Maria Kalamvoki

Circadian CLOCK histone acetyl transferase localizes at ND10 nuclear bodies and enables herpes simplex virus gene expression

Expression of herpes simplex virus genes at the initiation of replication involves two steps that take place at ND10 nuclear bodies. These are suppression of cellular repressors that attempt to silence viral DNA and remodeling of the viral chromatin to make it accessible for transcription. In earlier studies we reported on the mechanism by which viral proteins ICP0 and US3 protein kinase modify and disrupt the HDAC1/CoREST/REST/LSD1 repressor complex. The remodeling step requires in addition acetylation of histones bound to DNA. In an attempt to identify the enzyme, we took note of the observation that ICP0 physically and functionally interacts with Bmal1, a partner of the CLOCK histone acetyl transferase, and key members of the bHLH–PAS family of transcriptional factors. The Bmal11 and CLOCK heterodimer is best known as a regulator of the circadian oscillation in the mammalian CLOCK system. In this article we report the following: (i) in infected cells both Bmal1 and CLOCK localize at ND10 bodies; (ii) wild-type virus stabilizes the CLOCK protein; (iii) overexpression of CLOCK partially compensates for the absence of ICP0 and enables higher yields in cells infected with a ΔICP0 mutant and this activity is not expressed by CLOCK mutants lacking histone acetyl transferase activity; and (iv) depletion of CLOCK in cells infected with wild-type virus results in significant decrease in the expression of all viral proteins tested. We conclude that ICP0 interacts with Bmal1 and by extension with CLOCK histone acetyl transferase to remodel viral chromatin.

Cells infected with herpes simplex virus 1 export to uninfected cells exosomes containing STING, viral mRNAs, and microRNAs

Significance The stimulator of IFN genes (STING) is an important innate immune response to infection by herpes simplex virus 1 (HSV-1). STING’s impact may be deduced from the observation that HSV-1 replicates significantly better in normal immortalized cells depleted of STING. Nevertheless, published evidence shows that STING is stabilized by the virus in infected cells, and this report shows that STING is exported from infected cells in exosomes along with viral microRNAs (miRNAs) and mRNAs. Some miRNAs have been reported to suppress reactivation of latent virus. The results suggest that HSV-1 in special circumstances may control the spread of infection from cell to cell and support the hypothesis that HSV-1 controls its virulence to enable effective person-to-person transmission. STING (stimulator of IFN genes) activates the IFN-dependent innate immune response to infection on sensing the presence of DNA in cytosol. The quantity of STING accumulating in cultured cells varies; it is relatively high in some cell lines [e.g., HEp-2, human embryonic lung fibroblasts (HEL), and HeLa] and low in others (e.g., Vero cells). In a preceding publication we reported that STING was stable in four cell lines infected with herpes simplex virus 1 and that it was actively stabilized in at least two cell lines derived from human cancers. In this report we show that STING is exported from HEp-2 cells to Vero cells along with virions, viral mRNAs, microRNAs, and the exosome marker protein CD9. The virions and exosomes copurified. The quantity of STING and CD9 exported from one cell line to another was inoculum-size–dependent and reflected the levels of STING and CD9 accumulating in the cells in which the virus inoculum was made. The export of STING, an innate immune sensor, and of viral mRNAs whose major role may be in silencing viral genes in latently infected neurons, suggests that the virus has evolved mechanisms that curtail rather than foster the spread of infection under certain conditions.

