Human cytomegalovirus UL50 and UL53 recruit viral protein kinase UL97, not protein kinase C, for disruption of nuclear lamina and nuclear egress in infected cells

Abstract

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them.

FIG 1

Construction of HCMV UL50 and UL53 null mutants. (A) Organization of the HCMV genome. TR_L, terminal repeat long; U_L, unique long; IR_L/S, internal repeat long and neighboring internal repeat short; U_S, unique short; TR_S, terminal repeat short. Below the top schematic, the region of the viral genome from UL49 to UL54 is expanded, showing the overlaps between UL49 and UL50 and between UL52, UL53, and UL54. Below this, the mutations introduced into the UL50 and UL53 coding sequences for generation of 50N and 53N pBADGFP are shown. Amino acids that were mutated to stop codons (dashes) are underlined. Extra nucleotides are shown in bold. nt, nucleotide; aa, amino acid. (B) Construction of HCMV UL53-FLAG AD169-RV. Fusion of sequences encoding the FLAG tag to sequences encoding the C terminus of UL53 in a BAC of AD169 HCMV was performed. The UL52-UL53-UL54 region of the viral genome is expanded to show the details of the UL53 C-terminal sequence overlap with UL54 in the original wild-type (WT) genome. The stop codons for UL53 and UL54 are indicated by asterisks. The duplicated sequence is underlined, and the FLAG sequence is indicated in bold.

FIG 2

CLEM analysis of HCMV UL50 and UL53 null mutants. HFF cells were electroporated with UL50 null (50N) (A to D) or UL53 null (53N) (E to H) pBADGFP or the rescued derivative 50NR (I) or 53NR pBADGFP (J) and were seeded on gridded coverslips. Phase and fluorescence microscopy was performed on electroporated cells (A and E) on day 7 postelectroporation, and the cells were then processed for EM (B to D and F to J). The original GFP-positive cells (A and E) were traced back, and serial sections were scanned for nuclear and cytoplasmic nucleocapsids, for each sample. The insets in panels B, C, F, and G indicate regions magnified in the next panels (C, D, G, and H, respectively). Bars, 500 nm. N, nucleus; Cyt, cytoplasm; cB and cC, cytoplasmic B and C capsids, respectively. Arrows A, B, and C point to examples of nuclear A, B, and C capsids.

FIG 3

Nuclear lamina structure in the absence of HCMV UL50 or UL53. HFFs were mock electroporated (A to D) or electroporated with WT (E to H), UL50 null (50N) (I to L), or UL53 null (53N) (M to P) pBADGFP. Cells were fixed on day 7 and stained with antibody against lamin A/C (B, F, J, and N), the nucleus was stained with DAPI (C, G, K, and O), and cells were visualized using confocal microscopy, with GFP-positive cells (E, I, and M) identified by green fluorescence.

FIG 4

PKC distribution in HSV-1- or HCMV-infected cells. Vero cells were mock infected [Mock (V)] (A to C) or infected with HSV-1 (D to F) at an MOI of 1, and HFFs were mock infected [Mock (H)] (G to I) or infected with HCMV (J to L) at an MOI of 1. Vero cells were fixed at 16 h, and HFFs were fixed at 72 h postinfection. Samples were stained with a pan-PKC antibody (red) and DAPI (blue) and visualized by confocal microscopy.

FIG 5

Cellular distribution and nuclear rim localization of cellular kinases PKC and Cdk-1 during HCMV infection. (A) HFFs were mock infected (i to iv) or infected with HCMV (v to viii) at an MOI of 1. At 72 h p.i., cells were fixed and stained for lamin B (green) and PKC (red). The samples were visualized using confocal microscopy. (B) HFFs were mock infected (i) or infected with HCMV (ii and iii) at an MOI of 1. At 72 h p.i., cells were fixed and stained for lamin B (green) and Cdk-1 (red). The insets indicate magnified (×3) sections of the respective images. (C) Cells showing colocalization (shaded bars) or no colocalization (unshaded bars) for the cellular kinases and lamin B were counted among the infected (I) and mock-infected (M) samples. The data were analyzed using two-tailed Fisher's exact test, and the P values for the differences between the mock-infected and infected samples are shown.

FIG 6

Cellular distribution of PKC in the absence of HCMV UL50 or UL53. HFFs were mock electroporated (A to D) or electroporated with WT (E to H), UL50 null (50N) (I to L), or UL53 null (53N) (M to P) pBADGFP. Cells were fixed on day 7 and stained with antibody against PKC (red), and the nucleus was stained with DAPI (blue). Cells positive for GFP (green) were visualized by confocal microscopy.

FIG 7

Cellular distribution of UL97 in the absence of HCMV UL50 or UL53. (A) HFFs were mock electroporated (i to iv) or electroporated with WT (v to ix), UL50 null (50N) (x to xiv), or UL53 null (53N) (xv to xix) pBADGFP expressing FLAG epitope-tagged UL97. HFFs were also electroporated with rescued derivatives 50NR and 53NR. Cells were fixed on day 7 and stained with antibodies against FLAG (red) and lamin B (green). GFP-positive cells were located by confocal microscopy. Panels ix, xiv, and xix represent magnified (×3) sections of the insets in the panels to their left. White arrows point at regions of colocalization. (B) For statistical analysis, 10 infected (GFP-positive) cells from each electroporation were analyzed for whether they showed any points of colocalization (shaded bars) between FLAG-UL97 and lamin B or no observable colocalization (unshaded bars). The difference between the data for cells infected with 50N versus 50NR was analyzed using one-tailed Fisher's exact test, and the P value is shown. (C) The number of foci of FLAG-UL97 and lamin B colocalization was counted in representative optical sections of 10 cells/sample. Differences in these data between cells infected with a mutant versus its rescued derivative were analyzed using one-way analysis of variance (ANOVA) followed by Sidak's multiple-comparison test, and P values are shown.

FIG 8

Association of UL97 with UL50 and UL53. (A) HFFs were infected with FLAG-UL97 BADGFP at an MOI of 1. At 72 h p.i., cells were fixed and stained for FLAG (green) and either UL50 (red) (i to iv) or UL53 (red) (v to viii). The samples were visualized using confocal microscopy. The insets indicate magnified (×3) sections of the respective images. Arrows show areas of colocalization. (B) Nuclear lysates were obtained at 72 h p.i. from HFF cells infected with HCMV AD169-RV (WT) or UL53-FLAG AD169-RV (53F) at an MOI of 1. Lysates were precleared and incubated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads. Bound proteins were eluted using low pH and analyzed by Western blotting using antibodies against the proteins indicated to the right of the panel.