Hsc70 focus formation at the periphery of HSV-1 transcription sites requires ICP27

Abstract

Background: The cellular chaperone protein Hsc70, along with components of the 26S proteasome and ubiquitin-conjugated proteins have been shown to be sequestered in discrete foci in the nuclei of herpes simplex virus 1 (HSV-1) infected cells. We recently reported that cellular RNA polymerase II (RNAP II) undergoes proteasomal degradation during robust HSV-1 transcription, and that the immediate early protein ICP27 interacts with the C-terminal domain and is involved in the recruitment of RNAP II to viral transcription/replication compartments.

Methodology/principle findings: Here we show that ICP27 also interacts with Hsc70, and is required for the formation of Hsc70 nuclear foci. During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed. Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed. There was also a decrease in virus yields, indicating that proteasomal degradation of stalled RNAP II complexes during robust HSV-1 transcription and replication benefits viral gene expression.

Conclusions/significance: We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

Figure 1. ICP27 Is Required for the Formation of Hsc70 Nuclear Foci.

Vero cells or 2-2 cells were either mock-infected, infected with WT HSV-1 KOS or ICP27 null mutant 27-LacZ, or were first transfected with plasmid pSG130B/S, which expresses ICP27 , and were then infected 24 h later with 27-LacZ, as indicated. All infections were performed at a moi of 10. Cells were fixed at 6 h after infection and stained with anti-Hsc70, anti-SC35, anti-ICP4 and anti-ICP27 antibodies as indicated. Antibody to the nuclear splicing SR protein SC35 was used in the mock-infected sample to define the nucleus. Arrows mark sites of Hsc70 nuclear foci (green) or ICP4-containing pre-replication sites (red). In this figure, and in all subsequent immunofluorescence images, representative cells are shown at higher magnification to better illustrate the pattern of protein localization and to observe intracellular structures. In all cases, cells shown in the figures represent the pattern observed in greater than 75% of the cells visualized in at least 10 fields.

Figure 2. ICP27 Associates with Hsc70 in vitro.

A) GST-binding assays were performed with GST-Hsc70 (1-540) and in vitro translated WT ICP27 and mutants ΔN, D2ΔS5, S5, Δ26-100, H17 and ΔC. Asterisks mark the position of the ICP27 proteins that were seen to bind. Input ³⁵S-labeled proteins are shown in the middle panel. In the bottom panel, a schematic diagram of the 512 amino acid ICP27 coding region, including the leucine-rich amino terminus (LRR), nuclear localization signal (NLS), RGG box RNA binding motif (RGG), arginine-rich region (R2), three predicted KH domains and a zinc-finger-like motif (CCHC) in the C-terminus. The positions of the deletion mutations (dotted lines) are shown below. B) GST binding assays were performed with ³⁵S-labeled, in vitro-translated WT ICP27 and GST-tagged Hsc70 truncated proteins. The arrow shows the position of ICP27. Input GST-Hsc70 proteins are shown in the middle panel. A schematic of the Hsc70 coding region is shown illustrating the nucleotide binding domain (NBD-ATPase), the substrate binding domain (SBD) and the C-terminal domain (CTD).

Figure 3. The N- and C-termini of ICP27 Are Required for Interaction with Hsc70 during Infection.

