Herpes Simplex Virus Immediate-early Genes Research Articles

Protocol Optimization for the Production of the Non-Cytotoxic JΔNI5 HSV Vector Deficient in Expression of Immediately Early Genes

Non-toxic herpes simplex virus (HSV) vectors can be generated by functional deletion of all immediate-early (IE) genes, providing a benign vehicle with potential for gene therapy. However, deletion of multiple IE genes raises manufacturing concerns and thus limits clinical application of these vectors. To address this issue, we previously developed a novel production cell line, called U2OS-ICP4/27, by lentiviral transduction of human osteosarcoma U2OS cells with two essential HSV IE genes, ICP4 and ICP27. To optimize the process of vector manufacturing on this platform, we evaluated several cell culture parameters of U2OS-ICP4/27 for high-titer and -quality production of non-toxic HSV vectors, revealing that the yields and functionality of these vectors can be significantly influenced by culturing conditions. We also found that several chemical compounds can enhance the replication of non-toxic HSV vectors and their release from producer cells into the supernatants. Notably, the vector produced by our optimized protocol displayed a greatly improved vector yield and quality and showed elevated transgene expression in cultures of primary dorsal root ganglion neurons. Taken together, our optimized production approach emerges as a relevant protocol for high-yield and high-quality preparation of non-toxic HSV-based gene therapy vectors.

Transcriptional Elongation of HSV Immediate Early Genes by the Super Elongation Complex Drives Lytic Infection and Reactivation from Latency

The cellular transcriptional coactivator HCF-1 is required for initiation of herpes simplex virus (HSV) lytic infection and for reactivation from latency in sensory neurons. HCF-1 stabilizes the viral Immediate Early (IE) gene enhancer complex and mediates chromatin transitions to promote IE transcription initiation. In infected cells, HCF-1 was also found to be associated with a network of transcription elongation components including the super elongation complex (SEC). IE genes exhibit characteristics of genes controlled by transcriptional elongation, and the SEC-P-TEFb complex is specifically required to drive the levels of productive IE mRNAs. Significantly, compounds that enhance the levels of SEC-P-TEFb also potently stimulated HSV reactivation from latency both in a sensory ganglia model system and invivo. Thus, transcriptional elongation of HSV IE genes is a key limiting parameter governing both the initiation of HSV infection and reactivation of latent genomes.

Inhibition of O-Linked N-Acetylglucosamine Transferase Reduces Replication of Herpes Simplex Virus and Human Cytomegalovirus.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential cellular enzyme that posttranslationally modifies nuclear and cytoplasmic proteins via O-linked addition of a single N-acetylglucosamine (GlcNAc) moiety. Among the many targets of OGT is host cell factor 1 (HCF-1), a transcriptional regulator that is required for transactivation of the immediate-early genes of herpes simplex virus (HSV). HCF-1 is synthesized as a large precursor that is proteolytically cleaved by OGT, which may regulate its biological function. In this study, we tested whether inhibition of the enzymatic activity of OGT with a small molecule inhibitor, OSMI-1, affects initiation of HSV immediate-early gene expression and viral replication. We found that inhibiting OGT's enzymatic activity significantly decreased HSV replication. The major effect of the inhibitor occurred late in the viral replication cycle, when it reduced the levels of late proteins and inhibited capsid formation. However, depleting OGT levels with small interfering RNA (siRNA) reduced the expression of HSV immediate-early genes, in addition to reducing viral yields. In this study, we identified OGT as a novel cellular factor involved in HSV replication. Our results obtained using a small molecule inhibitor and siRNA depletion suggest that OGT's glycosylation and scaffolding functions play distinct roles in the replication cycle of HSV. Antiviral agents can target viral or host gene products essential for viral replication. O-GlcNAc transferase (OGT) is an important cellular enzyme that catalyzes the posttranslational addition of GlcNAc sugar residues to hundreds of nuclear and cytoplasmic proteins, and this modification regulates their activity and function. Some of the known OGT targets are cellular proteins that are critical for the expression of herpes simplex virus (HSV) genes, suggesting a role for OGT in the replication cycle of HSV. In this study, we found that OGT is required for efficient expression of viral genes and for assembly of new virions. Thus, we identify OGT as a novel host factor involved in the replication of HSV and a potential target for antiviral therapy.

