Significance of host cell kinases in herpes simplex virus type 1 egress and lamin-associated protein disassembly from the nuclear lamina

Abstract

The nuclear lamina is thought to be a steric barrier to the herpesvirus capsid. Disruption of the lamina accompanied by phosphorylation of lamina proteins is a conserved feature of herpesvirus infection. In HSV-1-infected cells, protein kinase C (PKC) alpha and delta isoforms are recruited to the nuclear membrane and PKC delta has been implicated in phosphorylation of emerin and lamin B. We tested two critical hypotheses about the mechanism and significance of lamina disruption. First, we show that 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 does not inhibit viral growth. Second, we show hyperphosphorylation of emerin by viral and cellular kinases is required for its disassociation from the lamina. These data support hypothesis that phosphorylation of lamina components mediates lamina disruption during HSV nuclear egress.

Figure 1. PKC activity required for HSV-1 replication

Replicate cultures of HEp-2 cells were infected at an MOI of 5 with HSV-1(F) A) Cells were treated with DMSO or 10uM pan-PKC inhibitor bis-indolylmaleimide (BIM I) beginning at five hours post infection (hpi). Titer was determined at 24 hpi. Virus yields are expressed as plaque forming units (PFU) per milliliter. Each data point represents the mean of five experiments. Error bars indicate standard deviations. p-values were determined via a paired Student T-Test.

Figure 2. Inhibition of PKC blocks extra-nuclear capsid accumulation

Shown are digital images of transmission electron micrographs (TEM) of Vero cells infected with five PFU/cell HSV-1(F). Cells were treated with vehicle beginning at five hpi with vehicle (A) or BIM I (B) fixed with glutaraldehyde at 16 hpi, and thin sectioned for TEM. The inset picture is approximately twice the magnification and shows an example of an empty capsid (A and B capsids) (arrow) and a full capsid (C capsid) (arrow head).

Figure 3. PKC inhibitor inhibits cellular but not viral protein synthesis

A) Digital images of western blots of HEp-2 unlabeled total cell lysates prepared from mock-infected cells or cells infected with five PFU/cell of HSV-1(F). At 16 hpi, total cell lysates were prepared from equal numbers of cells. Cells were DMSO (D) or BIM I (B) treated beginning at five hpi. Blots were probed with monoclonal antibodies to VP5, pUL34, gC, and US11. B) Autoradiogram of ³⁵S-methionine labeled HEp-2 cell lysates from A.

Figure 4. cPKCs nor PKC delta are uniquely required for HSV-1 replication

A) Cells were treated vehicle (DMSO) or 1 uM Ro-31-7549 beginning at five hpi. Titer was determined at 24 hpi. Virus yields are expressed as plaque forming units (PFU) per milliliter. Each data point represents the mean of six experiments. B) Vero cells were co-transfected for 24 hr with pCMVβ and either: pRR1072, pRR1072Rep, PRKCDwt-UL34 Duo or PRKCDdn-UL34 Duo. Complementation of the vRR1072(tk+) was performed five times as previously described (Bjerke et al., 2003). Error bars indicate standard deviations. p-values were determined via a paired Student T-Test. C) Digital images of western blots probed with polyclonal antibody to PKC delta (top) and monoclonal antibody to actin (bottom). Equal amounts of total protein from total cell lysates from MCF-7 or BT-549 cells were probed. D) Replicate cultures of MCF-7 and BT-549 cells were infected at an MOI of 5 with HSV-1(F). At 24 hpi total culture virus was titrated on Vero cells. Virus yields are expressed as plaque forming units (PFU) per milliliter. Each data point represents the mean of three experiments. Error bars indicate standard deviations.

Figure 5. Disruption of emerin localization in HSV-1-infected HEp-2 cells is sensitive to pUS3-mediated and Rottlerin sensitive kinase phosphorylation events but not BIM I treatment or PKC delta activity

A) Shown are digital confocal images of optical sections taken near the middle of the nuclei of HEp-2 cells that were uninfected (a–c) or infected with WT HSV-1(F) (d–i) or pUS3 kinase-dead (vRR1204) (j–o) with five PFU/cell for 20 hr and treated with vehicle (a–f and j–l) or Rottlerin (g–i and m–o) beginning at five hpi. Cells were stained with antibodies directed against emerin (left column) or pUL34 (center column). Emerin is represented in red and pUL34 in green. B) Digital image of western blot for emerin in HEp-2 cell nuclear lamina preparations. Cells were mock infected or infected with five PFU/cell of either WT or pUS3 kinase-dead (vRR1204) HSV-1(F). At five hpi, cells began treatment with vehicle (D), BIM I (B), or Rottlerin (R) and were harvested for lamina preparations at 16 hpi. The arrow in lane four indicates the predominant middle species, while the dots in lanes five, six, and seven indicate double or single emerin species. C) Shown are digital confocal images of optical sections taken near the middle of the nuclei of HEp-2 cells that were uninfected (a–c), HSV-1(F) (d–i), or pUS3 kinase-dead (vRR1204) (j–o) infected with five PFU/cell for 20 hr and treated with vehicle (a–f and j–k) or BIM I (g–i, and m–o) beginning at five hpi. Cells were stained with antibodies directed against emerin (left column) or pUL34 (center column). Emerin is represented in green and pUL34 in red. D) Digital confocal images taken near the center of nuclei of EGFP-Emerin HEp-2 B2 cells. Cells were un-transfected (a, b, c) or transfected with PRKCDwt-UL34 Duo (d, e, f) or PRKCDdn-UL34 Duo (g, h, i) for 24 hr. Cells were then infected with pUS3 kinase-dead (vRR1204) at an MOI of 20 for 20 hr. Cells were fixed with formaldehyde and stained with antibodies directed against FLAG-PKC delta (middle column) or pUL34 (right column). Emerin is represented in green, PKC delta in blue, and pUL34 in red.

Figure 6. Model of HSV-1 induced nuclear lamina disruption

(1) In the un-infected cell, emerin is bound to the lamins via interaction with lamin A/C. Emerin is intended to be a prototype LAP protein and may behave in the cell similarly to MAN1 or other LAPs. (2) Upon infection with HSV-1, pUL34 and pUL31 are expressed and accumulated as a complex at the inner nuclear membrane (INM). pUL34/pUL31 recruits pUS3, the alpha-herpesvirus viral kinase, PKCs, and Rottlerin Sensitive Kinases (RttSK) to the NE to phosphorylate emerin, lamins and pUL34/pUL31. Arrows indicate that pUS3 and RttSK are known to be necessary for phosphorylation of emerin, pUL34/pUL31, and lamin B. (3) Phosphorylation of lamins and emerin, prevents there binding and creates flexibility within the lamina necessary for nuclear egress. PKCs, RttSK, and pUS3 are depicted near the INM due to their localization at the NE during infection.