E4Orf6-E1B-55k-Dependent Degradation of De Novo-Generated Adeno-Associated Virus Type 5 Rep52 and Capsid Proteins Employs a Cullin 5-Containing E3 Ligase Complex

Although human adenovirus type 5 (Ad5) has been widely studied, relatively little work has been done with other human adenovirus serotypes. The Ad5 E4orf6 and E1B55K proteins form Cul5-based E3 ubiquitin ligase complexes to degrade p53, Mre11, DNA ligase IV, integrin ␣3, and almost certainly other targets, presumably to optimize the cellular environment for viral replication and perhaps to facilitate persistence or latency. As this complex is essential for the efficient replication of Ad5, we undertook a systematic analysis of the structure and function of corresponding E4orf6/E1B55K complexes from other serotypes to determine the importance of this E3 ligase throughout adenovirus evolution. E4orf6 and E1B55K coding sequences from serotypes representing all subgroups were cloned, and each pair was expressed and analyzed for their capacity to assemble the Cullin-based ligase complex and to degrade substrates following plasmid DNA transfection. The results indicated that all formed Cullin-based E3 ligase complexes but that heterogeneity in both structure and function existed. Whereas Cul5 was present in the complexes of some serotypes, others recruited primarily Cul2, and the Ad16 complex clearly bound both Cul2 and Cul5. There was also heterogeneity in substrate specificity. Whereas all serotypes tested appeared to degrade DNA ligase IV, complexes from some serotypes failed to degrade Mre11, p53, or integrin ␣3. Thus, a major evolutionary pressure for formation of the adenovirus ligase complex may lie in the degradation of DNA ligase IV; however, it seems possible that the degradation of as-yet-unidentified critical targets or, perhaps even more likely, appropriate combinations of substrates plays a central role for these adenoviruses.The human adenovirus type 5 (Ad5) early region 4 34-kDa product from open reading frame 6 (E4orf6) and the E1B55K protein have been known for some time to act in concert to carry out several important functions during the infectious cycle, including regulation of the activity and stability of p53 and, late in infection, the selective transport of viral mRNAs (2,14,18,19,37,46,51,52,56). Our group and others have shown that the cooperative functions of these Ad5 proteins, including the E4orf6-E1B55K interaction itself, appear to require formation of a Cul5-based E3 ubiquitin ligase complex (8). We showed that Ad5 E4orf6 recruits an E3 ubiquitin ligase complex containing the Cullin family member Cul5, Elongins B and C, and the RING protein Rbx1 (8,20). E1B55K appears to associate with the E4orf6 protein only in the context of this complex, and it is believed to function as the substrate recruitment component, introducing specific proteins for ubiquitination and degradation by proteasomes (8,11,28). The formation and function of this complex are essential to permit efficient viral replication. At one time p53 was its only known substrate (10,34,36,40,47,48); however, a growing list of additional targets is emerging, including the cellular proteins Mre11 (8, 49), DNA ligase IV (4), and ...

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confidence: 99%

The E4orf6/E1B55K E3 Ubiquitin Ligase Complexes of Human Adenoviruses Exhibit Heterogeneity in Composition and Substrate Specificity

Cheng

Gilson

Dallaire

et al. 2011

“…Recently, additional targets of the adenovirus ubiquitin ligase with no obvious role in the cellular DNA damage response have been identified. These include the nonstructural Rep52 and capsid proteins of adeno-associated virus type 5 (52). In view of the diverse requirements for the recruitment of specific substrates by the viral ubiquitin ligase (49,63), it would not be surprising that additional cellular targets of the adenovirus ubiquitin ligase remain to be identified and that one of these may be critical for viral late gene expression.…”

Section: Discussionmentioning

confidence: 99%

E4orf1 Limits the Oncolytic Potential of the E1B - 55K Deletion Mutant Adenovirus

Thomas

Broughton

Goodrum

et al. 2009

Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G 1 phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G 1 restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.

“…The stability of NS1 and NP1 proteins was assessed by cycloheximide inhibition of protein synthesis, as described previously (13). Briefly, WRD cells were plated in six-well dishes to 60 to 70% confluence and then infected with MVC at a plaque assayderived MOI of 10.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Nonstructural Proteins of the Bocavirus Minute Virus of Canines

Sukhu

Fasina

Burger

et al. 2013

Self Cite

bWe present a detailed characterization of a single-cycle infection of the bocavirus minute virus of canines (MVC) in canine WRD cells. This has allowed identification of an additional smaller NS protein that derives from an mRNA spliced within the NS gene that had not been previously reported. In addition, we have identified a role for the viral NP1 protein during infection. NP1 is required for read-through of the MVC internal polyadenylation site and, thus, access of the capsid gene by MVC mRNAs. Although the mechanism of NP1's action has not yet been fully elucidated, it represents the first parvovirus protein to be implicated directly in viral RNA processing.