A) HeLa cells were either mock-infected or infected with WT HSV-1 or ICP27 mutants as indicated. At 6 h after infection, nuclear extracts were prepared. Expression of endogenous Hsc70 in mock-infected cells and HSV-1 WT- and mutant-infected cells is shown by Western blot analysis of samples from nuclear extracts in the top panel. Immunoprecipitations with anti-ICP27 antibody followed by Western blot analysis with anti-Hsc70 antibody are shown in the middle panel. The bottom panel shows a Western blot of anti-ICP27 immunoprecipitated samples probed with anti-ICP27 antibody to show ICP27 expression. Asterisks (*) mark WT and mutant ICP27 protein bands. The + signs mark heavy chain IgG from the immunoprecipitations. B) Reverse immunoprecipitations were performed on samples from the nuclear extracts described above. The top panel shows the expression of WT and mutant ICP27 in the nuclear extracts. Immunoprecipitation of each sample was performed with anti-Hsc70 antibody and Western blot analysis was performed by probing with anti-ICP27 antibody, as shown in the middle panel. The + sign marks heavy chain IgG from the immunoprecipitation. The bottom panel shows a Western blot of anti-Hsc70 immunoprecipitated samples probed with anti-Hsc70 antibody. C) A schematic diagram of the ICP27 coding region is shown in the left panel with the positions of the deletions illustrated by dotted lines. The table in the right panel describes the residues deleted or mutated in each mutant virus.

Figure 4. Hsc70 Nuclear Focus Formation Is Delayed in Infections with ICP27 Mutants.

A) Vero cells were infected with WT HSV-1 or mutants dLeu, n406 or n504. At the times indicated, cells were fixed and stained with anti-Hsc70 and anti-ICP27 antibodies as indicated. Arrows mark Hsc70 nuclear foci. B) Vero cells were infected with WT HSV-1, 27-LacZ, dLeu, n406 or n504 for 6 h, at which time cells were fixed and stained with anti-Hsc70 and anti-ICP0 antibodies. The arrow marks an Hsc70 focus adjacent to an ICP0 speckle.

Figure 5. ICP27 N- and C-Terminal Mutants Show Poor Replication Compartment Formation and Reduced Transcription and DNA Replication.

A) Vero cells were infected with WT HSV-1, dLeu or n406 for 8 h, at which time cells were fixed and stained with anti-RNAP II antibody ARNA3, which recognizes all forms of RNAP II, and anti-ICP4 antibody. The arrows point to a fully formed replication compartment in WT-infected cells and to small pre-replication sites in dLeu and n406-infected cells. B) HeLa cells were infected with WT HSV-1, dLeu or n406 as indicated. At 8 h after infection total RNA was extracted and microarray analysis was performed as described previously against an array of HSV-1 transcript-specific probes . Hybridizations were performed in triplicate and the experiments were performed twice. Error bars represent the standard deviations. C) Vero cells were mock-infected or were infected with WT HSV-1, 27-LacZ, dLeu or n406 at a moi of 10. At 1 h and 12 h after infection, DNA was purified from cell lysates by phenol-chloroform extraction. Quantitative real-time PCR was performed with a probe for the glycoprotein C (gC) gene to determine viral DNA copy number.

Figure 6. Hsc70 Focus Formation Correlates with HSV-1 Replication Compartment Formation.

Vero cells were infected with WT HSV-1, 27-LacZ, dLeu or n406. At the times indicated, cells were fixed and stained with anti-Hsc70 and anti-ICP4 antibodies. Arrows point to Hsc70 foci (green) or pre-replication sites (red).

Figure 7. Hsc70 Nuclear Sequestration Correlates with the Loss of Phospho-serine 2 Form of RNAP II CTD in Fully Formed Replication Compartments.

Vero cells were mock-infected or infected with WT HSV-1, dLeu or n406 for the times indicated. In the left hand panels, cells were stained with anti-RNAP II antibody, H14, which recognizes phospho-serine 5 RNAP II CTD, and anti-Hsc70 antibody. Arrows show a replication compartment with an Hsc70 focus site at the periphery in the 8 h WT-infected cell, and a pre-replication site (red) next to a small Hsc70 focus site (merge) in the 12 h dLeu-infected cell. In the right panels, cells were stained with anti-Hsc70 antibody and anti-RNAP II antibody H5, which recognizes phospho-serine-2 RNAP II CTD. This antibody also cross-reacts with a phospho-epitope in splicing SR proteins , . The arrow points to an Hsc70 focus site (green) adjacent to a splicing speckle (red).

