Adenovirus Ubiquitin-Protein Ligase Stimulates Viral Late mRNA NuclearExport

Abstract

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 proteins are required together to stimulate viral late nuclear mRNA export to the cytoplasm and to restrict host cell nuclear mRNA export during the late phase of infection. Previous studies have shown that these two viral proteins interact with the cellular proteins elongins B and C, cullin 5, RBX1, and additional cellular proteins to form an E3 ubiquitin-protein ligase that polyubiquitinates p53 and probably one or more subunits of the MRE11-RAD50-NBS1 (MRN) complex, directing their proteasomal degradation. The MRN complex is required for cellular DNA double-strand break repair and induction of the DNA damage response by adenovirus infection. To determine if the ability of E1B-55K and E4orf6 to stimulate viral late mRNA nuclear export requires the ubiquitin-protein ligase activity of this viral ubiquitin-protein ligase complex, we designed and expressed a dominant-negative mutant form of cullin 5 in HeLa cells before infection with wild-type Ad5 or the E1B-55K null mutant dl1520. The dominantnegative cullin 5 protein stabilized p53 and the MRN complex, indicating that it inhibited the viral ubiquitinprotein ligase but had no effect on viral early mRNA synthesis, early protein synthesis, or viral DNA replication. However, expression of the dominant-negative cullin 5 protein caused a decrease in viral late protein synthesis and viral nuclear mRNA export similar to the phenotype produced by mutations in E1B-55K. We conclude that the stimulation of adenovirus late mRNA nuclear export by E1B-55K and E4orf6 results from the ubiquitin-protein ligase activity of the adenovirus ubiquitin-protein ligase complex.Two adenovirus type 5 (Ad5) early proteins, E1B-55K and E4orf6, function to stimulate nuclear export and translation of viral late mRNAs, inhibit host cell mRNA nuclear export, and inhibit the functions of p53 and the MRE11-RAD50-NBS1 (MRN) DNA double-strand break repair complex during wildtype (WT) Ad5 infection (3,4,6,11,18,30,35,47,50,64,83). Initial insight into how these two proteins are involved in these diverse processes came from characterizing adenoviral mutant forms of E1B-55K and/or E4orf6. Mutants defective in expressing either E1B-55K or E4orf6 or with a mutation in genes for both show similar phenotypes: They all have a defect in viral late nuclear message export to the cytoplasm and cannot inhibit host cell mRNA nuclear export to the cytoplasm, and synthesis of viral late proteins is reduced compared to that of WT Ad5 (3,4,18,29,30,47,64). These mutants are also unable to induce the degradation of p53 or subunits of the MRN complex (56,65,73,78).Much information has accumulated that is relevant to the functions of E1B-55K and E4orf6. The two viral proteins associate in vivo (68) and colocalize in Ad5-infected cell nuclei at viral DNA replication-transcription centers, as well as other regions of the nucleoplasm and cytoplasm during the late phase of infection (59). Both E1B-55K and E4orf6 have nuclear export signals and nuclear localization signals req...

Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection

“…4B), in agreement with previous findings. 40 However, Cul5 NTD did not stabilize p53 in Ad12-infected cells (lane 11 of Fig. 4B), suggesting that Cul5 is not required for p53 degradation in Ad12-infected cells.…”

“…Zhang et al reported that the HCMV ubiquitination and degradation of p53 and other substrates. 4,39,40 To assess whether Cul5 is involved in downregulation of Mdm2 and Mdm4, we infected SJSA-1 cells with recombinant adenoviruses carrying the full-length Cul5 cDNA (ψ5-Cul5) or the N-terminal domain of Cul5 (ψ5-Cul5 NTD). Cul5 NTD was shown to act as a dominant-negative mutant of Cul5.…”

“…Cul5 NTD was shown to act as a dominant-negative mutant of Cul5. 40 As a control, cells were infected with Ad-GFP. The cells were then superinfected with wt Ad12 or Ad5.…”

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.…”

“…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).…”