The late assembly (L) domain of retrovirus Gag, required in the final steps of budding for efficient exit from the host cell, is thought to mediate its function through interaction with unknown cellular factors. Here, we report the identification of the Nedd4-like family of E3 ubiquitin protein ligases as proteins that specifically interact with the Rous sarcoma virus (RSV) L domain in vitro and in vivo. We screened a chicken embryo cDNA expression library by using a peptide derived from the RSV p2b sequence, isolating two unique partial cDNA clones. Neither clone interacted with a peptide containing mutations known to disrupt in vivo RSV L domain function or with human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) L domain-derived peptides. The WW domain region of one of the clones, late domain-interacting protein 1 (LDI-1), but not the C2 domain, bound RSV Gag and inhibited RSV Gag budding from human 293 cells in a dominantnegative manner, functionally implicating LDI-1 in RSV particle budding from cells. RSV Gag can be coimmune precipitated from cell extracts with an antisera directed at an exogenously expressed hemagglutinin (HA)-tagged LDI-1 or endogenous Nedd4 proteins. These findings mechanistically link the cellular ubiquitination pathway to retrovirus budding.RSV ͉ Gag ͉ virus particle assembly O ne of the least understood aspects of viral replication is the assembly of virions and their exit from the host cell. However, it has been demonstrated that determinants of retroviral assembly and budding lie solely within the Gag polyprotein (1, 2). Mutational analyses of Gag revealed three distinct domains involved in virion assembly: a membrane-binding domain (M domain), found at the amino terminus of matrix (MA), which contains a myristoylation signal in most retroviruses (1, 3, 4); an internal Gag interaction domain (I domain), which maps to nucleocapsid (NC) in RSV (5), and is involved in the aggregation of Gag polyproteins necessary for particle formation; and a late assembly domain (L domain), required late in the budding process (6-10).For Gag particle budding, a functional L domain is not required on every Gag molecule (9); moreover, the L domain can function at other positions in Gag, suggesting that it is a protein interaction domain (10). The RSV Gag L domain maps specifically to a proline-rich sequence of p2b, PPPPYV, located between the MA and capsid (CA) proteins in Gag (9, 10). Referred to as the PY motif, this sequence is found in the Gag protein of many retroviruses, but not lentiviruses, and in numerous cellular proteins. Interestingly, this motif is also found in the structural proteins of other budding viruses, including rhabdoviruses (vesicular stomatitis virus) and filoviruses (Ebola and Marburg viruses; ref. 11). For VSV, this PY motif has recently been shown to function in the late steps of budding, analogous to the L domain of the retroviral Gag (12). The PY motif specifically binds to a protein-interaction domain found mostly in signaling and regula...

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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The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. In higher eukaryotes, ORC lacks sequence-specific DNA binding, and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr virus origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG-like motifs, referred to as LR1 and LR2, in the EBNA1 aminoterminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G-rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP.

Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences.