Title

Analysis of the cellular heat shock response and machinery during herpes simplex virus type I infection

Date of Completion

January 2010

Keywords

Biology, Molecular|Biology, Cell|Biology, Virology

Degree

Ph.D.

Abstract

HSV-1 infection results in formation of nuclear replication compartments (RC), sites of replication and encapsidation of the HSV-1 DNA genome. During infection, nuclear protein quality control (nPQC) machinery such as the 20S proteasome, chaperones such as Hsc70, putative misfolded proteins and polyubiquitinated proteins are reorganized into virus-induced chaperone-enriched (VICE) domains adjacent to RC. VICE domains resemble nuclear inclusion bodies that form in neurological disease states and sequester mutant huntingtin or ataxin-1 protein in addition to chaperones, the 20S proteasome and ubiquitin. VICE domains contain proteolytic activity, and Hsc70 rapidly exchanges between VICE domains and the nucleoplasm possibly to scan the nucleus for damaged proteins. VICE domain formation requires live virus and active proteasome machinery. VICE domains form when viral gene expression is robust; there formation is impaired in the absence of immediate-early proteins ICP0 and ICP27 that are required for efficient gene expression. ICP22 is essential for reorganization of Hsc70 to VICE domains. ICP0 and ICP22 are sufficient to reorganize Hsc70 in transfected cells suggesting that these proteins may actively reorganize Hsc70 during infection. VICE domains form adjacent to developing RC called prereplicative sites during the earliest stages of HSV-1 infection. Chaperones are important for HSV-1 infection; inhibition of chaperones by drugs, siRNA depletion or expression of dominant-negative chaperones impairs HSV-1 productivity. Hsc70 ATPase activity is required for its reorganization to VICE domains, replication compartment formation, nuclear import of an essential capsid protein and for efficient viral productivity. The inducible heat shock response is also required for infection. The master regulator of the heat shock response, HSF-1, is modified during infection and localizes to RC. HSF-1 activation normally results in increased Hsp70 protein levels however; during infection, Hsp70 protein levels remain constant suggesting partial activation of the heat shock response. An inhibitor of the heat shock response, KNK437, impairs HSV-1 gene expression, RC formation and viral productivity. KNK437 prevents modification of HSF-1 during infection. Overexpression of HSF-1 in KNK437-treated infected cells partially relieves the inhibitory effect of the drug suggesting that KNK437 may target HSF-1 activity. Efficient HSV-1 infection requires formation of nPQC centers and the inducible heat shock response. ^