Herpes simplex virus 1 gene expression is accelerated by inhibitors of histone deacetylases in rabbit skin cells infected with a mutant carrying a cDNA copy of the infected-cell protein no. 0 - PubMed (original) (raw)

Herpes simplex virus 1 gene expression is accelerated by inhibitors of histone deacetylases in rabbit skin cells infected with a mutant carrying a cDNA copy of the infected-cell protein no. 0

Alice P W Poon et al. J Virol. 2003 Dec.

Abstract

An earlier report showed that the expression of viral genes by a herpes simplex virus 1 mutant [HSV-1(vCPc0)] in which the wild-type, spliced gene encoding infected-cell protein no. 0 (ICP0) was replaced by a cDNA copy is dependent on both the cell type and multiplicity of infection. At low multiplicities of infection, viral gene expression in rabbit skin cells was delayed by many hours, although ultimately virus yield was comparable to that of the wild-type virus. This defect was rescued by replacement of the cDNA copy with the wild-type gene. To test the hypothesis that the delay reflected a dysfunction of ICP0 in altering the structure of host protein-viral DNA complexes, we examined the state of histone deacetylases (HDACs) (HDAC1, HDAC2, and HDAC3). We report the following. (i) HDAC1 and HDAC2, but not HDAC3, were modified in infected cells. The modification was mediated by the viral protein kinase U(S)3 and occurred between 3 and 6 h after infection with wild-type virus but was delayed in rabbit skin cells infected with HSV-1(vCPc0) mutant, concordant with a delay in the expression of viral genes. (ii) Pretreatment of rabbit skin cells with inhibitors of HDAC activity (e.g., sodium butyrate, Helminthosporium carbonum toxin, or trichostatin A) accelerated the expression of HSV-1(vCPc0) but not that of wild-type virus. We conclude the following. (i) In the interval in which HSV-1(vCPc0) DNA is silent, its DNA is in chromatin-like structures amenable to modification by inhibitors of histone deacetylases. (ii) Expression of wild-type virus genes in these cells precluded the formation of DNA-protein structures that would be affected by either the HDACs or their inhibitors. (iii) Since the defect in HSV-1(vCPc0) maps to ICP0, the results suggest that this protein initiates the process of divestiture of viral DNA from tight chromatin structures but could be replaced by other viral proteins in cells infected with a large number of virions.

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Figures

FIG. 1.

FIG. 1.

Electrophoretic profiles of HDAC1, HDAC2, and HDAC3 in wild-type HSV-1(F) virus-infected cells at early and late times after infection. Replicate cultures of HEp2, SK-N-SH, 143TK−, HFF, RSC, HeLa, or Vero cells in 25-cm2 flasks were either mock infected or infected with 5 PFU of HSV-1(F) per cell. The cells were harvested at early (3 h) or late (18 h [**] or 24 h [*]) times after infection. Proteins were solubilized in disruption buffer and electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, blocked with 5% nonfat milk, and reacted with polyclonal antibodies to HDAC1, HDAC2, and HDAC3 as described in Materials and Methods. The positions of posttranslationally modified protein bands are indicated by the solid circles to the right of the blots.

FIG. 2.

FIG. 2.

Electrophoretic profiles of HDAC1 and HDAC2 in RSC infected with wild-type and mutant viruses at early and late times after infection. Replicate cultures of RSC in 25-cm2 flasks were either mock infected (lane 7) or infected with wild-type HSV-1(F), HSV-1 (17), HSV-1(KOS), or HSV-2(G), mutant HSV-1(vCPc0), or repaired HSV-1(vCPc0)R. Cell cultures were infected with 10 PFU of virus per cell. Cells were harvested at 6.5 h (lanes 1 to 6) or 21 h (lanes 7 to 13) after infection. Proteins were solubilized in disruption buffer and electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, blocked with 5% nonfat milk, and reacted with polyclonal antibodies to HDAC1 and HDAC2 as described in Materials and Methods. The positions of HDAC1 and HDAC2 and of bands reacting with HDAC2 antibody (V) are indicated to the right of the gel.

FIG. 3.

FIG. 3.

Modification of HDAC1 and HDAC2. (A) Processing of HDAC1 and HDAC2 is enhanced by viral protein kinase US3. Replicate cultures of RSC in 25-cm2 flasks were either mock infected or infected with wild-type HSV-1(F) or mutant R7356 (ΔUL13), R7041 (ΔUS3), R7353 (ΔUL13/ΔUS3), or R7802 (Δα22). Cell cultures were infected with 5 PFU of virus per cell. At 3 h after infection, one set of cultures was replenished with fresh untreated medium (− lanes), whereas a second set was replenished with medium containing 10 μM MG132 (+ lanes). The cells were harvested 20 h after infection. Proteins were solubilized in disruption buffer, electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, blocked with 5% nonfat milk, and reacted with polyclonal antibodies to HDAC1 and HDAC2, and monoclonal antibody to ICP0. The positions of HDAC1 and HDAC2 and of bands reacting with HDAC2 antibody (V) and with monoclonal antibody to ICP0 are indicated to the right of the gel. (B) Complementation of ΔUS3 mutants by baculovirus expressing US3. Replicate RSC cultures in 25-cm2 flasks were transduced with baculoviruses that were either empty of inserts (lanes 3 and 4) or encoding the US3 protein kinase (lanes 5 to 9). At 12 h after transduction, cells were mock infected (lanes 3 and 5) or infected with wild-type HSV-1(F) (lanes 4 and 6) or mutant virus R7356 (ΔUL13), R7041 (ΔUS3), or R7353 (ΔUL13/ΔUS3) (lanes 7 to 9). Cell cultures were infected with 5 PFU of virus per cell. At 12 h after infection with wild-type HSV-1 or mutant viruses, the cultures were harvested and processed as described above except that cell lysates were reacted with anti-US11 antibody rather than ICP0. Lanes 1 and 2 show protein profiles of mock-infected and wild-type virus-infected cell cultures not previously exposed to baculoviruses.

FIG. 4.

FIG. 4.

Temporal pattern of accumulation of selected wild-type HSV-1(F) and mutant HSV-1(vCPc0) proteins in RSC treated with sodium butyrate (NaBu) (A) at the time of infection and (B) 11 h before infection. (A) Replicate cultures of RSC in 25-cm2 flasks were either mock infected (lanes 1 and 8) or infected with 5 PFU of wild-type HSV-1(F) per cell (lanes 2 to 7) or with 5 PFU of mutant HSV-1(vCPc0) per cell (lanes 9 to 14) in the absence (−) or presence (+) of 6 mM sodium butyrate. The cells were harvested at the indicated times after infection. Proteins were solubilized in disruption buffer, electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, blocked with 5% nonfat milk, and reacted with polyclonal antibody to ICP22 and monoclonal antibodies to ICP4 and US11. (B) Replicate cultures of RSC in 25-cm2 flasks were either mock treated (lanes 1 to 3, 7, and 9 to 11) or treated (lanes 4 to 6, 8, and 12 to 14) with 6 mM sodium butyrate. At 11 h after treatment, cells were either mock infected (lanes 7 and 8) or infected with 5 PFU of wild-type HSV-1(F) per cell (lanes 1 to 6) or with 5 PFU of mutant HSV-1(vCPc0) per cell (lanes 9 to 14) in the absence (−) or presence (+) of 6 mM sodium butyrate. Cells were harvested at the indicated times after infection. Proteins were solubilized in disruption buffer, electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, blocked with 5% nonfat milk, and reacted with polyclonal antibody to ICP22 and monoclonal antibodies to ICP4 and US11.

FIG. 5.

FIG. 5.

Temporal pattern of accumulation of selected wild-type HSV-1(F) and mutant HSV-1(vCPc0) proteins in RSC treated with H. carbonum (HC) toxin (A) and trichostatin A (B) 11 h before infection. (A) Replicate cultures of RSC in 25-cm2 flasks were either mock treated (−) or treated (+) with 70 ng of H. carbonum toxin per ml. At 11 h after treatment, cells were either mock infected (lanes 1 and 2) or infected with 5 PFU of wild-type HSV-1(F) per cell (lanes 3 to 8) or with 5 PFU of mutant HSV-1(vCPc0) per cell (lanes 9 to 16) in the absence (−) or presence (+) of 70 ng of H. carbonum toxin per ml. Cells were harvested at the indicated times after infection. Proteins were solubilized in disruption buffer, electrophoretically separated in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, blocked with 5% nonfat milk, and reacted with polyclonal antibody to ICP22 and monoclonal antibodies to ICP0, ICP4, and US11. (B) The experiment was repeated with 150 ng of trichostatin A (TSA) per ml, instead of H. carbonum toxin.

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References

    1. Ackermann, M., M. Sarmiento, and B. Roizman. 1985. Application of antibody to synthetic peptides for characterization of the intact and truncated α22 protein specified by herpes simplex virus 1 and the R325 α22− deletion mutant. J. Virol. 56:207-215. - PMC - PubMed
    1. Boutell, C., S. Sadis, and R. D. Everett. 2002. Herpes simplex virus type 1 immediate-early protein ICP0 and its isolated RING finger domain act as ubiquitin E3 ligases in vitro. J. Virol. 76:841-850. - PMC - PubMed
    1. Bryant, H., and P. J. Farrell. 2002. Signal transduction and transcription factor modification during reactivation of Epstein-Barr virus from latency. J. Virol. 76:10290-10298. - PMC - PubMed
    1. Chee, A. V., P. Lopez, P. P. Pandolfi, and B. Roizman. 2003. PML mediates interferon-based anti-herpes simplex virus 1 effects. J. Virol. 77:7101-7105. - PMC - PubMed
    1. Chelbi-Alix, M. K., and H. de The. 1999. Herpes virus induced proteasome-dependent degradation of the nuclear body-associated PML and Sp100 proteins. Oncogene 18:935-941. - PubMed

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