US3 of herpes simplex virus type 1 encodes a promiscuous protein kinase that phosphorylates and alters localization of lamin A/C in infected cells

The herpes simplex virus type 1 (HSV-1) U(S)3 gene encodes a serine/threonine kinase that, when inactivated, causes capsids to aggregate aberrantly between the inner and outer nuclear membranes (INM and ONM, respectively) within evaginations/extensions of the perinuclear space. In both Hep2 cells and an engineered cell line derived from Hep2 cells expressing lamin A/C fused to enhanced green fluorescent protein (eGFP-lamin A/C), lamin A/C localized mostly in a reticular pattern with small regions of the INM devoid of eGFP-lamin A/C when they were either mock infected or infected with wild-type HSV-1(F). Cells infected with HSV-1(F) also contained some larger diffuse regions lacking lamin A/C. Proteins U,31 and U,34, markers of potential envelopment sites at the INM and perinuclear virions, localized within the regions devoid of lamin A/C and also in regions containing lamin A/C. Similar to previous observations with Vero cells (S. L. Bjerke and R. J. Roller, Virology 347:261-276, 2006), the proteins U(L)34 and U(L)31 localized exclusively in very discrete regions of the nuclear lamina lacking lamin A/C in the absence of U(S)3 kinase activity. To determine how U(S)3 alters lamin A/C distribution, U(S)3 was purified and shown to phosphorylate lamin A/C at multiple sites in vitro, despite the presence of only one putative U(S)3 kinase consensus site in the lamin A/C sequence. U(S)3 kinase activity was also sufficient to invoke partial solubilization of lamin A/C from permeabilized Hep2 cell nuclei in an ATP-dependent manner. Two-dimensional electrophoretic analyses of lamin A/C revealed that lamin A/C is phosphorylated in HSV-infected cells, and the full spectrum of phosphorylation requires U(S)3 kinase activity. These data suggest that U(S)3 kinase activity regulates HSV-1 capsid nuclear egress at least in part by phosphorylation of lamin A/C.