A herpesviral ubiquitin ligase activates expression of a specific host miRNA cluster by degrading ZEB transcriptional repressors

Abstract

Herpes simplex virus 1 (HSV-1) is an important and widespread human pathogen infecting more than two-thirds of the human population and causing significant disease in many individuals, especially the immunocompromised. Upon infection with HSV-1, host cells undergo a stress response involving extensive changes in host gene expression. The methods for accomplishing these changes include the use of miRNAs - subtle regulators of gene expression that can potentially influence a wide range of cellular processes including the host response to pathogens. Several viruses, including HSV-1, also encode their own miRNAs which are used to regulate the viral lytic gene expression program.

The work presented in this thesis describes the induction of three host miRNAs, named the miR-183/96/182 cluster (miR-183 cluster) during HSV-1 productive infection. This is most clearly observed in primary cells and is much reduced or absent in highly transformed cell lines. I show that HSV-1 induced transcription of the miR-183 cluster is dependent on the viral immediate-early (IE) gene ICP0, an E3 ubiquitin ligase important for viral gene expression and the antagonism of host innate defenses. ICP0 is essential for reactivation from latency and for optimal viral replication in primary cells. Induction of the miR-183 cluster represents a new function for this intensively studied protein. ICP0 has been shown to polyubiquitinate a large number of cellular proteins and mark them for proteasomal degradation. Using ICP0-mutants, I show that the E3 ubiquitin ligase function is required for stimulation of the miR-183 cluster and that ICP0 must be targeted to the nucleus. Evidence is presented implicating the host ZEB transcriptional repressor proteins as the primary targets for ICP0-mediated miR-183 cluster induction. Binding sites for ZEB1 and ZEB2 are found upstream of the miR-183 cluster and steady-state levels of both proteins are reduced in the presence of ICP0 during HSV-1 infection. Inhibition or overexpression of the miR-183 cluster miRNAs did not significantly alter HSV-1 replication in primary fibroblasts, and may reflect choice of cell type or limitations in the assay. However, replication of both wild type and mutant HSV-1 strains was increased by siRNA-mediated knockdown of ZEB1, suggesting that additional changes in host gene expression beyond the increased expression of miR-182, miR-96 and miR-183 can have a positive impact on viral replication. This work provides the first example of host miRNAs that are directly regulated by HSV-1 during infection and identifies the viral factor, an E3 ubiquitin ligase, as responsible.