Role of emerin and protein kinase C in herpes simplex nuclear egress

The nuclear lamina consists of a mesh-like network of lamin proteins anchored to the inner nuclear membrane by interactions with integral membrane proteins such as emerin. Emerin binding to lamin A/C is one of the interactions that connect the inner nuclear membrane to the lamina. Infection by herpesviruses results in changes in the organization of the nuclear lamina, perhaps in order to facilitate envelopment of capsids at the inner nuclear membrane. In HSV-1 infected cells, alterations to the lamin proteins have been shown to involve pUL34, pUL31, and pUS3 proteins, which are also required for normal nuclear envelopment. We tested hypotheses about the mechanism and significance of lamina disruption. This thesis presents the following data. Infection of multiple cell types induced emerin hyperphosphorylation that was dependent on the presence of pUL34 and kinase active pUS3 proteins. The pUL34-dependent component was also sensitive to Rottlerin treatment suggesting that cellular kinases sensitive to Rottlerin were involved in emerin modification. LAP2 (another lamin associated protein) was de-modified (perhaps de-phosphorylated) in a pUS3 and pUL34 independent manner. Emerin was not required for growth of HSV-1. Hyperphosphorylation of emerin was required for its disassociation from the lamina.

PKC family members have been implicated in the disruption of the nuclear lamina during herpesvirus nuclear egress. To test the hypothesis that PKC activity was required for viral replication, PKC activity was blocked with PKC inhibitors and dominate negative PKC constructs. Chemical inhibition of all PKC isoforms reduced viral growth five-fold and inhibited capsid egress from the nucleus. However, specific inhibition of either conventional PKCs or PKC delta did not inhibit viral growth. In addition to lamin associated proteins, lamin localization is also disrupted during herpesvirus infections. Emerin and lamin A/C are binding partners and the localization of both proteins is disrupted by pUS3 and cellular kinase mediated phosphorylation. To test the hypothesis that HSV-1-induced lamin A/C disruption is mediated via a mechanism similar to emerin's, pUS3 and Rottlerin Sensitive Kinases were inhibited and lamin A/C localization was observed. Unlike emerin, HSV-1-induced disruption of lamin A/C was not altered by Rottlerin Sensitive Kinase inhibition suggesting that HSV has multiple mechanisms for disruption of the lamina.

Phosphorylation of lamina components, by Rottlerin Sensitive Kinases, may be a required event prior to primary envelopment. To test this hypothesis, growth of HSV-1 was tested in Rottlerin treated infected cells. Although the inhibitor Rottlerin, did reduce viral growth, it was also was also associated with severe depression of viral late-gene expression. TEM analysis suggested that Rottlerin Sensitive Kinases(s) were required for: (i) nuclear egress and (ii) capsid accumulation or formation supporting the hypothesis that the capsids were made in the presence of Rottlerin were unable to leave the nucleus. pUS3 is a multi-functional protein in alpha-herpesviruses. It has been implicated in lamina disruption, protecting the infected cell from apoptosis, and de-envelopment at the outer nuclear membrane. In BT-549 cells, a breast cancer cell line with low PKC delta expression, the hypothesis was tested that in the absence of cellular lamina disrupting kinases, an US3-null virus would be blocked at the lamina disruption step. In BT-549 cells, the US3-null (vRR1202) virus was 10-fold decreased above the typical 10-fold decrease, compared to WT virus, to produce a 100-fold decrease in infectious PFU yet apoptosis was not increased. Lamin A/C disruption occurred via similar mechanism in both breast cancer cell lines: BT-549 and MCF-7. Interestingly, in the BT-549 cells, emerin was extensively hyperphosphorylated in an US3-null infection, yet was not redistributed along the NE. These data support a model that one or more specific residue(s) must be phosphorylated for emerin disconnection from lamina.