Definition of herpes simplex virus helper functions for the replication of adeno-associated virus type 5.

Adeno-associated virus (AAV) type 5 represents the genetically most distant AAV serotype and the only one isolated directly from human tissue. Seroepidemiological evidence suggests herpes simplex virus (HSV) as a helper virus for human AAV5 infections, underlining the in vivo relevance of the AAV-herpesvirus relationship. In this study we analysed, for the first time, HSV helper functions for productive AAV5 replication, and compared these to AAV2. Using a combination of HSV strains and plasmids for individual genes, the previously defined HSV helper functions for AAV2 replication were shown to induce AAV5 gene expression, DNA replication and production of infectious progeny. The helper functions comprise the replication genes for ICP8 (UL29), helicase-primase (UL5/8/52), and DNA polymerase (UL30/42). HSV immediate-early genes for ICP0 and ICP4 further enhanced AAV5 replication, mainly by induction of rep gene expression. In the presence of HSV helper functions, AAV5 Rep co-localized with ICP8 in nuclear replication compartments, and HSV alkaline exonuclease (UL12) enhanced AAV5 replication, similarly to AAV2. UL12, in combination with ICP8, was shown to induce DNA strand exchange on partially double-stranded templates to resolve and repair concatemeric HSV replication intermediates. Similarly, concatemeric AAV replication intermediates appeared to be processed to yield AAV unit-length molecules, ready for AAV packaging. Taken together, our findings show that productive AAV5 replication is promoted by the same combination of HSV helper functions as AAV2.

A Novel Selective LSD1/KDM1A Inhibitor Epigenetically Blocks Herpes Simplex Virus Lytic Replication and Reactivation from Latency

ABSTRACTCellular processes requiring access to the DNA genome are regulated by an overlay of epigenetic modifications, including histone modification and chromatin remodeling. Similar to the cellular host, many nuclear DNA viruses that depend upon the host cell’s transcriptional machinery are also subject to the regulatory impact of chromatin assembly and modification. Infection of cells with alphaherpesviruses (herpes simplex virus [HSV] and varicella-zoster virus [VZV]) results in the deposition of nucleosomes bearing repressive histone H3K9 methylation on the viral genome. This repressive state is modulated by the recruitment of a cellular coactivator complex containing the histone H3K9 demethylase LSD1 to the viral immediate-early (IE) gene promoters. Inhibition of the activity of this enzyme results in increased repressive chromatin assembly and suppression of viral gene expression during lytic infection as well as reactivation from latency in a mouse ganglion explant model. However, available small-molecule LSD1 inhibitors are not originally designed to inhibit LSD1, but rather monoamine oxidases (MAO) in general. Thus, their specificity for and potency to LSD1 is low. In this study, a novel specific LSD1 inhibitor was identified that potently repressed HSV IE gene expression, genome replication, and reactivation from latency. Importantly, the inhibitor also suppressed primary infection of HSV in vivo in a mouse model. Based on common control of a number of DNA viruses by epigenetic modulation, it was also demonstrated that this LSD1 inhibitor blocks initial gene expression of the human cytomegalovirus and adenovirus type 5.IMPORTANCE Epigenetic mechanisms, including histone modification and chromatin remodeling, play important regulatory roles in all cellular processes requiring access to the genome. These mechanisms are often altered in disease conditions, including various cancers, and thus represent novel targets for drugs. Similarly, many viral pathogens are regulated by an epigenetic overlay that determines the outcome of infection. Therefore, these epigenetic targets also represent novel antiviral targets. Here, a novel inhibitor was identified with high specificity and potency for the histone demethylase LSD1, a critical component of the herpes simplex virus (HSV) gene expression paradigm. This inhibitor was demonstrated to have potent antiviral potential in both cultured cells and animal models. Thus, in addition to clearly demonstrating the critical role of LSD1 in regulation of HSV infection, as well as other DNA viruses, the data extends the therapeutic potential of chromatin modulation inhibitors from the focused field of oncology to the arena of antiviral agents.

