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. 2007 Oct 23;104(43):17134-9.
doi: 10.1073/pnas.0707266104. Epub 2007 Oct 15.

Herpes simplex virus-infected cell protein 0 blocks the silencing of viral DNA by dissociating histone deacetylases from the CoREST-REST complex

Haidong Gu  1 , Bernard Roizman
Affiliations

Herpes simplex virus-infected cell protein 0 blocks the silencing of viral DNA by dissociating histone deacetylases from the CoREST-REST complex

Haidong Gu et al. Proc Natl Acad Sci U S A. .

Abstract

A preeminent phenotype of the infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is that it acts as a promiscuous transactivator. In most cell lines exposed to DeltaICP0 mutant virus at low ratios of virus per cell infection, alpha genes are expressed but the transition to beta and gamma gene expression does not ensue, but can be enhanced by inhibitors of histone deacetylases (HDACs). Earlier studies have shown that ICP0 interacts with CoREST and displaces HDAC1 from the CoREST-REST-HDAC1/2 complex. HDAC1 and CoREST are then independently translocated to the cytoplasm. Here, we test the hypothesis that ICP0 blocks the silencing of HSV DNA by displacing HDAC1 from the CoREST-REST complex. Specifically, first, mapping studies led us to construct a truncated CoREST (CoREST(146-482)) that in transfected cells displaced HDAC1 from the CoREST-REST complex. Second, we constructed two viruses. In BACs encoding the entire HSV-1, we replaced the gene encoding ICP0 with AmpR to yield a DeltaICP0 mutant R8501. We also replaced ICP0 with CoREST(146-482) to yield recombinant R8502. The yield of R8502 mutant virus in Vero, HEp-2, and human embryonic lung cells exposed to 0.1 pfu of virus per cell was 100-, 10-, and 10-fold higher, respectively, than those of R8501 mutant virus. In Vero cells, the yield of R8502 was identical with that of wild-type virus. We conclude that CoREST(146-482) functionally replaced ICP0 and that, by extension, ICP0 acts to block the silencing of viral DNA by displacing HDAC1/2 from the CoREST-REST complex.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic representation of early events in HSV-1-infected cells. (A) Cell infected with a ΔICP mutant at low pfu per cell. The schematic diagram illustrates that VP16 enters the nucleus concomitantly with the release of viral DNA from capsids at the nuclear pore. VP16 associates with cellular proteins Oct1 and HCF and transactivates α gene promoters. α proteins are made, but the expression of β and γ genes does not ensue. (B) Cell infected with wild-type virus. As reported earlier, ICP0 interacts with CoREST and displaces HDAC1 from the CoREST–REST complex. HDAC1 and CoREST are phosphorylated and translocated to the cytoplasm. β and γ genes are expressed. (C) Predicted role of a dominant-negative CoREST that dissociates HDAC1 from the CoREST–REST complex. If ICP0 enables the transition from α to β and γ gene expression by dissociating HDAC1/2 from the CoREST–REST complex, then a dominant-negative CoREST that displaces HDAC1 from the complex would substitute for ICP0 to enable enhanced replication of ΔICP0 mutant virus.
Fig. 2.
Fig. 2.
The position of key domains in CoREST and ICP0 relevant to these studies. (A) The numbers 1–3 identify the residues encoded in exons 1–3. The numbers above the bar refer to residue numbers. The sequence homologous to residue 3–79 of CoREST is at position 537–613. The binding site for CoREST determined on the basis of SI Fig. 7 is between residues 668 and 718. (B) Location of key domains in CoREST. The site of ICP0 homologous sequences (residues 3–79) are also the site of binding to ICP0 on the basis of the data shown in SI Fig. 8. The numbers below the bar indicate amino acid number. The binding site for HDAC1 is based on results shown in SI Fig. 9.
Fig. 3.
Fig. 3.
CoREST146–482 polypeptide displaces HDAC1 from the HDAC1–CoREST–REST complex. HEK 293 cells were transfected with plasmids encoding the truncated CoREST polypeptides tagged with Myc. After 40 h of incubation, the cells were harvested and lysed. The immune precipitates (IPs) obtained with polyclonal anti-HDAC1 antibody (A) or polyclonal CoREST antibody (B) were collected as described in Experimental Procedures, solubilized, electrophoretically separated in a denaturing gel, and reacted with monoclonal antibody against CoREST (A) or anti-HDAC1 or anti-REST antibodies (B).
Fig. 4.
Fig. 4.
Construction and verification of recombinant viruses. (A) Schematic representation of wild-type HSV-1, the sequence arrangements of the α0 gene encoding ICP0. The third line shows the replacement of ICP0 with AmpR. The fourth line shows the replacement of ICP0 with a Myc-tagged CoREST146–482 gene. The procedures were as described in Experimental Procedures. (B) Expression of CoREST146–482 in Vero cells infected with 10 pfu of R8501 or R8502 mutant virus per cell. As shown, lysates of R8502 reacted with antibodies to the truncated and endogenous CoREST, whereas the lysates of cells infected with the R8501 mutant reacted only with antibody to endogenous CoREST. (C) Electrophoretically separated lysates of cells harvested at times shown after infection with 10 pfu per cell were probed with antibodies to ICP0, ICP4, or ICP27.
Fig. 5.
Fig. 5.
The replication of wild-type HSV-1 and of mutant viruses R8501 and R8502 in Vero, HEp-2, and HEL cells. Replicate cultures were exposed to 0.1 pfu per cell. The cultures were harvested at 3, 11, 24, and 48 h after infection for A; 3, 7, 24, and 48 h for B; 5, 8, 12, 24, 40, and 64 h for C; 3, 11, 24, and 48 h for D; and 3, 11, 24, and 48 h for E. The yields shown are based on titrations on USOS cells.
Fig. 6.
Fig. 6.
Schematic representations of the interactions between ICP0 and HDAC1/2–CoREST–REST complex and the predicted structure of ICP0. (A) The sequence of ICP0 showing the residues in the binding sites for CoREST (red) and the sequence homologous to residues 3–79 of CoREST (brown). (B) Hypothetical structure of ICP0 in the absence of CoREST (closed conformation) and in the presence of CoREST (open conformation).
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