Using the E4orf6-Based E3 Ubiquitin Ligase as a Tool To Analyze the Evolution of Adenoviruses

Abstract

E4orf6 proteins from all human adenoviruses form Cullin-based ubiquitin ligase complexes that, in association with E1B55K, target cellular proteins for degradation. While most are assembled with Cul5, a few utilize Cul2. BC-box motifs enable all these E4orf6 proteins to assemble ligase complexes with Elongins B and C. We also identified a Cul2-box motif used for Cul2 selection in all Cul2-based complexes. With this information, we set out to determine if other adenoviruses also possess the ability to form the ligase complex and, if so, to predict their Cullin usage. Here we report that all adenoviruses known to encode an E4orf6-like protein (mastadenoviruses and atadenoviruses) maintain the potential to form the ligase complex. We could accurately predict Cullin usage for E4orf6 products of mastadenoviruses and all but one atadenovirus. Interestingly, in nonhuman primate adenoviruses, we found a clear segregation of Cullin binding, with Cul5 utilized by viruses infecting great apes and Cul2 by Old/New World monkey viruses, suggesting that a switch from Cul2 to Cul5 binding occurred during the period when great apes diverged from monkeys. Based on the analysis of Cullin selection, we also suggest that the majority of human adenoviruses, which exhibit a broader tropism for the eye and the respiratory tract, exhibit Cul5 specificity and resemble viruses infecting great apes, whereas those that infect the gastrointestinal tract may have originated from monkey viruses that share Cul2 specificity. Finally, aviadenoviruses also appear to contain E4orf6 genes that encode proteins with a conserved XCXC motif followed by, in most cases, a BC-box motif. IMPORTANCETwo early adenoviral proteins, E4orf6 and E1B55K, form a ubiquitin ligase complex with cellular proteins to ubiquitinate specific substrates, leading to their degradation by the proteasome. In studies with representatives of each human adenovirus species, we (and others) previously discovered that some viruses use Cul2 to form the complex, while others use Cul5. In the present study, we expanded our analyses to all sequenced adenoviruses and found that E4orf6 genes from all mast-and atadenoviruses encode proteins containing the motifs necessary to form the ligase complex. We found a clear separation in Cullin specificity between adenoviruses of great apes and Old/New World monkeys, lending support for a monkey origin for human viruses of the Human mastadenovirus A, F, and G species. We also identified previously unrecognized E4orf6 genes in the aviadenoviruses that encode proteins containing motifs permitting formation of the ubiquitin ligase.

Adenoviruses across the animal kingdom: a walk in the zoo

“…The atadenovirus genomes do not contain obvious homologues of the E1 and E3 regions, and genes of protein V and IX (known from mastadenoviruses only). Nonetheless, in the overall genomic organization, the squamate‐originated atadenoviruses show closer relation to mastadenoviruses than to members of any other genera .…”

Virus–Host Coevolution with a Focus on Animal and Human DNA Viruses

“…The most ancient lineages are the prosimian and New World monkey adenoviruses (Podgorski et al 2018). On the next branches we can find the Old World monkey adenoviruses including members of Human mastadenovirus (HAdV) A, F and G (Gilson et al 2016;Pantó et al 2015;Podgorski et al 2016) (Fig. 1).…”

Enhanced replication of mouse adenovirus type 1 following virus-induced degradation of protein kinase R (PKR)

“…We examined whether PKR is ubiquitinated by immunoblot. We detected ubiquitination of a positive control, mouse p53, which is degraded in the presence of MAV-1 proteins (66). However, even with the use of epitope-tagged ubiquitin (67) and MG132 treatment, we were unable to detect PKR ubiquitination during infection (Fig.…”

“…More likely, the reduction in PKR steady-state mRNA levels at the late infection time points can be attributed to other effects from viral infection, including the degradation or inhibition of proteins that induce PKR expression. For example, p53 is capable of binding to the PKR promoter and inducing its expression (73), but MAV-1 proteins cause p53 proteolysis (66). Together our results in C57BL/6 MEFs and macrophages suggest the virus does not cause PKR protein depletion by reducing PKR steady-state mRNA levels.…”

“…In human adenovirus, E4orf6 interacts with another early hAd protein, E1B 55K, to participate in an E3 ligase complex that ubiquitinates and degrades p53 via proteasomal degradation (77, 78). When MAV-1 E4orf6, E1B 55K, and mouse p53 are introduced by transfection into human cells, all three proteins interact and mouse p53 is degraded (66). If MAV-1 E4orf6 and E1B 55K form a similar complex in mouse cells, it may also degrade PKR.…”