Crosstalk between O-GlcNAcylation and proteolytic cleavage regulates the host cell factor-1 maturation pathway.

Host Cell Factor 1 (HCF-1) plays critical roles in regulating gene expression in a plethora of physiological processes. HCF-1 is first synthesized as a precursor, and subsequently specifically proteolytically cleaved within a large middle region termed the proteolytic processing domain (PPD). Although the underlying mechanism remains enigmatic, proteolysis of HCF-1 regulates its transcriptional activity and is important for cell cycle progression. Here we report that HCF-1 proteolysis is a regulated process. We demonstrate that a large proportion of the signaling enzyme O-linked-N-acetylglucosaminyl transferase (OGT) is complexed with HCF-1 and this interaction is essential for HCF-1 cleavage. Moreover, HCF-1 is, in turn, required for stabilizing OGT in the nucleus. We provide evidence indicating that OGT regulates HCF-1 cleavage via interaction with and O-GlcNAcylation of the HCF-1 PPD. In contrast, although OGT also interacts with the basic domain in the HCF-1 amino-terminal subunit, neither the interaction nor the O-GlcNAcylation of this region are required for proteolysis. Moreover, we show that OGT-mediated modulation of HCF-1 impacts the expression of the herpes simplex virus immediate-early genes, targets of HCF-1 during the initiation of viral infection. Together the data indicate that O-GlcNAcylation of HCF-1 is a signal for its proteolytic processing and reveal a unique crosstalk between these posttranslational modifications. Additionally, interactions of OGT with multiple HCF-1 domains may indicate that OGT has several functions in association with HCF-1.

Curcumin inhibits herpes simplex virus immediate-early gene expression by a mechanism independent of p300/CBP histone acetyltransferase activity

Curcumin, a phenolic compound from the curry spice turmeric, exhibits a wide range of activities in eukaryotic cells, including antiviral effects that are at present incompletely characterized. Curcumin is known to inhibit the histone acetyltransferase activity of the transcriptional coactivator proteins p300 and CBP, which are recruited to the immediate early (IE) gene promoters of herpes simplex virus type 1 (HSV-1) by the viral transactivator protein VP16. We tested the hypothesis that curcumin, by inhibiting these coactivators, would block viral infection and gene expression. In cell culture assays, curcumin significantly decreased HSV-1 infectivity and IE gene expression. Entry of viral DNA to the host cell nucleus and binding of VP16 to IE gene promoters was not affected by curcumin, but recruitment of RNA polymerase II to those promoters was significantly diminished. However, these effects were observed using lower curcumin concentrations than those required to substantially inhibit global H3 acetylation. No changes were observed in histone H3 occupancy or acetylation at viral IE gene promoters. Furthermore, p300 and CBP recruitment to IE gene promoters was not affected by the presence of curcumin. Finally, disruption of p300 expression using a short hairpin RNA did not affect viral IE gene expression. These results suggest that curcumin affects VP16-mediated recruitment of RNA polymerase II to IE gene promoters by a mechanism independent of p300/CBP histone acetyltransferase activity.

Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV gene products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli β-galactosidase induced durable β-gal-specific IgG and CD8 + T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes.

