate immune system. This activity has been attributed to the dsRNA exonuclease activity of in the C-terminal domain of NP. NP is proposed to degrade viral dsRNA, thereby eliminating its recognition by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), and preventing the subsequent signaling cascade. However, we discovered that NP blocks transcription from interferon-stimulated response element (ISRE) promoters downstream of RIG-I and MDA5, and that the exonuclease activity of NP is not required for this inhibition. We propose the exonuclease activity of NP may be conserved across mammarenaviruses because it functions in concatemer resolution during genome replication.

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.

Coronaviruses, like nearly all studied positive sense single-stranded RNA viruses, rely on host cell internal membranes to produce structures that support viral replication. These structures form in the cytoplasm and are called double membrane vesicles (DMVs) and convoluted membranes (CMs). DMVs are ~100-300 nm in diameter and are usually found in clustered groups. They are interconnected with regions of CM that can vary in size and extent. These membranous structures are the localized sites of viral genome replication and transcription within the infected cell. DMVs and CMs are distinct from structures that form later in the infection life cycle for purposes of virion assembly and budding. Ultrastructural studies have confirmed that the walls of DMVs and CMs are composed of two lipid bilayers in close proximity to each other and that the membranes are at least somewhat continuous with the membrane of the endoplasmic reticulum (ER). However, they do not stain for canonical ER markers. Despite the work that has been performed to discern the intricacies of DMV and CM assembly in coronavirus infected cells, the specific mechanisms and pathways involved have remained unclear. We present here the results of work done to elucidate both the viral proteins responsible for DMV and CM formation as well as work to decipher which host cellular proteins and pathways might be important. We used plasmid constructs expressing nonstructural proteins (nsps) from the severe acute respiratory syndrome (SARS) coronavirus to determine that the combination of nsp3, nsp4, and nsp6 is sufficient for producing structures similar to DMVs and CMs induced during SARS-coronavirus infection. We also characterized structures induced by each of the three nsps individually and in paired combinations to theorize a general putative mechanism by which DMV and CM structures form. Additionally, we used inhibitors and siRNA technology to determine that host cellular fatty acid synthase (FASN) is an important protein for murine hepatitis virus replication and that, in the absence of FASN activity, DMVs and CMs are not observed. This work adds to the body of evidence characterizing membrane rearrangement by coronaviruses and will aid ultimately in a full description of the process.

Immunoglobulin (Ig) gene class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which requires activation-induced cytidine deaminase (AID), underpins autoantibody class-switching from the non-harmful, protective IgM class to the potentially pathogenic IgG and IgA classes. CSR replaces the Ig heavy chain (IgH) constant (CH) region, e.g., Cμ of IgM, with Cγ, Cε or Cα, thereby producing IgG, IgE or IgA antibodies. Aberrant CSR results in a variety of diseases, including atopic IgE reactions (e.g., in asthma), hyper-IgM syndrome, and systemic or organ-specific autoimmunity.

I have explored the in-depth molecular mechanisms underlying antibody class-switching to IgG, IgA or IgE, with emphasis on the targeting and induction of CSR factors. Our work seeks to establish a paradigm that intervention at any of these two levels of regulation can modulate class-switching. Such a paradigm will be critical in shaping research in vaccines, whose efficacies depends on generation of high-affinity antibodies and on therapeutics to inhibit unwanted class-switching in a variety of clinical conditions. We first explored CSR inhibition mechanisms by quantifying direct interaction between 14-3-3, AID, PKA or Ung and viral protein R (Vpr). As we have shown in activated B cells, 14-3-3 adaptors specifically recruit AID on IgH switch (S) region DNA. Our research has been focused on using a naturally occurring viral protein R (Vpr) to disrupt 14-3-3-mediated recruitment of the AID-centered CSR machinery to S region DNA, which inhibited CSR. We then focused on using a small molecule compound for therapeutic treatment in preventing high titers of autoantibodies in systemic lupus erythematosus (SLE). MRL/Faslpr/lpr mice treated with CID 1067700, the only known inhibitor of Rab7, displayed reduced decreased glomerular hypercellularity and proteinuria, virtually abolished skin lesions, and extended life span. Accordingly, serum titers of anti-dsDNA IgG1, IgG2a, IgG2b and IgG3 autoantibodies were reduced in treated mice, in association with reduced generation of IgG+ B cells. These mice, however, showed increased proportion of IgM+ B cells, but normal proportions of CD4+ and CD8+ T cells as well as CD138+ plasma cells. These together with reduced AID expression in treated mice indicates that CID 1067700 selectively inhibits B cell class-switching. Indeed, the CID 1067700 treatment blunted the antibody response NP-LPS, a T cell-independent antigen, and inhibited CSR to IgG1 and IgA in purified B cells in vitro, but did not affect B cell proliferation, survival or differentiation into plasma cells. Overall, our data have further confirmed the specific role of Rab7 in mediating AID expression and CSR, and strongly suggest Rab7 as a therapeutic target for systemic lupus.

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