HSV-1 degrades, stabilizes, requires, or is stung by STING depending on ICP0, the US3 protein kinase, and cell derivation

Significance STING (stimulator of IFN genes) and IFI16 are sensors of DNA in cytoplasm and the nucleus, respectively. Both signal activation of innate immune responses. Both proteins are stable in wild-type virus-infected cells. The key finding with broad implications is that in HSV-1–infected cells STING and IFI16 are actively stabilized inasmuch as they are degraded in cells lacking functional ICP0 or protein kinase US3. Moreover, STING was required for optimal replication in HEp-2 or HeLa cells derived from cancers but was inimical in HEL or HEK293 cells derived initially from normal tissues and in the case of HEK293 cells transformed with viral oncogenes. The requirements for stabilization of STING and IFI16 were not covariant. STING (stimulator of IFN genes) activates the IFN pathway in response to cytosolic DNA. Knockout of STING in mice was reported to exacerbate the pathogenicity of herpes simplex virus 1 (HSV-1). Here we report the following: (i) STING is stable in cancer-derived HEp-2 or HeLa cells infected with wild-type HSV-1 but is degraded in cells infected with mutants lacking the genes encoding functional infected cell protein 0 (ICP0), ICP4, or the US3 protein kinase (US3-PK). In HEp-2 cells, depletion of STING by shRNA results in a decrease in the yields of wild-type or ΔICP0 viruses. (ii) STING is stable throughout infection with either wild-type or ICP0 mutant viruses in human embryonic lung cells (HEL) or HEK293T cells derived from normal tissues. In these cells, depletion of STING results in higher yields of both wild-type and ΔICP0 viruses. (iii) The US3-PK is also required for stabilization of IFI16, a nuclear DNA sensor. However, the stability of IFI16 does not correlate positively or negatively with that of STING. IFI16 is stable in STING-depleted HEL cells infected with wild-type virus. In contrast to HEL cells, IFI16 was undetectable in STING-depleted HEp-2 cells, and hence the role of HSV-1 in maintaining IFI16 could not be ascertained. The results indicate that in HSV-1–infected cells the stability of IFI16 and the function and stability of STING are dependent on cell derivation, the functional integrity of ICP0, and US3-PK, an indication that in wild-type virus-infected cells both proteins are actively stabilized. In HEp-2 cells, the stability of IFI16 requires STING.

The Histone Acetyltransferase CLOCK Is an Essential Component of the Herpes Simplex Virus 1 Transcriptome That Includes TFIID, ICP4, ICP27, and ICP22

ABSTRACT Studies published elsewhere have shown that the herpes simplex virus regulatory protein ICP0 interacts with BMAL1, a partner and regulator of circadian histone acetyltransferase CLOCK, that both proteins localize at ND10 bodies and are stabilized by viral proteins, that enzymatically active CLOCK partially complements ΔICP0 mutants, and that silencing of CLOCK suppresses the expression of viral genes. Here we report that CLOCK is a component of the transcriptional complex that includes TFIID, ICP4, ICP27, and ICP22. The results suggest that the CLOCK histone acetyltransferase is a component of the viral transcriptional machinery throughout the replicative cycle of the virus and that ICP27 and ICP22 initiate their involvement in viral gene expression as components of viral transcriptome.

Nuclear retention of ICP0 in cells exposed to HDAC inhibitor or transfected with DNA before infection with herpes simplex virus 1

The α (immediate early) protein ICP0 of herpes simplex virus 1 enhances the expression of genes introduced by infection or transfection. Early in infection it performs two key functions: It blocks the silencing of the viral DNA by cellular proteins and it blocks the IFN stimulated host response to infection. Between 5 and 9 h after infection, ICP0 is translocated to the cytoplasm but remains dynamically associated with proteasomes. In this report we show that in permissive cells that are poor expressors of transfected genes (HEp-2, U2OS, etc.), ICP0 is retained in the nucleus if the cells had been transfected with DNA and then infected. The retention is DNA dose- and size-dependent but not DNA type-dependent. Retention of ICP0 is also a consequence of infection with wild-type virus concomitant with exposure of cells to sodium butyrate. ICP0 is not retained in transfected/infected cells that efficiently express transfected genes (HEK293, rabbit skin cells). The retention of ICP0 in the nucleus is concordant with failure to degrade PML and disperse ND10 structures, and delays in the transition to post α genes expression, translocation of components of the CoREST/REST/HDAC1 complex and histone relocation in the infected cell. The data suggest that (i) retention of ICP0 is linked to its function to remodel acetylated DNA but not DNA in heterochromatin. This function is independent of response elements embedded in the DNA and (ii) transfection-resistant cells do take up DNA but process it differently than cells that readily express transfected genes.