Combinatorial Transcription of Herpes Simplex Virus and Varicella Zoster Virus Immediate Early Genes Is Strictly Determined by the Cellular Coactivator HCF-1

The mammalian transcriptional coactivator host cell factor-1 (HCF-1) functions in concert with Oct-1 and VP16 to assemble the herpes simplex virus (HSV) immediate early (IE) transcription enhancer core complexes that mediate the high level transcription of these genes upon infection. Although this transcriptional model has been well characterized in vitro, the requirements and significance of the components have not been addressed. Oct-1 was previously determined to be critical but not essential for HSV IE gene expression. In contrast, RNA interference-mediated depletion of HCF-1 resulted in abrogation of HSV IE gene expression. The HSV IE gene enhancer domain is a model of combinatorial transcription and consists of the core enhancer and multiple binding sites for factors such as Sp1 and GA-binding protein. It was striking that HCF-1 was strictly required for VP16-mediated transcriptional induction via the core enhancer as well as for basal level transcription mediated by GA-binding protein and Sp1. HCF-1 was also found to be essential for the induction of varicella zoster virus IE gene expression by ORF10, the VZV ortholog of the HSV IE transactivator VP16, and the autostimulatory IE62 protein. The critical dependence upon HCF-1 demonstrates that this cellular component is a key factor for control of HSV and VZV IE gene expression by functioning as the common element for distinct factors cooperating at the IE gene enhancers. The requirements for this protein supports the model whereby the regulated transport of HCF-1 from the cytoplasm to the nucleus in sensory neurons may control IE gene expression and reactivation of these viruses from the latent state.

HCF-1 Functions as a Coactivator for the Zinc Finger Protein Krox20

HCF-1 is a transcriptional cofactor required for activation of herpes simplex virus immediate-early genes by VP16 as well as less clearly defined roles in cell proliferation, cytokinesis, and spliceosome formation. It is expressed as a large precursor that undergoes proteolysis to yield two subunits that remain stably associated. VP16 uses a degenerate 4-amino acid sequence, known as the HCF-binding motif, to bind to a six-bladed beta-propeller domain at the N terminus of HCF-1. Functional HCF-binding motifs are also found in LZIP and Zhangfei, two cellular bZIP transcription factors of unknown function. Here we show that the HCF-binding motif occurs in a wide spectrum of DNA-binding proteins and transcriptional cofactors. Three well characterized examples were further analyzed for their ability to use HCF-1 as a coactivator. Krox20, a zinc finger transcription factor required for Schwann cell differentiation, and E2F4, a cell cycle regulator, showed a strong requirement for functional HCF-1 to activate transcription. In contrast, activation by estrogen receptor-alpha did not display HCF dependence. In Krox20, the HCF-binding motif lies within the N-terminal activation domain and mutation of this sequence diminishes both transactivation and association with the HCF-1 beta-propeller. The activation domain in the C-terminal subunit of HCF-1 contributes to activation by Krox20, possibly through recruitment of p300. These results suggest that HCF-1 is recruited by many different classes of cellular transcription factors and is therefore likely to be required for a variety of cellular processes including cell cycle progression and development.

Proteolytic processing is necessary to separate and ensure proper cell growth and cytokinesis functions of HCF-1.

HCF-1 is a highly conserved and abundant chromatin-associated host cell factor required for transcriptional activation of herpes simplex virus immediate-early genes by the virion protein VP16. HCF-1 exists as a heterodimeric complex of associated N- (HCF-1(N)) and C- (HCF-1(C)) terminal subunits that result from proteolytic processing of a precursor protein. We have used small-interfering RNA (siRNA) to inactivate HCF-1 in an array of normal and transformed mammalian cells to identify its cellular functions. Our results show that HCF-1 is a broadly acting regulator of two stages of the cell cycle: exit from mitosis, where it ensures proper cytokinesis, and passage through the G(1) phase, where it promotes cell cycle progression. Proteolytic processing is necessary to separate and ensure these two HCF-1 activities, which are performed by separate HCF-1 subunits: the HCF-1(N) subunit promotes passage through the G(1) phase whereas the HCF-1(C) subunit is involved in proper exit from mitosis. These results suggest that HCF-1 links the regulation of exit from mitosis and the G(1) phase of cell growth, possibly to coordinate the reactivation of gene expression after mitosis.

