Placement of the plastid-division machinery at the organelle midpoint requires a plastid-targeted form of MinD. The results are consistent with a model whereby assembly of the division apparatus is initiated inside the chloroplast by the plastidic form of FtsZ, and suggest that positioning of the cytosolic components of the apparatus is specified by the position of the plastidic components.

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), has significant sequence homology to Epstein-Barr virus (EBV). In cell culture, HHV8 is primarily latent, and viral genes associated with lytic replication are not expressed. Two lytic origins of DNA replication (oriLyt) are present within the HHV8 genome and are composed of an AT-rich region adjacent to GC-rich DNA sequences. We have now identified essential cis- and trans-acting elements required for oriLyt-dependent DNA replication. The transient replication assay was used to show that two AT-rich elements, three consensus AP1 transcription factor-binding sites, an ORF50 response element (RE), and a consensus TATA box motif are essential for efficient origin-dependent DNA replication. Transient transfection of luciferase reporter constructs indicated that the downstream region of the HHV8 oriLyt responds to ORF50 and suggests that part of the oriLyt may be an enhancer/promoter. In addition, a transient cotransfection-replication assay elucidated the set of trans-acting factors required for lytic DNA replication. These factors consist of homologues to the core replication proteins: ORF6 (ssDNA binding protein), ORF9 (DNA polymerase), ORF40-41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor) common to all herpesviruses along with ORF50 (K-Rta) and K8 (K-bZIP).

Human cytomegalovirus (HCMV) UL84 is a phosphoprotein that shuttles from the nucleus to the cytoplasm and is required for oriLyt-dependent DNA replication and viral growth. UL84 was previously shown to interact with IE2 (IE86) in infected cells, and this interaction down-regulates IE2-mediated transcriptional activation in transient assays. UL84 and IE2 were also shown to cooperatively activate a promoter within HCMV oriLyt. UL84 alone can interact with an RNA stem-loop within oriLyt and is bound to this structure within the virion. In an effort to investigate the binding partners for UL84 in infected cells, we pulled down UL84 from protein lysates prepared from HCMV-infected human fibroblasts by using a UL84-specific antibody and resolved the immunoprecipitated protein complexes by two-dimensional gel electrophoresis. We subsequently identified individual proteins by matrix-assisted laser desorption ionization-tandem time of flight analysis. This analysis revealed that UL84 interacts with viral proteins UL44, pp65, and IE2. In addition, a number of cell-encoded proteins were identified, including ubiquitin-conjugating enzyme E2, casein kinase II (CKII), and the multifunctional protein p32. We also confirmed the interaction between UL84 and IE2 as well as the interaction of UL84 with importin ␣. UL44, pp65, and CKII interactions were confirmed to occur in infected and cotransfected cells by coimmunoprecipitation assays followed by Western blotting. Ubiquitination of UL84 occurred in the presence and absence of the proteasome activity inhibitor MG132 in infected cells. The identification of UL84 binding partners is a significant step toward the understanding of the function of this significant replication protein.Human cytomegalovirus (HCMV) lytic DNA replication requires the cis-acting oriLyt sequence, six core replication proteins, and, in human fibroblasts, the immediate-early protein IE2 and the multifunctional protein UL84 (4, 29, 30). Recent evidence indicates that UL84 may have several roles in the virus life cycle. Also, a number of activities have been associated with UL84. These include a shuttling function, inhibition of IE2-mediated transcriptional activation, RNA binding, and UTPase activity (9,12,17,43). The UL84-IE2 protein complex can activate a strong bidirectional promoter within oriLyt, presumably to initiate a transcription event that triggers initiation of DNA synthesis. Evidence that UL84 is the initiator protein for lytic DNA synthesis comes from recent data demonstrating that UL84 binds to a stem-loop RNA structure within oriLyt (7). In vitro binding assays suggest that UL84 can change the conformation of RNA stem-loop structures and possibly allow for the assembly of the replication complex. UL84 was shown to be a component of the virion (40) and is bound to viral DNA at the lytic origin within isolated virions (7). This suggests that UL84 can play a positive or a negative role in initiation of lytic DNA synthesis. Both the wide range of activities associated with UL84 and analysis of the...