Interwoven Roles of Cyclin D3 and cdk4 Recruited by ICP0 and ICP4 in the Expression of Herpes Simplex Virus Genes

ABSTRACT Elsewhere this laboratory reported that (i) ICP0 interacts with cyclin D3 but not D1 or D2. The 3 cyclins independently partially rescue ΔICP0 mutants. (ii) Interaction with cyclin D3 is required for the switch from nuclear to cytoplasmic accumulation of ICP0. (iii) In infected cells cdk4 is activated whereas cdk2 is not. Inhibition of cdk4 results in nuclear retention of ICP0. Overexpression of cyclin D3 reverses the effect of the inhibitor. Here we report the following. (i) cdk4 interacts with ICP0, ICP4, and possibly with ICP8. This interaction is required to recruit cdk4 initially to ND10 and later to the viral replication compartments. (ii) cdk4 inhibitor I reduced or delayed the transcription and ultimately translation of mRNAs of ICP4, ICP27, or ICP8 and to a lesser extent that of the ICP0 gene in wild-type virus-infected cells. (iii) Overexpression of cyclin D3 resulted in a more rapid transcription of these genes. In the presence of inhibitor, the rates of accumulation of the products of these genes resemble those of wild-type virus in the absence of inhibitor. (iv) Overexpression of cyclin D3 also results in mobilization of cdk6 in nuclei of infected cells. We conclude that ICP0 encodes a function that enhances the recruitment of cyclin D3 to ND10 structures to activate cdk4 and that ICP0 along with other viral proteins recruits cdk4 to ND10 structures and ultimately to replication compartments for enhanced expression of viral genes and viral DNA synthesis.

Translocation and Colocalization of ICP4 and ICP0 in Cells Infected with Herpes Simplex Virus 1 Mutants Lacking Glycoprotein E, Glycoprotein I, or the Virion Host Shutoff Product of the UL41 Gene

ABSTRACT In wild-type herpes simplex virus 1-infected cells, the major regulatory protein ICP4 resides in the nucleus whereas ICP0 becomes dynamically associated with proteasomes and late in infection is translocated and dispersed in the cytoplasm. Inhibition of proteasomal function results in retention or transport of ICP0 to the nucleus. We report that in cells infected with mutants lacking glycoprotein E (gE), glycoprotein I (gI), or the product of the UL41 gene, both ICP4 and ICP0 are translocated to the cytoplasm and coaggregate in small dense structures that, in the presence of proteasomal inhibitor MG132, also contain proteasomal components. Gold particle-conjugated antibody to ICP0 reacted in thin sections with dense protein aggregates in the cytoplasm of mutant virus-infected cells. Similar aggregates were present in the nuclei but not in the cytoplasm of wild-type virus-infected cells. Exposure of cells early in infection to MG132 does not result in retention of ICP0 as in wild-type virus-infected cells. The results suggest that the retention of ICP4 and ICP0 in the nucleus is a dynamic process that involves the function of other viral proteins that may include the Fc receptor formed by the gE/gI complex and is not merely the consequence of expression of a nuclear localization signal. It is noteworthy that in ΔUL41-infected cells gE is retained in the trans-Golgi network and is not widely dispersed in cellular membranes.

