Modification and reorganization of the cytoprotective cellular chaperone Hsp27 during herpes simplex virus type 1 infection

Abstract

Chaperone-enriched domains are formed in the nuclei of cells lytically infected with herpes simplex virus type 1 (HSV-1). These domains, called VICE, for virus induced chaperone enriched, contain Hsc70, Hsp70, Hsp40, Hsp90, polyubiquitinated proteins, and components of the proteasome machinery. Accumulating evidence indicates that these sites may be utilized during infection to sequester misfolded, modified, or otherwise unwanted proteins away from viral replication compartments, sites of robust transcription, DNA synthesis, and capsid maturation. To further explore the role of cellular chaperones and VICE domains during HSV-1 infection, we have analyzed the cytoprotective chaperone Hsp27. Here we present evidence that Hsp27, which is known to possess several antioxidant functions, is rapidly reorganized and modified at early stages in response to HSV-1 infection and signaling from the mitogen-activated protein kinase p38. Immunofluorescence analysis and fractionation experiments reveal disparate subcellular localizations of nonphosphorylated and phosphorylated forms of Hsp27 during wild-type HSV-1 infection. Unmodified forms of Hsp27 are localized in nuclear foci that are outside of replication compartments, adjacent to VICE domains, and in the cytoplasm. Conversely, we find that phosphorylated forms of Hsp27 are localized exclusively in the cytoplasm. Last, in cells depleted of all forms of Hsp27, virus replication is significantly reduced.

FIG. 1.

Hsp27 localization in HSV-1-infected cells. (A) Immunofluorescence analysis of cellular chaperones at 6 h in uninfected cells (a to d) or HSV-1-infected cells (e to h). Shown are staining profiles for DNA using DAPI (blue in frames a and e and the merged images), the host chaperone Hsc70—a marker of VICE domains (red in frames b and f and the merged images), and Hsp27 (green in frames c and g and the merged images). The Hsp27 antibody used in this experiment recognizes all forms of Hsp27 (unmodified and phosphorylated). Composite images showing the three signals merged are shown in frames d and h. The inset in frame h shows a magnified view of VICE domains and Hsp27 foci. (B) Time course immunofluorescence analysis of Hsp27. Staining profiles of Hsp27 at 2, 4, 6, and 12 h (a to d, respectively) after HSV-1 infection are shown. White arrows show Hsp27 foci in the nucleus during viral infection.

FIG. 2.

Hsp27 focus formation and modification and p38 activity during HSV-1 infection. (A) Merged images of staining profile for the cellular chaperone and marker for VICE domains, Hsc70 (red) and Hsp27 species (green), in uninfected (a and c) or HSV-1-infected cells (b and d) fixed at 6 h are shown. Cells shown in frames c and d have been treated with the p38 inhibitor SB during the time of infection (6 h). White arrows show Hsp27 foci. (B) Western blotting analysis of phosphorylated Hsp27 (Ser15) (upper panel) in HSV-1-infected or mock-infected Vero cell lysates. Cells were either left untreated (−SB) or treated with SB. Samples for 1-, 2-, 4-, 6-, and 24-h time points were collected. The slower-migrating phospho-reactive form is indicated by a black arrowhead. The viral ICP8 protein (middle panel) is used as a marker for infection, and the cellular L26 protein is used as a loading control (bottom panel). (C) Western blotting of phosphorylated Hsp27 (Ser78 and Ser82) of HSV-1-infected Vero cell lysates. Cells were either left untreated (−SB) or treated with 10 μM SB (+SB). Samples for 1-, 2-, 4-, 6-, and 24-h time points were collected. The slower-migrating phospho-reactive form is indicated by a black arrowhead. A faster-migrating cross-reacting species detected in ppHsp27Ser15 Western blots is indicated with an asterisk. Molecular weight markers are indicated on the right. “α” indicates antibody.

FIG. 3.

Immunofluorescence analysis of phospho-reactive species of Hsp27 in HSV-1-infected cells. Merged images of staining profiles for the viral single-strand DNA binding protein (ICP8; red) and phospho-reactive Hsp27 species (ppHsp27; green) in uninfected (a, d, and g) or HSV-1-infected (b, e, and h) cells collected at 6 h are shown. Deconvolved images of each ppHsp27 species are shown in panels c, f, and i.

FIG. 4.

Immunofluorescence time course analysis of Hsp27 and phosphorylated Hsp27 in virus-infected cells. Immunofluorescence analysis of cellular chaperones at various time points in uninfected cells (top row, Mock) or HSV-1-infected cells (bottom three rows) is shown. Infected cells were collected at 4, 6, and 10 h after infection with HSV-1. Uninfected cells were fixed at 10 h after the start of the infection. Staining profiles for Hsp27 (red), for phospho-reactive Hsp27 species (ppHsp27Ser15; green), and for DNA using DAPI (blue) are shown. Merged images are shown in the last column. Hsp27 localization at nuclear foci is observed as early as 6 h after infection (white arrows in frames i and l).

FIG. 5.

Localization of phosphorylated Hsp27 during HSV-1 infection. Nuclear (N; lanes 1, 3, and 5) and cytosolic (C; lanes 2, 4, and 6) fractions from HSV-1-infected (lanes 1 to 4) or uninfected (lanes 5 and 6) cells were collected and subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. Western blots of viral (ICP8) or cellular (ppHsp27Ser15/78/82, Hsp27, and γ-tubulin) proteins are shown. The viral ICP8 protein is used as a marker for infection and an indicator of the quality of fractionation because it is a nuclear protein. “α” indicates antibody.

FIG. 6.

Hsp27 knockdown and HSV-1 virus yield in Hsp27-depleted cells. (A) Western blot analysis of Hsp27 and γ-tubulin proteins in cells treated with either Hsp27 siRNA (lanes 3 and 4), irrelevant siRNA against cyclophilin D (irr. si, lane 1), or a nonspecific siRNA (ns si, lane 2). Untreated infected (lane 5) or uninfected (lane 6) cells are shown. (B) Western blot analysis of Hsp27, ppHsp27Ser15, and γ-tubulin proteins in untreated cells (UT, lanes 1, 3, and 5) or in cells treated with Hsp27 siRNA (si, lanes 2, 4, and 6). Western blot analysis was performed on infected-cell lysates collected at 4 h (lanes 3 and 4) or 12 h (lanes 5 and 6). (C) Immunofluorescence analysis (IFA) of Hsp27 levels in untreated (left) or Hsp27 siRNA-treated (right) cells. (D) Multistep growth analysis of HSV-1 (MOI, 0.1 PFU/ml) in no siRNA-treated, nontargeting siRNA-treated, or Hsp27 siRNA-treated cells at 24, 48, or 72 h postinfection. The asterisks represent statistically significant differences in growth of HSV-1 in the Hsp27-depleted cells compared to that with the other treatments (P values of <0.05) from two independent experiments. “α” indicates antibody.