Faculty Bibliography

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

HCF-1 functions as a coactivator for herpes simplex virus VP16 and a number of mammalian transcription factors. Mature HCF-1 is composed of two subunits generated by proteolytic cleavage of a larger precursor at six centrally-located HCF(PRO) repeats. The resulting N- and C-terminal subunits remain tightly associated via two complementary pairs of self-association domains: termed SAS1N-SAS1C and SAS2N-SAS2C. Additional HCF proteins have been identified in mammals (HCF-2) and Caenorhabditis elegans (CeHCF). Both contain well-conserved SAS1 domains but do not undergo proteolytic processing. Thus, the significance of the cleavage and self-association of HCF-1 remains enigmatic. Here, we describe the isolation of the Drosophila HCF homologue (dHCF) using a genetic screen based on conservation of the SAS1 interaction. The N-terminal beta-propeller domain of dHCF supports VP16-induced complex formation and is more similar to mammalian HCF-1 than other homologues. We show that full-length dHCF expressed in Drosophila cells undergoes proteolytic cleavage giving rise to tightly associated N- and C-terminal subunits. As with HCF-1, the SAS1N and SAS1C elements of dHCF are separated by a large central region, however, this sequence lacks obvious homology to the HCF(PRO) repeats required for HCF-1 cleavage. The conservation of HCF processing in insect cells argues that formation of separate N- and C-terminal subunits is important for HCF function

In herpes simplex virus, lytic replication is initiated by the viral transactivator VP16 acting with cellular cofactors Oct-1 and HCF-1. Although this activator complex has been studied in detail, the role of HCF-1 remains elusive. Here, we show that HCF-1 contains an activation domain (HCF-1(AD)) required for maximal transactivation by VP16 and its cellular counterpart LZIP. Expression of the VP16 cofactor p300 augments HCF-1(AD) activity, suggesting a mechanism of synergy. Infection of cells lacking the HCF-1(AD) leads to reduced viral immediate-early gene expression and lowered viral titers. These findings underscore the importance of HCF-1 to herpes simplex virus replication and VP16 transactivation

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

When herpes simplex virus infects permissive cells, the viral regulatory protein VP16 forms a specific complex with HCF-1, a preexisting nuclear protein involved in cell proliferation. The majority of HCF-1 in the cell is a complex of associated amino (HCF-1(N))- and carboxy (HCF-1(C))-terminal subunits that result from an unusual proteolytic processing of a large precursor polypeptide. Here, we have characterized the structure and function of sequences required for HCF-1(N) and HCF-1(C) subunit association. HCF-1 contains two matched pairs of self-association sequences called SAS1 and SAS2. One of these matched association sequences, SAS1, consists of a short 43-amino-acid region of the HCF-1(N) subunit, which associates with a carboxy-terminal region of the HCF-1(C) subunit that is composed of a tandem pair of fibronectin type 3 repeats, a structural motif known to promote protein-protein interactions. Unexpectedly, the related protein HCF-2, which is not proteolyzed, also contains a functional SAS1 association element, suggesting that this element does not function solely to maintain HCF-1(N) and HCF-1(C) subunit association. HCF-1(N) subunits do not possess a nuclear localization signal. We show that, owing to a carboxy-terminal HCF-1 nuclear localization signal, HCF-1(C) subunits can recruit HCF-1(N) subunits to the nucleus

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

Human herpesvirus 8 (HHV-8; also known as Kaposi's sarcoma-associated herpesvirus) is the causative agent of Kaposi's sarcoma and certain B-cell lymphomas. In most infected cells, HHV-8 establishes a latent infection characterized by the expression of latency-associated nuclear antigen (LANA) encoded by open reading frame 73. Although unrelated by sequence, there are functional similarities between LANA and the EBNA-1 protein of Epstein-Barr virus. Both accumulate as subnuclear speckles and are required for maintenance of the viral episome. EBNA-1 also regulates viral gene expression and is required for cell immortalization, suggesting that LANA performs similar functions in the context of HHV-8 infection. Here we show that LANA forms stable dimers, or possibly higher-order multimers, and that this is mediated by a conserved region in the C terminus. By expressing a series of truncations, we show that both the N- and C-terminal regions localize to the nucleus, although only the C terminus accumulates as nuclear speckles characteristic of the intact protein. Lastly, we show that LANA can function as a potent transcriptional repressor when tethered to constitutively active promoters via a heterologous DNA-binding domain. Domains in both the N and C termini mediate repression. This suggests that one function of LANA is to suppress the expression of the viral lytic genes or cellular genes involved in the antiviral response

Host Cell Factor-1 (HCF-1, C1) was first identified as a cellular target for the herpes simplex virus transcriptional activator VP16. Association between HCF and VP16 leads to the assembly of a multiprotein enhancer complex that stimulates viral immediate-early gene transcription. HCF-1 is expressed in all cells and is required for progression through G(1) phase of the cell cycle. In addition to VP16, HCF-1 associates with a cellular bZIP protein known as LZIP (or Luman). Both LZIP and VP16 contain a four-amino acid HCF-binding motif, recognized by the N-terminal beta-propeller domain of HCF-1. Herein, we show that the N-terminal 92 amino acids of LZIP contain a potent transcriptional activation domain composed of three elements: the HCF-binding motif and two LxxLL motifs. LxxLL motifs are found in a number of transcriptional coactivators and mediate protein-protein interactions, notably recognition of the nuclear hormone receptors. LZIP is an example of a sequence-specific DNA-binding protein that uses LxxLL motifs within its activation domain to stimulate transcription. The LxxLL motifs are not required for association with the HCF-1 beta-propeller and instead interact with other regions in HCF-1 or recruit additional cofactors

Herpes simplex virus infection is initiated by VP16, a viral transcription factor that activates the viral immediate-early (IE) genes. VP16 does not recognize the IE gene promoters directly but instead forms a multiprotein complex with Oct-1 and HCF-1, a ubiquitous nuclear protein required for progression through the G1 phase of the cell cycle. The functional significance of recruiting HCF-1 to the VP16-induced complex is not understood. Here we describe the identification of a second HCF-like protein, designated HCF-2. HCF-2 is smaller than HCF-1 but shares three regions of strong amino acid sequence homology, including the beta-propeller domain required for association with VP16. HCF-2 is expressed in many tissues, especially the testis, and shows a more dynamic pattern of subcellular localization than HCF-1. Although HCF-2 associates with VP16 and can support complex assembly with Oct-1 and DNA, it is significantly less efficient than HCF-1. A similar preference is shown by LZIP, a cellular counterpart of VP16. Analysis of chimeric proteins showed that differences between the fifth and sixth kelch repeats of the beta-propeller domains from HCF-1 and HCF-2 dictate this selectivity. These results reveal an unexpected level of specificity in the recruitment of HCF-1 to the VP16-induced complex, paralleling the preferential selection of Oct-1 rather than the closely related POU domain protein Oct-2. Implications for regulation of the viral life cycle are discussed