Characterization of viral mutants for the functional analysis of ppUL69 during human cytomegalovirus replication.
2009-11
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Characterization of viral mutants for the functional analysis of ppUL69 during human cytomegalovirus replication.
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2009-11
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Abstract
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.
Description
University of Minnesota M.S. thesis. November 2009. Major: Microbiology, Immunology and Cancer Biology. Advisor: Wade A Bresnahan, Ph.D. 1 computer file (PDF); xi, 102 pages. Ill. (some col.)
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Kronemann, Daniel Aaron. (2009). Characterization of viral mutants for the functional analysis of ppUL69 during human cytomegalovirus replication.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/59922.
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