Browsing by Subject "Mutant"

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    Characterization of viral mutants for the functional analysis of ppUL69 during human cytomegalovirus replication.
    (2009-11) Kronemann, Daniel Aaron
    Human cytomegalovirus (HCMV) is a β-herpesvirus that infects over 80% of the human population. Although disease is rare in immunocompetent individuals, severe disease is common in immunocompromised patients, including neonates, transplant recipients and acquired immunodeficiency syndrome (AIDS) patients. HCMV UL69 is a viral protein packaged in the tegument of the virion and, as such, is present from the earliest moments of infection. Using transient transfection assays, UL69 has been shown to bind the cellular splicing and mRNA export factor U2AF65 associated protein 56 (UAP56), shuttle between the nucleus and the cytoplasm, and bind the cellular chromatin remodeling and transcriptional elongation factor Suppressor of Ty 6 (Spt6). In previously published work, these characteristics were shown to be required for transactivating the major immediate/early promoter (MIEP) and promoting the export of an intron-containing chloramphenicol acetyltransferase (CAT) reporter transcript. These results, in addition to the fact that most herpesviral transcripts are intronless, have led to a model for UL69 function during HCMV infection as a viral mRNA export factor. However, recently published work using a UL69 deletion viral mutant, termed TNsubUL69, has demonstrated that during viral infection, UL69 is not required for expression of immediate/early (IE) or early (E) genes. In contrast, deletion of UL69 results in a severe defect in late (L) gene expression. Additionally, the UL69 deletion mutant exhibits a defect in viral DNA replication and a MOI-dependent replication defect. These results demonstrate the importance of examining the functional role of UL69 in the context of a viral infection, in the presence of a full complement of viral factors at physiologically relevant levels. The goal of this thesis is to characterize viral mutants that contain mutations in the UL69 open reading frame (ORF) which have been previously described to abolish either binding to UAP56 (UL69mUAP), nucleocytoplasmic shuttling (UL69P603 or UL69E618), or binding to Spt6 (UL69C496). We demonstrate our own ΔUL69 viral mutant exhibits a similar replication phenotype, consistent with previously published results. We demonstrate that the UL69mUAP mutation abolishes UL69 binding of UAP56 but does not affect viral replication. The UL69P603 mutation, but not the UL69E618 mutation, abolishes UL69 shuttling and results in a defect in viral replication similar to the deletion mutant virus. However, the UL69P603 viral mutant is also defective for every other UL69 characteristic for which we have assayed, suggesting the UL69P603 mutation affects a core domain in the UL69 ORF and results in a global UL69 defect during viral infection. A similar result has been obtained for the UL69C496 viral mutant, which is defective for UL69 binding of Spt6, but also results in a defect in other UL69 functions. Using an shRNA strategy to further examine the role of Spt6 during HCMV replication, we demonstrate that partial knockdown of Spt6 negatively affects WT HCMV replication. Taken together, we conclude that UL69 binding of UAP56 is not required efficient HCMV replication, but are unable to make strong conclusions about UL69 shuttling or binding of Spt6 during viral replication. Since both UL69 shuttling and UL69 binding of UAP56 are required for export of an introncontaining transcript, and given that UL69 binding of UAP56 is not required for efficient viral replication, we predict that UL69 shuttling is also not important for efficient viral replication. Thus we propose three models for UL69 function, which center on UL69 binding of Spt6. Through its interaction with Spt6, UL69 functions as either a viral mRNA export factor, a chromatin remodeling factor, or a transcriptional elongation factor.
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    Genetic and Genomic Analysis of Nonhost Resistance to Wheat Stem Rust in Brachypodium distachyon
    (2016-08) Della Coletta, Rafael
    Wheat stem rust, caused by the fungus Puccinia graminis f.sp. tritici (Pgt), is a devastating disease that has been under control for decades. However, new races of this pathogen have emerged that overcome many important wheat stem rust resistance genes, and their spread toward important areas of wheat production threatens global wheat production. Nonhost resistance in plants, which provides durable and broad-spectrum resistance to non-adapted pathogens, may hold great potential to help in the control of wheat stem rust, but the genetic and molecular basis of nonhost resistance is poorly understood. This research project employed the model plant Brachypodium distachyon (Brachypodium), a nonhost of Pgt, for genetic analysis to map loci associated with nonhost wheat stem rust resistance. Using bulked segregant analysis, next-generation sequencing, and bioinformatics approaches, seven quantitative trait loci were found to contribute to nonhost stem rust resistance in a recombinant inbred population derived from a cross between two Brachypodium genotypes with differing levels of resistance. In a second study, analysis of a Brachypodium recombinant inbred population segregating for an induced mutation that confers susceptibility to wheat stem rust led to the identification of a one base pair deletion in a gene that may be the cause of the mutant’s susceptibility. The gene is a homolog of the Arabidopsis gene TIME FOR COFFEE (TIC), which plays a role both in circadian clock regulation and jasmonate signaling. Collectively, the findings of this research project advance our understanding of the genetic basis of nonhost resistance to wheat stem rust, and will guide future research aiming to identify genes essential to the nonhost resistance response, as well as their mechanisms of action.