Host Cell Factor (HCF-1) is a conserved, essential protein initially identified as a co-regulator for the Herpes Simplex Virus transactivator VP16. HCF-1 is variously involved in regulating transcription, splicing, cell proliferation and cytokinesis; however, its mechanisms of action remain unknown. HCF-1 function is manifested through an increasing assortment of cellular factors that target different regions of the protein. Several HCF-1 partners target the amino-terminal kelch domain of HCF-1 (residues 1-380) via a consensus HCF-binding motif (HBM) comprising the tetrapeptide (D/E)HXY. Searches of sequence databases indicated that this motif is present in E2F1 and E2F4, two members of the E2F family of cell cycle regulators. We show here that E2F4 specifically and directly interacts with HCF-1. Mutational analysis showed E2F4 independently targets the kelch domain and the basic domain (residues 450-902) of HCF-1, both of which are required for normal cell-cycle progression via separate determinants. The HBM-containing domain of E2F4 was necessary for interaction with the kelch domain of HCF-1 but not for interaction with the basic domain. Mutations in the HCF-1 kelch domain known to block cell growth abrogated E2F4 binding to the kelch domain in the absence but not in the presence of the juxtaposed basic region. Functionally, HCF-1 co-activated E2F4/DP-1 in transient transfection assays, while E2F4 blocked HCF-1-dependent rescue of a cell line that harbors a temperature sensitive mutant of HCF-1 that causes growth arrest. Our findings show that HCF-1 and E2F4 interact via multiple determinants and suggest a linkage between E2F4 and HCF-1 cell growth pathways.

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 8MA⌬Sma showed that VP16(L344A) was able to complement the growth of 8MA⌬Sma 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.Herpes simplex virus type 1 (HSV-1) is a large, enveloped DNA virus whose genome encodes some 80 genes. These genes fall into three broad kinetic classes, depending on their order of appearance during a lytic infection: immediate early (IE or ␣), early (E or ␤), and late (L or ␥). Members of each class are coordinately and temporally regulated in a cascade fashion, primarily at the transcriptional level, by interactive networks that involve both virus and host factors. HSV-1 is noteworthy in that several important viral regulatory proteins exist as preformed structural components of the virion. These factors are delivered into the host cell by the infecting virus particle and are thus poised to affect the earliest events of viral infection and initiation of replication (reviewed in reference 41). The most prominent of these is the viral transactivator VP16, an abundant 490-amino-acid phosphoprotein contained in the viral tegument, an amorphous protein layer present between the viral capsid and envelope (5). VP16 possesses a potent C-terminal transactivation domain and triggers the lytic cycle by initiating IE gene expression via conserved cis-acting TAATGARAT elements present in the promoter regions of all the IE genes (2, 33, 38, 42, 51). VP16 on its own has only weak DNA binding activity, and efficient recruitment to promoter target sites relies on the assembly of a multicomponent protein/DNA binding transc...

The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. A number of distinct properties of VP16 have been implicated in the assembly of the VP16-induced complex (VIC). These include its independent association with VCAF-1 and, under appropriate conditions, its ability to bind to DNA or to DNAbound Oct-1 in the absence of VCAF-1. In order to probe the requirements of these individual interactions in the functional asembly of VIC, we mutated selected charged amino acids in two subdomains of VP16 previously shown to be important in protein-DNA complex formation. Purified VP16 proteins were analyzed for their ability to direct protein-DNA complex formation and to interact directly with VCAF-1. Several classes of mutants that were differentially compromised in VCAF-1 interaction, direct DNA binding, and/or association with DNA-bound Oct-1 were obtained. Interestingly, all of the derivatives were still capable of generating the VIC complex in vitro and activating transcription in vivo. Our findings indicate that the cooperative assembly of functional VP16-containing complexes can occur by pathways that do not necessarily require the prior interaction of VP16 with VCAF-1 or the ability of VP16 to bind directly to DNA or associate with DNA-bound Oct-1.

The herpes simplex virus transactivator VP16 and the virion host shutoff protein vhs are viral structural components that direct the activation of immediate-early gene expression and the arrest of host protein synthesis, respectively, during an infection. Recent studies show that VP16 and vhs physically interact with each other in vitro and in infected cells, suggesting that their respective regulatory functions are coupled. In this report, we used the yeast two-hybrid system and affinity chromatography with purified VP16 fusion proteins to precisely map a region in vhs that directs interaction with VP16. Deletion analysis of vhs demonstrated that a 21-amino-acid-long domain spanning residues 310 to 330 (PAAGGTEMRVSWTEILTQQIA) was sufficient for directing complex formation with VP16 in vivo and in vitro when fused to a heterologous protein. Site-directed mutagenesis of this region identified tryptophan 321 as a crucial determinant for interaction with VP16 in vitro and in vivo and additional residues that are important for stable complex formation in vitro. These findings indicate that vhs residues 310 to 330 constitute an independent and modular binding interface that is recognized by VP16.