Control of mRNA Export by Adenovirus E4orf6 and E1B55K Proteins during Productive Infection Requires E4orf6 Ubiquitin Ligase Activity

Abstract

During the adenovirus infectious cycle, the early proteins E4orf6 and E1B55K are known to perform several functions. These include nuclear export of late viral mRNAs, a block of nuclear export of the bulk of cellular mRNAs, and the ubiquitin-mediated degradation of selected proteins, including p53 and Mre11. Degradation of these proteins occurs via a cellular E3 ubiquitin ligase complex that is assembled through interactions between elongins B and C and BC boxes present in E4orf6 to form a cullin 5-based ligase complex. E1B55K, which has been known for some time to associate with the E4orf6 protein, is thought to bind to specific substrate proteins to bring them to the complex for ubiquitination. Earlier studies with E4orf6 mutants indicated that the interaction between the E4orf6 and E1B55K proteins is optimal only when E4orf6 is able to form the ligase complex. These and other observations suggested that most if not all of the functions ascribed to E4orf6 and E1B55K during infection, including the control of mRNA export, are achieved through the degradation of specific substrates by the E4orf6 ubiquitin ligase activity. We have tested this hypothesis through the generation of a virus mutant in which the E4orf6 product is unable to form a ligase complex and indeed have found that this mutant behaves identically to an E4orf6 ؊ virus in production of late viral proteins, growth, and export of the late viral L5 mRNA.The late phase of an adenoviral infection, typified by human adenovirus type 5 (Ad5), is characterized by a massive production of progeny virions. To support this production, the virus takes control of the cellular machinery to produce abundant amounts of its own late proteins to the detriment of the synthesis of cellular proteins. This host cell shutoff is achieved via several mechanisms. The translation of cellular mRNAs is blocked by the action of the L4-100K protein on the ribosomal machinery (15, 16) so that only the late viral mRNAs containing the tripartite leader sequence can be translated (26). At the same time, export of cellular mRNAs to the cytoplasm is blocked by the action of the early viral proteins E4orf6 and E1B55K (3, 6, 23, 42). These same proteins were also shown to be required for the export of the late viral mRNAs (4,20,23,42,53). The E4orf6 and E1B55K proteins were shown to interact during infection (48) and to function in the same pathway, as a viral mutant with defects in both of these products was seen to have essentially the same phenotype as mutants affecting only one of these species (3,17,23,42). The role that E4orf6 and E1B55K proteins play in the control of mRNA export has yet to be defined.It has been known for some time that the E4orf6 and E1B55K proteins play a role in the degradation of the p53 tumor suppressor, and recently the mechanism for such degradation has been elucidated by our group. Expression of the virus E1A protein results in increased levels of p53 (10, 34); however, during infection, in the presence of both E4orf6 and E1B55K products, p53 is degraded ...

Distinct Requirements of Adenovirus E1b55K Protein for Degradation of Cellular Substrates

“…BC-box motifs in E4orf6 have been identified previously as important for binding elongins B and C (5,15,43). It has been suggested that all functions of the E1b55K/E4orf6 complex are due to the degradation of cellular proteins by the ubiquitin ligase activity (6,16,70). Since E1b55K alone can physically associate with p53 (13,34,41,56,71,73), DNA ligase IV (2), and the Mre11/Rad50/Nbs1 (MRN) complex (12), it is believed to mediate substrate recognition, while both E4orf6 and E1b55K are required for proteasome-mediated degradation (12,13,49,50,56,69).…”

Viral-mediated stabilization of AU-rich element containing mRNA contributes to cell transformation

“…So far, several proteins were identified as a target of this ubiquitin ligase, such as p53 (Querido et al, 2001), Mre11 (Stracker et al, 2002), DNA ligase IV (Baker et al, 2007) and integrin a3 (Dallaire et al, 2009); we have no evidence about which protein or additional another protein is required as a substrate for the stabilization of ARE-mRNA. As the E4orf6 E3 ubiquitin ligase activity was reported to be necessary for the export of viral late mRNAs (Woo and Berk, 2007;Blanchette et al, 2008), this activity is involved in the regulation of both cellular and viral mRNAs.…”

“…To confirm this, we constructed the E4orf6 (BCÀ) mutant by introducing specific point mutations in BC boxes 1 and 2 (Figure 2a), which are necessary for E4orf6 to associate with Cullin5, a complex containing E3 ubiquitin ligase bound with E1B55k, using mutant adenovirus H5pm4139 (a generous gift from T Dobner) as a template (Blanchette et al, 2008). We confirmed whether this mutant is functional by measuring its ability to degrade p53 (Supplementary Figure 1).…”

“…The complementary DNA of the luciferase-ARE cÀfos fusion gene was inserted into the pTRE vector to produce a tet-off system according to the manufacturer's (Clontech, Palo Alto, CA, USA) instructions. pcDNA-E4orf6 BC(À) was made by amplifying segments of the E4orf6-coding region using the primer 5 0 -CGGGATCCC ATGACTACGTCCGGCGTT-3 0 , together with a second primer 5 0 -GAATTCCTACATGGGGGTAGAGTCATA-3 0 using mutant adenovirus H5pm4139 (a generous gift from T Dobner) as a template (Blanchette et al, 2008). The PCRamplified fragments were inserted into the BamHI/EcoRI site of pcDNA3.…”

“…E4orf6 forms a complex with adenovirus E1B55k (Sarnow et al, 1984), and this complex assembles into E3 ubiquitin ligase through the domain of E4orf6 that binds with elongins B and C (Querido et al, 2001). It is suggested that most, if not all, of the functions of the E1B55k/E4orf6 complex, including the control of mRNA export, are achieved through the degradation of target proteins by E4orf6 via its E3 ligase activity (Woo and Berk, 2007;Blanchette et al, 2008).…”