Figure 8. Preventing Phospho-serine 2 RNAP II Degradation Also Prevents Hsc70 Focus Formation.

Vero cells that were mock-infected or were infected with WT HSV-1 or 27-LacZ were untreated (left panels) or were treated with 50 µM MG132 added 1 h after infection (right panels). Cells were fixed at 8 h after infection and stained with anti-Hsc70 antibody, antibody H5 and anti-ICP4 antibody as indicated. Arrows mark Hsc70 foci (green) or replication or pre-replication sites (red).

Figure 9. An Hsc70 Dominant Negative Mutant Can Prevent RNAP II Phospho-serine 2 Degradation and Hsc70 Focus Formation.

A) Vero cells were transfected with pGFP-Hsc70 and were subsequently infected with WT HSV-1 for 8 h. Cells were stained with H5, anti-ICP4 or anti-ICP8 antibodies. GFP fluorescence was viewed directly. Arrows point to Hsc70 foci. B) Vero cells were transfected with the dominant negative mutant GFP-Hsc70K71M and then infected with WT HSV-1 for 8 h. Cells were stained with H5, anti-ICP4 or anti-ICP8 antibodies. Arrows point to GFP Hsc70K71M expressing cells showing diffuse nuclear staining with H5, and to pre-replication sites in cells stained for ICP4 and ICP8. The yellow arrows in the ICP4 and ICP8 stained panels point to a fully formed replication compartment.

Figure 10. Dominant Negative Mutant Hsc70 K71M Prevents Proteasomal Degradation of Phospho-Serine 2 RNAP II CTD.

A) RSF cells were transfected with vector alone (-), Myc-tagged Hsc70 or Myc-tagged Hsc70K71M and 24 h later were either mock-infected or were infected with WT HSV-1 or 27-LacZ. Nuclear extracts were prepared 8 h after infection. Western blots were probed with antibody H5 to detect the phospho-serine 2 form of RNAP II; anti-myc antibody to detect myc-tagged Hsc70; anti-ICP27 antibody to monitor ICP27 expression, and anti-YY1 antibody as a loading control. YY1 is a cellular transcription factor. B) HeLa cells were either mock-infected or infected with WT HSV-1. MG132 (50 µM) was added at the times indicated and nuclear extracts were prepared at 8 after infection. Western blots were probed with H5 antibody to detect RNAP II phospho-serine 2; ARNA3 antibody, which recognizes all forms of RNAP II, both phosphorylated and unphosphorylated; anti-ICP27 antibody, and anti-YY1 antibody. C) RSF cells were transfected with empty vector alone (control), myc-tagged Hsc70 or myc-tagged Hsc70K71M as indicated, and were infected 24 h later with WT HSV-1. Nuclear extracts were prepared 8 h after infection, and samples were fractionated directly by SDS-PAGE for Western blot analysis (left panel, NE) or were immunoprecipitated with anti-ubiquitin antibody (right panel, IP- anti-Ub). Immunoprecipitated complexes were fractionated by SDS-PAGE and the Western blot was probed with anti-RNAP II antibody ARNA3. Arrows point to slower migrating ubiquitinated forms of RNAP II. D) RSF cells were transfected with empty vector alone (control), myc-tagged Hsc70 or myc-tagged Hsc70K71M and were infected 24 h later with WT HSV-1. Cells were harvested 24 h after infection and viral titers were determined by plaque assays.

Figure 11. Model of RNAP II Degradation during HSV-1 Transcription.

ICP27 interacts with the CTD of RNAP II and recruits RNAP II to a viral replication compartment , , which is represented in magenta. Elongating RNAP II complexes on opposite DNA strands may collide causing one or both complexes to stall. At least one of the stalled complexes may then be ubiquitinated and degraded by the proteasome. The other transcription complex is now free to continue. Hsc70 foci, which form at the same time as replication compartments, and which lie at their periphery, may aid this process by providing a ready source of chaperone proteins as well as other components of the 26S proteasome , .