VP16-dependent association of chromatin-modifying coactivators and underrepresentation of histones at immediate-early gene promoters during herpes simplex virus infection

Abstract

During infection by herpes simplex virus type 1 (HSV-1), the virion protein VP16 activates the transcription of viral immediate-early (IE) genes. Genetic and biochemical assays have shown that the potent transcriptional activation domain of VP16 can associate with general transcription factors and with chromatin-modifying coactivator proteins of several types. The latter interactions are particularly intriguing because previous reports indicate that HSV-1 DNA does not become nucleosomal during lytic infection. In the present work, chemical cross-linking and immunoprecipitation assays were used to probe the presence of activators, general transcription factors, and chromatin-modifying coactivators at IE gene promoters during infection of HeLa cells by wild-type HSV-1 and by RP5, a viral strain lacking the VP16 transcriptional activation domain. The presence of VP16 and Oct-1 at IE promoters did not depend on the activation domain. In contrast, association of RNA polymerase II, TATA-binding protein, histone acetyltransferases (p300 and CBP), and ATP-dependent remodeling proteins (BRG1 and hBRM) with IE gene promoters was observed in wild-type infections but was absent or reduced in cells infected by RP5. In contrast to the previous evidence for nonnucleosomal HSV-1 DNA, histone H3 was found associated with viral DNA at early times of infection. Interestingly, histone H3 was underrepresented on IE promoters in a manner dependent on the VP16 activation domain. Thus, the VP16 activation domain is responsible for recruiting general transcription factors and coactivators to IE promoters and also for dramatically reducing the association of histones with those promoters.

FIG. 1.

Detection of VP16 and Oct-1 at viral IE promoters during infection. ChIP assays were performed using antibodies against VP16 or Oct-1 in lysates of cells infected for 2 h by KOS (wild-type virus) (A) or RP5 (lacking sequences encoding the VP16 AD) (B). Controls include precipitations performed without specific antisera (No Ab) and aliquots of samples prior to precipitation (0.5, 0.1, and 0.02% input). Samples were analyzed by PCR detecting viral IE gene promoters (ICP0, ICP4), a viral L gene promoter (VP16), and the promoter of the cellular U3 snRNA gene. Negative images of ethidium bromide-stained gels are shown.

FIG. 2.

GTFs are recruited to IE promoters by the VP16 AD. (A) RT-PCR analysis of steady-state mRNA levels of viral IE (ICP27), DE (TK), and L (VP16) genes in cells infected with KOS or RP5 (top panels) or in the presence or absence of cycloheximide (CHX) (lower panels). (B) ChIP assays using antibodies against TBP and Pol II in lysates of cells infected for 2 h with KOS or RP5. Immunoprecipitated samples were analyzed by PCR detecting viral IE gene promoters ICP0, ICP27, and ICP4, and the cellular U3 snRNA promoter. Controls include precipitations performed without specific antisera (No Ab) and aliquots of samples prior to precipitation (0.5, 0.1, and 0.02% input). (C) ChIP assay performed as for panel B for KOS infections in the presence or absence of cycloheximide, using PCR to detect promoters of the viral genes ICP0, TK, and VP16.

FIG. 3.

Recruitment of chromatin-modifying coactivator proteins to IE promoters during HSV infection. (A) ChIP assays using antibodies specific for CBP or p300 in lysates of cells infected with KOS (K) or RP5 (R). (B) ChIP assays using antibodies specific for BRG1 or BRM in lysates of cells infected with KOS or RP5.

FIG. 4.

Histone H3 associates with HSV-1 DNA during infection but is underrepresented at transcriptionally active IE gene promoters. ChIP assays were performed using antibodies specific for histone H3 acetylated at lysines 9 and 14 (AcH3) or for a carboxyl-terminal epitope of histone H3 in lysates of cells infected for 2 h by KOS (A) or RP5 (B). Immunoprecipitated samples were analyzed by PCR detecting viral IE (ICP0, ICP4, and ICP27), DE (TK), or L (VP16 and gC) gene promoters, the cellular U3 snRNA or IFN-β promoter, or the coding region (ORF) of ICP27. Controls include precipitations performed without specific antisera (No Ab) and aliquots of samples prior to precipitation (0.5, 0.1, and 0.02% input).