Inhibition of apoptosis by the Us3 protein kinase of herpes simplex virus 1

Abstract

The Herpes Simplex Virus 1 (HSV-1) US3 protein kinase is part of the anti-apoptotic arsenal that salvage HSV-1-infected cells from damage caused by different stimuli. The Us3 gene encodes a protein kinase that phosphorylates serine/threonine residues within a specific arginine-rich consensus sequence. Us3 was shown to block apoptosis induced by the virus infection itself. The aim of this thesis was to study the mechanisms by which Us3 blocks apoptosis induced by cytolytic lymphocytes and exogenous stimuli.

Us3 was shown to protect HSV-1-infected cells from lysis by MHC class I-restricted CD8T cells by acting downstream of antigen presentation. Expression of Us3 was associated with inhibition of CD8T cell-induced activation of caspases-3, -8 and -9 and a significant reduction in the cleavage of the pro-apoptotic protein Bid. Us3 selectively targeted the processing of Bid since recombinant human granzyme B (GrB) failed to cleave Bid in cytosolic extracts from Us3 positive cells and recombinant human Bid served as substrate for Us3 phosphorylation in vitro.

The role of Us3 in infected cells against cytotoxicity mediated by NK and LAK cells was also tested since these cells share common mechanisms in inducing death of virusinfected cells. In contrast to their lower sensitivity to CD8T cell lysis, HSV-1-infected cells were lysed by NK or LAK cells as efficiently as the uninfected controls. Both MHCrestricted and non-restricted cytolytic effects were significantly dependent on the activity of GrB and were efficiently blocked by treatment with a GrB inhibitor. However, in contrast to a significant induction of Bid cleavage mediated by CD8T cells, Bid was not cleaved when targets were co-cultured with LAK or NK cells suggesting that these effectors induce lysis by a mechanism that does not involve cleavage of Bid.

The ability of Us3 to block apoptosis induced by exogenous stimuli was also studied. We used a set of stable HSV-1 Us3 transfected cell lines with constitutive and inducible expression of the protein to show that Us3 alone is sufficient in inhibiting apoptosis induced by virus infection, sorbitol and Fas antibody treatment. Us3 blocked the activation of caspase-3 and caspase-9 in virus-infected cells. Bad was rapidly phosphorylated in cells expressing high levels of Us3 following virus infection or induction from a tetracycline-regulated plasmid, and Bad served as substrate for Us3 phosphorylation in vitro.

Taken together our data illustrate that modification of cellular pro-apoptotic proteins by Us3 presents a new viral strategy to prolong survival of the infected host cells. The ability of NK and LAK cells to overcome the anti-apoptotic function exerted by Us3 further demonstrates the intricate interplay between the virus and the immune system to control HSV-1 infection.