A Novel 50-Kilodalton Fragment of Host Cell Factor 1 (C1) in G0 Cells

Upon herpes simplex virus (HSV) infection, the viral protein VP16 (also called αTIF and vmw65) is released from the virion particle and forms a complex with two cellular proteins, Oct-1 and host cell factor 1 (HCF-1; also called C1 and VCAF) (2, 7, 16, 19). This complex, termed the C1 complex, directs specific transcription from the alpha/immediate-early (α/IE) promoter element of the viral genome (8). Oct-1 is a member of the POU domain family of proteins and normally regulates transcription from octamer and related elements (5′-ATGCAAAT-3′) found in the promoters of a diverse array of cellular genes (5, 21). In contrast, the complex of Oct-1 and VP16 has specificity for the viral α gene promoters, with Oct-1 contacting the 5′ half of the α/IE promoter element (5′-ATGCTAAT-3′), and VP16 making contacts in the 3′ half (8). HCF-1 stabilizes the interaction between VP16 and Oct-1 and may also contact the DNA (8, 23). HCF-1 consists of an array of polypeptides, varying in length from 100 to 230 kDa (6, 9, 23). All of the observed peptides are derived from the proteolysis of a single 2,035-amino-acid, 230-kDa protein (6, 23). After translation, HCF-1 is imported into the nucleus, where it is cleaved at one or more of the six near-perfect 26-amino-acid repeats found near the center of the protein. The cleavage is specific and occurs between a glutamic acid and a threonine (6, 25). Neither this 26-amino-acid sequence nor a related motif has been identified as a cleavage site in other proteins, but this repeat can direct the cleavage of a heterologous protein when inserted into its coding sequence (25). After cleavage, the N and C termini of HCF-1 remain strongly associated (6, 25). The N terminus of HCF-1 contains a series of six kelch repeats (named after the Drosophila egg chamber protein kelch, in which they were originally recognized). These repeats are found in other unrelated proteins and are predicted to fold into a barrel-like β-propeller structure (22). The first 360 amino acids of HCF-1 (HCF1–360), encompassing the kelch repeat region, are sufficient for binding to VP16 and for formation of a fast-mobility C1-like complex in vitro (12, 22). The cellular protein luman, or LZIP, a member of the basic leucine zipper family of DNA-binding proteins, has also been shown to bind HCF-1 in this region (1, 13). VP16 and luman share a short region of amino acid homology that, when mutated, greatly reduces both proteins' ability to bind HCF-1 (1, 14). A proline-to-serine mutation in the third kelch repeat (P134S) confers a temperature-sensitive phenotype on HCF-1. BHK cells carrying this mutation in their only copy of HCF-1 arrest in a G0-like state. These cells divide for 36 h after a shift to the nonpermissive temperature, then arrest and remain highly viable for several days. If they are shifted back to the permissive temperature during this time, they will reenter the G1 phase of the cell (3). This suggests that a function of HCF-1 is required for cells to cycle. Consistent with this hypothesis, HCF-1 is most abundant in cycling cells, including fetal tissue and immortalized cell lines (6, 24). It has therefore been proposed that VP16 interacts with HCF-1 to allow the infecting virus to sense the proliferative state of the host cell (3). At the nonpermissive temperature, the expression levels and proteolytic profiles of the P134S mutant HCF-1 are identical to those of the wild-type protein, and the mutant N terminus is still able to associate with the cleaved C terminus. However, mutant HCF-1 is unable to support VP16-mediated transcription from an α/IE reporter element in vivo and does not support C1 complex formation in vitro (3, 22). Nearly the entire N terminus of HCF-1 (HCF1–902) is required for complementation of the temperature-sensitive phenotype; deletion of as few as 66 additional amino acids from the C terminus of this fragment renders it unable to prevent G0 arrest at the nonpermissive temperature (22). The role of HCF-1 in controlling HSV entry into either a replicative or latent state is not known. The amount of VP16 that enters the nucleus may influence the balance toward one type of infection or the other (20). La Boissiere et al. recently reported that VP16 entry into the nucleus depends on the HCF-1 nuclear localization signal (NLS), which is found within the C terminus of HCF-1. When the NLS was deleted, transfected HCF-1 remained in the cytoplasm, along with a large portion of the cotransfected VP16 (11). In extracts from dorsal root ganglia, a cell type in which HSV is able to establish latency, a faster-mobility form of the C1 complex has been observed, suggesting that an altered form of HCF-1 may be present (4). Interestingly, immunofluorescent staining of HCF-1 in trigeminal ganglia shows the protein to be sequestered in cytoplasmic granules, and conditions which activate HSV from latency rapidly induce the appearance of HCF-1 in the nucleus of these cells (10). We have investigated the structure of HCF-1 in primary cells and describe a truncated form of HCF-1 that is present in resting, or G0, cells. This N-terminal fragment is able to bind VP16 and is present in cytoplasmic extracts from these cells.