During latency, Epstein-Barr virus (EBV) is stably maintained as a circular plasmid that is replicated once per cell cycle and partitioned at mitosis. Both these processes require a single viral protein, EBV nuclear antigen 1 (EBNA1), which binds two clusters of cognate binding sites within the latent viral origin, oriP. EBNA1 is known to associate with cellular metaphase chromosomes through chromosome-binding domains within its amino terminus, an association that we have determined to be required not only for the partitioning of oriP plasmids but also for their replication. One of the chromosome-binding domains of EBNA1 associates with a cellular nucleolar protein, EBP2, and it has been proposed that this interaction underlies that ability of EBNA1 to bind metaphase chromosomes. Here we demonstrate that EBNA1's chromosome-binding domains are AT hooks, a DNA-binding motif found in a family of proteins that bind the scaffold-associated regions on metaphase chromosomes. Further, we demonstrate that the ability of EBNA1 to stably replicate and partition oriP plasmids correlates with its AT hook activity and not its association with EBP2. Finally, we examine the contributions of EBP2 toward the ability of EBNA1 to associate with metaphase chromosomes in human cells, as well as support the replication and partitioning of oriP plasmids in human cells. Our results indicate that it is unlikely that EBP2 directly mediates these activities of EBNA1 in human cells.The latent Epstein-Barr virus (EBV) genome is stably retained in cells extrachromosomally as a circular plasmid. Stable replication of the EBV genome relies on the latently expressed viral protein EBV nuclear antigen 1 (EBNA1) binding to both the family of repeats (FR) within oriP, as well as to cellular chromosomes. This tethering of viral plasmids to cellular chromosomes is hypothesized to be necessary for their replication during S phase and partitioning during mitosis.Homodimers of EBNA1 are bound via a carboxy-terminal DNA-binding domain (DBD; amino acids [aa] 451 to 640) to two clusters of binding sites within oriP (60) called the FR and the dyad symmetry element (DS), whereas the amino terminus of EBNA1 (aa 1 to 450) is required for an association with cellular chromosomes (37, 48). During S phase, in concert with cellular chromosomes, oriP-plasmids are semiconservatively replicated (1, 2, 11, 47, 61), a process mediated by EBNA1's binding at the DS (7). However, the DBD of EBNA1 is by itself insufficient to recruit the licensed cellular replication apparatus to DS (28) but requires that the amino terminus of EBNA1 contain domain(s) that associate with mitotic chromosomes (48). We have recently demonstrated that the entire amino terminus of EBNA1 can be replaced by a cellular protein that specifically associates with mitotic chromosomes and that such a fusion supports the stable replication and partitioning of oriP plasmids similarly to wild-type EBNA1. In contrast, a fusion in which the amino terminus associates with interphase chromatin, but not mitotic ...

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Rhizomania is an important virus disease of sugar beet and is caused by Beet necrotic yellow vein virus (BNYVV). During 2002-03, several sugar beet fields with cultivars partially resistant to BNYVV grown in the Imperial Valley of California were observed with severe rhizomania symptoms, suggesting that resistance conditioned by Rz1 had been compromised. Soil testing with sugar beet baiting plants followed by enzyme-linked immunosorbent assay (ELISA) was used to diagnose virus infection. Resistant varieties grown in BNYVV-infested soil from Salinas, CA, were ELISA-negative. In contrast, when grown in BNYVV-infested soil collected from the Imperial Valley, CA, all resistant varieties became infected and tested positive by ELISA. Based on host reaction, eight distinct BNYVV isolates have been identified from Imperial Valley soil (IV-BNYVV) by single local lesion isolation. Reverse transcription-polymerase chain reaction (RT-PCR) assays showed that the eight IV-BNYVV isolates did not contain RNA-5. Singlestrand conformation polymorphism banding patterns for the IV-BNYVV isolates were identical to A-type and different from P-type. Sequence alignments of PCR products from BNYVV RNA-1 near the 3′ end of IV-BNYVV isolates revealed that both IV-BNYVV and Salinas BNYVV isolates were similar to A-type and different from B-type. Our results suggest that the resistancebreaking BNYVV isolates from Imperial Valley likely evolved from existing A-type isolates.

Epstein-Barr Virus (EBV) infects resting B cells, within which it establishes latency as a stable, circular episome with only two EBV components, the cis element oriP and the latently expressed protein EBNA1. It is believed that EBNA1's ability to tether oriP episomes to metaphase chromosomes is required for its stable replication. We created fusions between the DNA-binding domain (DBD) of EBNA1 and the cellular chromatin-binding proteins HMGA1a and HMG1 to determine the minimal requirements for stable maintenance of an oriP-based episome. These two proteins differ in that HMGA1a can associate with metaphase chromosomes but HMG1 cannot. Interestingly, coinciding with metaphase chromosome association, HMGA1a-DBD but not HMG1-DBD supported both the transient replication and stable maintenance of oriP plasmids, with efficiencies quantitatively similar to that of EBNA1. However, HMGA1a-DBD activated transcription from EBNA1-dependent episomal reporter to only 20% of the level of EBNA1. Furthermore, EBNA1 but not HMGA1a-DBD activated transcription from a chromosomally integrated EBNA1-dependent transcription reporter. This indicates that EBNA1 possesses functional domains that support transcription activation independent of its ability to tether episomal oriP plasmids to cellular chromosomes. We provide evidence that metaphase chromosome tethering is a fundamental requirement for maintenance of an oriP plasmid but is insufficient for EBNA1 to activate transcription. Epstein-Barr virus (EBV) is a gammaherpesvirus that infectsresting B cells and also epithelial cells. Upon infection, the double-stranded, linear EBV genome circularizes and persists as a stable episome, infrequently integrating into the human genome. In rapidly dividing cells, stable maintenance of the EBV episome at copy numbers between 1 and 100 (48) requires the replication and partitioning of its genome through ensuing cell cycles. During latency, EBV replicates only once per cell cycle semiconservatively (1, 47), utilizing the cellular replication machinery for its own DNA synthesis (2, 9, 41).Stable maintenance of an EBV-based episomal plasmid requires only the origin of latent replication (in cis) oriP (46), and the latently expressed viral protein (in trans) EBNA1 (48). EBNA1 homotypically interacts with two clusters of imperfect, repetitive binding sites, the family of repeats (FR) and the dyad symmetry element (DS), found within the cis element oriP. Four binding sites within the DS, a formal origin of bidirectional replication, contribute to the initiation of DNA synthesis; the 20 binding sites comprising the FR support stable plasmid maintenance (2, 29) and transactivation by EBNA1 (39).The amino terminus of EBNA1 has two positively charged regions, which we term A (amino acids 33 to 89) and B (amino acids 328 to 378), that are required for EBNA1 to attach to cellular chromosomes, activate transcription, support the stable replication of oriP plasmids, and link DNAs. EBNA1 participates in all these processes without possessing any known enzym...