ICP0 enables and monitors the function of D cyclins in herpes simplex virus 1 infected cells

The herpes simplex virus 1 ICP0 is a regulatory protein. Early in infection ICP0 localizes in ND10 bodies and performs two functions: As an E3 ligase in conjunction with E2 UbcH5a conjugating enzyme, it degrades the ND10 components PML and SP100. Concurrently, it suppresses the silencing of viral DNA by dispersing the HDAC1/CoREST/REST/LSD1 repressor complex. Subsequently, ICP0 is exported to the cytoplasm. In cells treated with HDAC inhibitors or transfected with irrelevant DNA, the export is delayed in a DNA dose-dependent fashion. Here, we follow up an observation that ICP0 binds cyclin D3 and that ICP0 mutants unable to bind cyclin D3 are not exported. Moreover, in infected cells cdk4 is activated, but cdk2 is not. We report that (i) cyclin D1, D2, or D3 colocalize with ND10 bodies and ICP0 early in infection and ultimately become incorporated into viral replication compartments, (ii) each of the D cyclins partially rescues ΔICP0 mutants, and (iii) inhibition of cdk4 by inhibitor I sequesters ICP0 in the nucleus. A key finding is that overexpression of cyclin D3 enables the transport of ICP0 to the cytoplasm. We conclude that (i) ICP0 facilitates the recruitment of cyclin D3 to the sites of viral DNA synthesis, (ii) until its functions are completed, ICP0 is retained in the nucleus, and (iii) a common signal that results in the export of ICP0 to the cytoplasm is the accumulation of a viral DNA-synthesis-dependent late protein.

Role of herpes simplex virus ICP0 in the transactivation of genes introduced by infection or transfection: a reappraisal.

ABSTRACT ICP0, a promiscuous transactivator that enhances the expression of genes introduced by infection or transfection, functions in both nucleus and cytoplasm. The nuclear functions include degradation and dispersal of ND10 bodies and suppression of silencing of viral DNA. Subsequently, ICP0 shifts to the cytoplasm. Transfection of DNA prior to infection has no effect on the localization of ICP0 in cells that are efficient expressers of transgenes (e.g., Vero and HEK293) but results in delayed cytoplasmic localization of ICP0 in cells (e.g., HEp-2 and HEL) that are poor transgene expressers. Here, we examined by real-time PCR (qPCR) the accumulation of a transgene and of viral gI mRNAs in Vero or HEp-2 cells that were transfected and then infected with wild-type or ΔICP0 mutant viruses. The accumulation of transgene mRNA was unaffected by a ΔICP0 mutant, gradually increased in HEp-2 cells, but increased and then decreased in Vero cells infected with wild-type virus. In both cell lines, accumulation of gI mRNA increased with time and was less affected by the transfected DNA in Vero cells than in HEp-2 cells. The relative kinetics of mRNA accumulation reflected continued synthesis and degradation of the transgene and gI mRNAs. We conclude that the role of ICP0 is to render the DNA templates introduced by transfection or infection accessible by transcriptional factors, that the two cell lines differ with respect to the transcription-ready status of entering foreign DNA in the nucleus, and that ICP0 is not per se the recruiter of transcriptional factors to the accessible DNA templates.

Overexpression of the Ubiquitin-Specific Protease 7 Resulting from Transfection or Mutations in the ICP0 Binding Site Accelerates Rather than Depresses Herpes Simplex Virus 1 Gene Expression

ABSTRACT Earlier studies reported that ICP0, a key regulatory protein encoded by herpes simplex virus 1 (HSV-1), binds ubiquitin-specific protease 7 (USP7). The fundamental conclusion of these studies is that depletion of USP7 destabilized ICP0, that ICP0 mediated the degradation of USP7, and that amino acid substitutions in ICP0 that abolished binding to USP7 significantly impaired the ability of HSV-1 to replicate. We show here that, indeed, depletion of USP7 leads to reduction of ICP0 and that USP7 is degraded in an ICP0-dependent manner. However, overexpression of USP7 or substitution in ICP0 of a single amino acid to abolish binding to USP7 accelerated the accumulation of viral mRNAs and proteins at early times after infection and had no deleterious effect on virus yields. A clue as to why USP7 is degraded emerged from the observation that, notwithstanding the accelerated expression of viral genes, the plaques formed by the mutant virus were very small, implying a defect in virus transmission from cell to cell.