A single amino acid substitution in herpes simplex virus type 1 VP16 inhibits binding to the virion host shutoff protein and is incompatible with virus growth.

In addition to its well-established role in the activation of herpes simplex virus immediate-early gene transcription, VP16 interacts with and downregulates the function of the virion host shutoff protein (vhs), thereby attenuating vhs-mediated destruction of viral mRNAs and translational arrest at late times of infection. We have carried out two-hybrid analysis in vivo and protein-protein interaction assays in vitro to identify determinants in VP16 necessary for interaction with vhs. The minimal amino-terminal subfragment of VP16 capable of binding to vhs encompassed residues 1 to 345. Alteration of a single leucine at position 344 to alanine (L344A) in the context of the amino-terminal fragment of VP16 containing residues 1 to 404 was sufficient to abolish interaction with vhs in vitro and in vivo. Leu344 could be replaced with hydrophobic amino acids (Ile, Phe, Met, or Val) but not by Asn, Lys, or Pro, indicating that hydrophobicity is an important property of binding to vhs. VP16 harboring a loss-of-function mutation at L344 was not compromised in its ability to interact with host cell factor (HCF-1) or to activate transcription of viral immediate-early genes in transient-transfection assays. Virus complementation assays using the VP16-null virus 8MA and the VP16/vhs double-mutant virus 8MAdeltaSma showed that VP16(L344A) was able to complement the growth of 8MAdeltaSma but not 8MA. Thus, a single point mutation in VP16 uncouples binding to vhs from other functions of VP16 required for virus growth and indicates that direct physical association between VP16 and vhs is necessary to sustain a productive infection.

Interaction of HCF-1 with a Cellular Nuclear Export Factor

HCF-1 is a cellular protein required by VP16 to activate the herpes simplex virus (HSV) immediate-early genes. VP16 is a component of the viral tegument and, after release into the cell, binds to HCF-1 and translocates to the nucleus to form a complex with the POU domain protein Oct-1 and a VP16-responsive DNA sequence. This VP16-induced complex boosts transcription of the viral immediate-early genes and initiates lytic replication. In uninfected cells, HCF-1 functions as a coactivator for the cellular transcription factors LZIP and GABP and also plays an essential role in cell proliferation. VP16 and LZIP share a tetrapeptide HCF-binding motif recognized by the beta-propeller domain of HCF-1. Here we describe a new cellular HCF-1 beta-propeller domain binding protein, termed HPIP, which contains a functional HCF-binding motif and a leucine-rich nuclear export sequence. We show that HPIP shuttles between the nucleus and cytoplasm in a CRM1-dependent manner and that overexpression of HPIP leads to accumulation of HCF-1 in the cytoplasm. These data suggest that HPIP regulates HCF-1 activity by modulating its subcellular localization. Furthermore, HPIP-mediated export may provide the pool of cytoplasmic HCF-1 required for import of virion-derived VP16 into the nucleus.

Spontaneous Reversion of tsBN67 Cell Proliferation and Cytokinesis Defects in the Absence of HCF-1 Function

Mammalian HCF-1 is a highly conserved and abundant chromatin-bound protein that plays a role in both herpes simplex virus (HSV) immediate-early (IE) gene transcription and cell proliferation. Its role in cell proliferation has been evidenced through the analysis of a temperature-sensitive hamster cell line called tsBN67. When placed at nonpermissive temperature, tsBN67 cells undergo a stable and reversible proliferation arrest after a lag of 36–48 h. This phenotype results from a single point mutation in HCF-1, which disrupts HCF-1 association with both chromatin and the HSV IE transactivator VP16 at nonpermissive temperature. Here, we report the isolation and characterization of spontaneous tsBN67 growth-revertant cells that are able to proliferate at nonpermissive temperatures. These cells retain the tsBN67 HCF-1 point mutation and grow in the absence of HCF-1 chromatin association, demonstrating that complete restoration of tsBN67 HCF-1 functions is not essential for cell proliferation. Phenotypic analysis of both mutant and revertant tsBN67 cells shows that, in addition to a cell proliferation defect, these cells display a conspicuous multinucleated phenotype in a significant population of arrested cells. This defect in cytokinesis is also a result of loss of HCF-1 function, suggesting that HCF-1 plays a role in cell exit from mitosis. The revertant tsBN67 cells display a coincident restoration of cell proliferation and suppression of the cytokinetic defect, suggesting that HCF-1 plays a shared role in cell proliferation and cytokinesis.

