Date of Award

Summer 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

James C. Alwine

Abstract

Human cytomegalovirus (HCMV) regulates the unfolded protein response, including specifically inducing the ER chaperone BiP. The increase in BiP protein during infection is the result of promoter activation by the HCMV MIEPs and La autoantigen activation of the internal ribosome entry site of BiP mRNA. To determine the effects of BiP on HCMV replication, BiP was depleted using the SubAB subtilase cytotoxin or short hairpin RNAs. Depletion of BiP had little effect on viral protein synthesis. However, progeny virion formation was significantly inhibited, suggesting that BiP is important for virion formation. Electron microscopic analysis showed that infected cells were resistant to the toxin and showed none of the cytotoxic effects seen in uninfected cells. However, all viral activity in the cytoplasm ceased. During infection, BiP localizes in two cytoplasmic structures, regions of condensed ER near the periphery of the cell and the viral cytoplasmic assembly compartment, where it interacts with the viral proteins pp28 and TRS1. Depletion of BiP causes the assembly compartment to dissociate and the cytoplasm to return to a more normal morphology, indicating that BiP is important for assembly compartment integrity and confirming its role in viral cytoplasmic activity. Furthermore, depletion of BiP and the corresponding disruption of assembly compartment integrity results in the loss of virally-induced nuclear lamina rearrangement and a decrease in lamin phosphorylation. These results are due to an interaction between BiP and pUL50. Thus, BiP depletion affects both nuclear and cytoplasmic viral activity. These data and the following observations support an intricate link between vial nuclear and cytoplasmic activity. The inhibition of the molecular motor dynein results in the loss of both assembly compartment integrity and the nuclear morphology characteristic of an HCMV infection. Furthermore, the nuclear periphery is dramatically altered adjacent to the assembly compartment, where the nuclear lamina is rearranged, the outer nuclear membrane is altered, and the nucleus becomes permeable to large molecules. The perinuclear space is also enlarged, a result of decreasing the SUN proteins during infection. These results support a model for a highly integrated assembly-egress continuum, linking viral nuclear and cytoplasmic activity.

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