Amplification of the human cytomegalovirus (HCMV) lytic origin (oriLyt) in human fibroblasts is dependent upon six core replication proteins and UL84, IE2, and UL36-38. Using a telomerase-immortalized human fibroblast cell line (T-HFs), oriLyt-dependent DNA replication no longer required the gene products of UL36-38. To determine the role of IE2 in DNA replication in human fibroblasts, we examined potential IE2-binding sites within HCMV oriLyt. We now show that a strong bidirectional promoter (oriLyt PM ) (nucleotides 91754 to 92030) is located in the previously identified core region of the origin and is required for efficient amplification of oriLyt. It was determined that a 14-bp novel DNA motif (oriLyt promoter activation element), which was initially identified as a binding element for the immediate-early protein IE2, was essential for oriLyt PM activity. In Vero cells the oriLyt PM was constitutively active and strongly repressed by IE2, but it was reactivated by UL84. In contrast, transfection of the oriLyt PM into human fibroblasts resulted in a very low basal level of promoter activity that was dramatically up-regulated upon infection with HCMV. Cotransfection assays demonstrated that the transfection of UL84 along with IE2 transactivated the oriLyt PM in human fibroblasts. Further activation was observed upon cotransfection of the set of plasmids expressing the entire replication complex. Efficient oriLyt amplification in the absence of IE2 in human fibroblasts was observed by replacing the oriLyt PM with the simian virus 40 early promoter. Under these conditions, however, UL84 was still required for amplification of oriLyt. These results suggest that the mechanism of initiation of HCMV lytic replication in part involves transcriptional activation.Human cytomegalovirus (HCMV) contains a single lytic origin for DNA replication, oriLyt, located near the center of the U L region and upstream of the open reading frame (ORF) encoding the single-stranded DNA-binding protein ppUL57 (1,19,40). The HCMV oriLyt is remarkable among viral replication origins for its apparent size and complexity. The entire oriLyt region of HCMV is located from nucleotides (nt) 90500 to 93930 and is composed of a core (nt 91751 to 93299) which contains two essential regions (1,40,66). These essential regions (I and II) contain a pyrimidine-rich sequence (Y-block), various reiterated sequences, several transcription factor-binding sites, direct and inverted repeat sequences, base composition biases and strand asymmetries, and RNA-DNA hybrid structures and is a site of active transcription (the small replication transcript [SRT]) (1,22,40,49,66). Despite the apparent exhaustive investigation of elements within oriLyt that contribute to DNA synthesis, few data exist with respect to the actual function of these elements in initiation of DNA replication.Some information concerning the initiation of DNA synthesis can be inferred from the required virus-encoded transacting factors necessary to replicate oriLyt. In human fibroblasts, oriLy...

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.A common feature of herpesvirus lytic DNA replication is the requirement for six core replication proteins. These proteins-a DNA polymerase, helicase, primase, primase-associated factor, DNA processivity factor, and a single-stranded DNA-binding protein-carry out the necessary enzymatic processes to efficiently synthesize DNA (11,21,23). These enzymatic replication events follow the obligatory initiation of DNA synthesis that is mediated by proteins that are unique to each herpesvirus system and have been traditionally referred to as initiator proteins or origin-binding proteins (OBPs). In some cases, these initiator proteins themselves have an inherent catalytic property that may unwind a specific region of DNA and facilitate the assembly of the entire core replication machinery.Although the exact mechanism by which these OBPs initiate DNA synthesis has not been elucidated, several common features have been defined. (i) It appears that all characterized initiator proteins interact directly or indirectly with DNA within the origin of lytic replication. (ii) All identified initiator proteins have been shown to interact with themselves in transfected cells and contain leucine zipper motifs (5,7,8,14,15). Stable self-interaction of these proteins is seemingly necessary for their intrinsic enzymatic activity and may serve to regulate the initiation of DNA synthesis.For HCMV, the transient-cotransfection replication as...