The E6AP ubiquitin ligase catalyzes the high-risk human papillomaviruses' E6-mediated ubiquitylation of p53, contributing to the neoplastic progression of cells infected by these viruses. Defects in the activity and the dosage of E6AP are linked to Angelman syndrome and to autism spectrum disorders, respectively, highlighting the need for precise control of the enzyme. With the exception of HERC2, which modulates the ubiquitin ligase activity of E6AP, little is known about the regulation or function of E6AP normally. Using a proteomic approach, we have identified and validated several new E6AP-interacting proteins, including HIF1AN, NEURL4, and mitogen-activated protein kinase 6 (MAPK6). E6AP exists as part of several different protein complexes, including the proteasome and an independent high-molecular-weight complex containing HERC2, NEURL4, and MAPK6. In examining the functional consequence of its interaction with the proteasome, we found that UBE3C (another proteasome-associated ubiquitin ligase), but not E6AP, contributes to proteasomal processivity in mammalian cells. We also found that E6 associates with the HERC2-containing high-molecular-weight complex through its binding to E6AP. These proteomic studies reveal a level of complexity for E6AP that has not been previously appreciated and identify a number of new cellular proteins through which E6AP may be regulated or functioning. E6AP was originally discovered as the cellular protein that mediates the binding of the E6 protein of the high-risk human papillomaviruses (HPVs) to the tumor suppressor p53 (35). Subsequently, E6AP was shown to catalyze the E6-dependent transfer of ubiquitin to p53, targeting it for degradation by the 26S proteasome. As such, E6AP was the first mammalian ubiquitin (Ub) ligase to be described (36,37,80).E6AP is a 100-kDa protein containing a conserved carboxyterminal domain spanning 350 amino acids, referred to as the HECT domain (homologous to E6AP carboxy terminus), that defines a family of E3 Ub ligases (34). This domain participates directly in the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a conserved cysteine residue through a thiolester bond (34). E6AP then directs the transfer of ubiquitin from this active-site cysteine to a lysine residue of an associated substrate. Replacement of the active-site cysteine with alanine renders E6AP unable to accept ubiquitin and therefore unable to catalyze the transfer of ubiquitin to the target protein. This C/A substitution mutant is inactive and functions in a dominant-negative (DN) manner (88). Three different isoforms of E6AP that are generated by differential splicing from the same gene have been described (98). These three isoforms differ in their amino-terminal sequences, and it is not yet known whether they differ in function or substrate specificity.The E6AP protein is encoded by the UBE3A gene located in an imprinted region on chromosome 15q11-q13. As a consequence of the imprinting, only the maternal UBE3A allele is expressed in certain areas of the br...

Viruses and other pathogens can diverge in their evolved host-use strategies because of exposure to different host types and conflicts between within-host reproduction and between-host survival. Most host-pathogen studies have emphasized the role of intrahost reproduction in the evolution of pathogen virulence, whereas the role of extra-host survival has received less attention. Here, we examine the evolution of free-living virion survival in RNA virus populations differing in their histories of host use. To do so, we used lineages of vesicular stomatitis virus (VSV) that were experimentally evolved in laboratory tissue culture for 100 generations on cancer-derived cells, noncancerous cells, or alternating passages of the two host types. We observed that free-living survival improved when VSV populations specialized on human epithelial carcinoma (HeLa) cells, whereas this trait was not associated with selection on noncancer cells or combinations of the cell types. We attributed this finding to shorter-lived HeLa monolayers and/or rapid cell-to-cell spread of viruses on HeLa cells in tissue culture, both of which could select for enhanced virus stability between host-cell replenishment. We also showed evidence that increases in virion survival were associated with decreases in virulence, which suggests a trade-off between survival and virulence for the VSV populations on one cell type. Our results shed new light on the causes and consequences of "sit and wait" infection strategies in RNA viruses.