DNA recognition by the herpes simplex virus transactivator VP16: a novel DNA-binding structure.

Upon infection, the herpes simplex virus (HSV) transcriptional activator VP16 directs the formation of a multiprotein-DNA complex-the VP16-induced complex-with two cellular proteins, the host cell factor HCF-1 and the POU domain transcription factor Oct-1, on TAATGARAT-containing sequences found in the promoters of HSV immediate-early genes. HSV VP16 contains carboxy-terminal sequences important for transcriptional activation and a central conserved core that is important for VP16-induced complex assembly. On its own, VP16 displays little, if any, sequence-specific DNA-binding activity. We show here that, within the VP16-induced complex, however, the VP16 core has an important role in DNA binding. Mutation of basic residues on the surface of the VP16 core reveals a novel DNA-binding surface with essential residues which are conserved among VP16 orthologs. These results illuminate how, through association with DNA, VP16 is able to interpret cis-regulatory signals in the DNA to direct the assembly of a multiprotein-DNA transcriptional regulatory complex.

Loss of HCF-1-chromatin association precedes temperature-induced growth arrest of tsBN67 cells.

Human HCF-1 is a large, highly conserved, and abundant nuclear protein that plays an important but unknown role in cell proliferation. It also plays a role in activation of herpes simplex virus immediate-early gene transcription by the viral regulatory protein VP16. A single proline-to-serine substitution in the HCF-1 VP16 interaction domain causes a temperature-induced arrest of cell proliferation in hamster tsBN67 cells and prevents transcriptional activation by VP16. We show here that HCF-1 is naturally bound to chromatin in uninfected cells through its VP16 interaction domain. HCF-1 is chromatin bound in tsBN67 cells at permissive temperature but dissociates from chromatin before tsBN67 cells stop proliferating at the nonpermissive temperature, suggesting that loss of HCF-1 chromatin association is the primary cause of the temperature-induced tsBN67 cell proliferation arrest. We propose that the role of HCF-1 in cell proliferation is to regulate gene transcription by associating with a multiplicity of DNA-bound transcription factors through its VP16 interaction domain.

Herpes simplex virus type 1 ICP0 protein does not accumulate in the nucleus of primary neurons in culture.

Infected-cell protein 0 (ICP0), the product of the herpes simplex virus (HSV) immediate-early (IE) alpha0 gene, is a promiscuous transactivator of viral early (E) and late (L) gene expression. HSV mutants lacking ICP0 function are severely deficient in viral growth and protein synthesis, particularly at low multiplicities of infection. Early in the infectious process in vitro, ICP0 protein accumulates in distinct domains within the nucleus to form characteristic structures active in the transcription of viral genes. However, following infection of primary trigeminal ganglion cells in vitro with a recombinant HSV mutant that expresses only ICP0, we observed that ICP0 protein accumulated in the characteristic intranuclear distribution only in the nuclei of Schwann cells; neurons in the culture did not accumulate ICP0 despite expression of ICP0 RNA in those cells. The same phenomenon was observed in PC12 cells differentiated to assume a neuronal phenotype. In primary neurons in culture, the amount of ICP0 protein could be increased by pharmacologic inhibition of calcium-activated protease (calpain) activity or by inhibition of protein phosphatase 2B (calcineurin). The failure of ICP0 protein to accumulate in the nucleus of neurons suggests that one mechanism which may impair efficient replication of the virus in neurons, and thus favor the establishment of viral latency in those cells, may be found in the cell-specific processing of that IE gene product.

Zhangfei: a second cellular protein interacts with herpes simplex virus accessory factor HCF in a manner similar to Luman and VP16.

Host cell factor (HCF, C1, VCAF or CFF) is a cellular protein that is required for transcription activation of herpes simplex virus (HSV) immediate-early (IE) genes by the virion protein VP16. The biological function of HCF remains unclear. Recently we identified a cellular transcription activator, Luman. As with VP16, the transactivation function of Luman is also regulated by HCF. Here we report a second human protein, Zhangfei (ZF) that interacts with HCF in a fashion similar to Luman and VP16. Although ZF shares no significant sequence homology with Luman, the two proteins have some structural similarities. These include: a basic domain-leucine zipper (bZIP) region, an acidic activation domain and a consensus HCF-binding motif. Unlike Luman, or most other bZIP proteins, ZF by itself did not appear to bind consensus bZIP-binding sites. It was also unable to activate promoters containing these response elements. Although in transient expression assays ectopically expressed ZF was unable to block transactivation by VP16 of a HSV IE promoter, ZF could prevent the expression of several HSV proteins in cells infected with the virus. The ability of ZF to block the synthesis of the HSV IE protein ICP0 relied on its binding to HCF, since a mutant of ZF that was unable to bind HCF was also unable to prevent viral IE protein expression.

A novel 50-kilodalton fragment of host cell factor 1 (C1) in G(0) cells.

Host cell factor 1 (HCF-1; also called C1) is a 230-kDa protein which is cleaved posttranslationally into separate but associated N- and C-terminal polypeptides. These polypeptides are components of the C1 complex, along with Oct-1 and the viral protein VP16. The C1 complex is formed when herpes simplex virus (HSV) infects a cell and is responsible for transcription of the HSV immediate-early genes. A temperature-sensitive mutation in the N-terminal kelch domain of HCF-1 reversibly arrests cells in a G(0)-like state when grown at the nonpermissive temperature, and the same domain interacts with VP16 in the formation of the C1 complex. The form of HCF-1 in primary G(0) cells was investigated by using peripheral blood mononucleocytes and serum-arrested human primary fibroblasts. A novel 50-kDa N-terminal fragment of HCF-1 encompassing the kelch domain was identified in the cytoplasm of these cells. This fragment arises by proteolysis of the full-length HCF-1 protein and is able to associate with VP16.

Mutations in host cell factor 1 separate its role in cell proliferation from recruitment of VP16 and LZIP.

Host cell factor 1 (HCF-1) is a nuclear protein required for progression through G(1) phase of the cell cycle and, via its association with VP16, transcriptional activation of the herpes simplex virus immediate-early genes. Both functions require a six-bladed beta-propeller domain encoded by residues 1 to 380 of HCF-1 as well as an additional amino-terminal region. The beta-propeller domain is well conserved in HCF homologues, consistent with a critical cellular function. To date, the only known cellular target of the beta-propeller is a bZIP transcription factor known as LZIP or Luman. Whether the interaction between HCF-1 and LZIP is required for cell proliferation remains to be determined. In this study, we used directed mutations to show that all six blades of the HCF-1 beta-propeller contribute to VP16-induced complex assembly, association with LZIP, and cell cycle progression. Although LZIP and VP16 share a common tetrapeptide HCF-binding motif, our results reveal profound differences in their interaction with HCF-1. Importantly, with several of the mutants we observe a poor correlation between the ability to associate with LZIP and promote cell proliferation in the context of the full HCF-1 amino terminus, arguing that the HCF-1 beta-propeller domain must target other cellular transcription factors in order to contribute to G(1) progression.