E2F transcriptional regulators control human-cell proliferation by repressing and activating the transcription of genes required for cell-cycle progression, particularly the S phase. E2F proteins repress transcription in association with retinoblastoma pocket proteins, but less is known about how they activate transcription. Here, we show that the human G1 phase regulator HCF-1 associates with both activator (E2F1 and E2F3a) and repressor (E2F4) E2F proteins, properties that are conserved in insect cells. Human HCF-1-E2F interactions are versatile: their associations and binding to E2F-responsive promoters are cell-cycle selective, and HCF-1 displays coactivator properties when bound to the E2F1 activator and corepressor properties when bound to the E2F4 repressor. During the G1-to-S phase transition, HCF-1 recruits the mixed-lineage leukemia (MLL) and Set-1 histone H3 lysine 4 methyltransferases to E2F-responsive promoters and induces histone methylation and transcriptional activation. These results suggest that HCF-1 induces cell-cycle-specific transcriptional activation by E2F proteins to promote cell proliferation.

The hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The biology and pathogenesis of HEV remain poorly understood. We have used in vitro binding assays to show that the HEV ORF3 protein (pORF3) binds to a number of cellular signal transduction pathway proteins. This includes the protein tyrosine kinases Src, Hck, and Fyn, the p85␣ regulatory subunit of phosphatidylinositol 3-kinase, phospholipase C␥, and the adaptor protein Grb2. A yeast two-hybrid assay was used to further confirm the pORF3-Grb2 interaction. The binding involves a proline-rich region in pORF3 and the src homology 3 (SH3) domains in the cellular proteins. Competition assays and computer-assisted modeling was used to evaluate the binding surfaces and interaction energies of the pORF3⅐SH3 complex. In pORF3-expressing cells, pp60 src was found to associate with an 80-kDa protein, but no activation of the Src kinase was observed in these cells. However, there was increased activity and nuclear localization of ERK in the pORF3-expressing cells. These studies suggest that pORF3 is a viral regulatory protein involved in the modulation of cell signaling. The ORF3 protein of HEV appears to be the first example of a SH3 domain-binding protein encoded by a virus that causes an acute and primarily self-limited infection.Hepatitis E virus (HEV), 1 the causative agent for hepatitis E, is a waterborne pathogen endemic to much of the developing world where it causes rampant sporadic infections and large scale epidemics (1-4). While the infection is self-limited with no associated chronicity, a fraction of the patients progress to fulminant hepatitis (5, 6), the most severe form of acute hepatitis. High mortality rates of 20 -30% reported for HEV infection during pregnancy (7,8) are also the result of fulminant hepatitis. The reasons for this and the mechanisms of viral pathogenesis are not known. The studies on HEV biology and pathogenesis have been severely restricted by the lack of a reliable cell culture system and small animal models of viral infection. We have used subgenomic expression strategies to study the properties and functions of individual HEV gene products toward understanding viral replication and pathogenicity (9 -12).The HEV genome is a ϳ7.5-kilobase polyadenylated, positive-sense RNA that contains three open reading frames (ORFs) designated ORF1, ORF2, and ORF3 (13). The ORF3 of HEV encodes a protein of ϳ13.5 kDa, called pORF3, for which no function has been assigned. When expressed in animal cells, pORF3 is phosphorylated at a single serine residue (Ser 80 ) in its 123-amino acid primary sequence (11). In vitro phosphorylation experiments suggested that pORF3 may be a substrate for the mitogen-activated protein (MAP) kinase, and subcellular fractionation revealed its association with the cytoskeleton (11). Recent results using inhibitors, activators, and dominant negative alleles show that pORF3 is a substrate for the extracellular signal-regulated kinase (ERK) as well as the stressactivated pr...

Hepatitis E virus (HEV) is a human RNA virus contain-ing three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein, which exists in a glycosylated and non-glycosylated form; and ORF3 codes for a phosphoprotein of undefined function. Using fluorescence-based colocalization, yeast two-hybrid experiments, transiently transfected COS-1 cell co-immunoprecipitation, and cell-free coupled transcription-translation techniques, we have shown that the ORF3 protein interacts with the ORF2 protein. The domains involved in this ORF2-ORF3 association have been identified and mapped. Our deletion analysis showed that a 25-amino acid region (residues 57-81) of the ORF3 protein is required for this interaction. Using a Mexican HEV isolate, site-directed mutagenesis of ORF3, and a phosphatase digestion assay, we showed that the ORF2-ORF3 interaction is dependent upon the phosphorylation at Ser 80 of ORF3. Finally, using COS-1 cell immunoprecipitation experiments, we found that the phosphorylated ORF3 protein preferentially interacts with the non-glycosylated ORF2 protein. These findings were confirmed using tunicamycin inhibition, point mutants, and deletion mutants expressing only non-glycosylated ORF2. ORF3 maps in the structural region of the HEV genome and now interacts with the major capsid protein, ORF2, in a post-translational modification-dependent manner. Such an interaction of ORF2 with ORF3 suggests a possible well regulated role for ORF3 in HEV structural assembly.Hepatitis E is an acute disease endemic in many countries throughout developing parts of the world, in particular on the continents of Africa and Asia, where it causes epidemics and sporadic infections. The causative agent, hepatitis E virus (HEV), 1 is transmitted via the fecal-oral route, predominantly through contaminated water (1). HEV is an RNA virus with a positive-sense genome ϳ7.2 kb in length with three open reading frames (ORF1, ORF2, and ORF3) encoding three different proteins (2-4). ORF1 (5079 bp) is at the 5Ј-end of the genome and is predicted to code for putative nonstructural proteins with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (3-6). In the absence of a reliable in vitro culture system for HEV, fundamental studies on its replication and expression strategy have not been undertaken. ORF2 and ORF3 have been expressed in Escherichia coli, animal cells, baculovirus, and yeast and in vitro in a coupled transcriptiontranslation system (7-11). ORF2 encodes the major HEV structural protein, which has been shown to be an 88-kDa glycoprotein that is expressed intracellularly as well as on the cell surface. It is synthesized as a precursor and is processed through signal sequence cleavage into the mature protein, which is capable of self-association (12, 13). When expressed through the baculoviral expression system, ORF2 was shown to assemble into virus-like particles (VLPs), which were cell-associated as w...

Mixed-lineage leukemia (MLL), along with multisubunit (WDR5, RbBP5, ASH2L, and DPY30) complex catalyzes the trimethylation of H3K4, leading to gene activation. Here, we characterize a chromatin-independent role for MLL during mitosis. MLL and WDR5 localize to the mitotic spindle apparatus, and loss of function of MLL complex by RNAi results in defects in chromosome congression and compromised spindle formation. We report interaction of MLL complex with several kinesin and dynein motors. We further show that the MLL complex associates with Kif2A, a member of the Kinesin-13 family of microtubule depolymerase, and regulates the spindle localization of Kif2A during mitosis. We have identified a conserved WDR5 interaction (Win) motif, so far unique to the MLL family, in Kif2A. The Win motif of Kif2A engages in direct interactions with WDR5 for its spindle localization. Our findings highlight a non-canonical mitotic function of MLL complex, which may have a direct impact on chromosomal stability, frequently compromised in cancer.