Herpes simplex virus type 1 infection induces activation and recruitment of protein kinase C to the nuclear membrane and increased phosphorylation of lamin B

Abstract

We report that herpes simplex virus type 1 (HSV-1) infection leads to the recruitment of protein kinase C (PKC) to the nuclear rim. In HEp-2 cells, PKC recruitment to the nuclear rim was initiated between 8 h and 12 h postinfection. PKCdelta, a proapoptotic kinase, was completely recruited to the nuclear rim upon infection with HSV-1. PKCalpha was less dramatically relocalized mostly at the nuclear rim upon infection, although some PKCalpha remained in the cytoplasm. PKCzeta-specific immunofluorescence was not significantly relocated to the nuclear rim. The UL34 and UL31 proteins, as well as their association, were each required for PKC recruitment to the nuclear rim. The HSV-1 US3 protein product, a kinase which regulates the phosphorylation state and localization of UL34, was not required for PKC recruitment to the nuclear rim; however, it was required for proper localization along the nuclear rim, as PKC appeared unevenly distributed along the nuclear rim of cells infected with US3 null and kinase-dead mutants. HSV-1 infection induced the phosphorylation of both lamin B and PKC. Elevated lamin B phosphorylation in HSV-1-infected cells was partially reduced by inhibitors of PKC. The data suggest a model in which kinases that normally disassemble the nuclear lamina during apoptosis are recruited to the nuclear membrane through functions requiring UL31 and UL34. We hypothesize that the recruitment of PKC functions to phosphorylate lamin B to help modify the nuclear lamina and promote budding of nucleocapsids at the inner nuclear membrane.

FIG. 1.

Digital images of confocal fluorescence micrographs of mock-infected (A to D and I to L) and HSV-1 (F)-infected (E to H and M to P) HEp-2 cells (A to H) and HeLa cells (I to P). Cells were fixed in methanol at 16 h postinfection and incubated with anti-PKC, anti-lamin B, and anti-ICP4 antibody. Anti-PKC antibody was stained with FITC-conjugated secondary antibody (green channel), anti-lamin B antibody was stained with Texas Red-conjugated secondary antibody (red channel), and ICP4 was stained with Cy5-conjugated secondary antibody (blue channel).

FIG. 2.

Time course of PKC upregulation and recruitment to the nuclear rim. Digital images of confocal fluorescence micrographs were taken of HEp-2 cells infected with HSV-1 (F). At 0 h (A to C), 4 h (D to F), 8 h (G to I), 12 h (J to L), and 16 h (M to O) postinfection, cells were fixed and stained for PKC (FITC; green channel), lamin B (Texas Red; red channel), and ICP4 (Cy5; blue channel).

FIG. 3.

Digital confocal images of mock-infected or HSV-1 (F)-infected HEp-2 cells immunostained with PKC-specific antibodies. At 16 h postinfection, cells were fixed with methanol and incubated with anti-lamin B antibody, anti-ICP4 antibody, and one of the following PKC-specific antibodies: anti-PKCα (A to D), anti-PKCδ (E to H), anti-PKCζ (I to L). Anti-PKC antibodies were stained with FITC-conjugated secondary antibody (green channel), anti-lamin B antibody was stained with Texas Red-conjugated secondary antibody (red channel), and ICP4 was stained with Cy5-conjugated secondary antibody (blue channel).

FIG. 4.

Effects of U_L31 and U_L34 and their association on HSV-1-induced PKC recruitment. Digital confocal images show mock-infected HEp-2 cells (A to C) or HEp-2 cells infected with HSV-1 (F) (D to F), v3161 U_L31-null HSV-1 mutant (G to I), vRR1072 U_L34-null HSV-1 mutant (J to L), or v3480 U_L34-U_L31 association-defective mutant (M to O). At 16 h postinfection, cells were fixed and stained for PKC (FITC; green channel), lamin B (Texas Red; red channel), and ICP4 (Cy5; blue channel).

FIG. 5.

Digital images of confocal fluorescence micrographs of mock-infected HEp-2 cells (A to C) or HEp-2 cells infected with HSV-1 (F) (D to F), R7037 U_S3-null HSV-1 mutant (G to I), or vRR1204 U_S3-kinase-defective HSV-1 mutant (J to L). At 16 h postinfection, cells were fixed and immunostained for PKC (FITC; green channel), lamin B (Texas Red; red channel), and ICP4 (Cy5; blue channel).

FIG. 6.

Digital images of autoradiographs and immunoblots of immunoprecipitated lamin B and PKC labeled with [³²P]orthophosphate. HEp-2 cells were infected with HSV-1 (F), pulse-labeled with [³²P]orthophosphate, treated with PKC-specific inhibitors (C), lysed at 16 h postinfection, and immunoprecipitated with antibodies specific for lamin B (A and C) and PKC (D). Samples were electrophoretically resolved on an SDS denaturing polyacrylamide gel and processed for autoradiography. To confirm that lysates of mock-infected and HSV-1 (F)-infected cells contained nearly equal amounts of lamin B and PKC, immunoblots (B and E) of replicate crude lysates using antibodies against lamin B and PKC, respectively, were prepared according to standard protocols.

FIG. 7.

Digital images of confocal fluorescence micrographs of HSV-1 (F)-infected HeLa cells. At 16 h postinfection, cells were fixed and immunostained for lamin B (Texas Red; red channel), lamin A (FITC; green channel), and ICP4 (Cy5; blue channel).