Analyses of single-amino-acid substitution mutants of adenovirus type 5 E1B-55K protein

The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus dl309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.     

An activity associated with human chromosome 21 permits nuclear colocalization of the adenovirus E1B-55K and E4orf6 proteins and promotes viral late gene expression

The adenovirus E1B-55K and E4orf6 proteins cooperate during virus infection while performing several tasks that contribute to a productive infection, including the selective nucleocytoplasmic transport of late viral mRNA. Previous studies have shown that the E4orf6 protein retains the E1B-55K protein in the nucleus of human and monkey cells, but not in those of rodents, suggesting that primate-specific cellular factors contribute to the E4orf6-mediated retention of the E1B-55K protein in the nucleus. In an effort to identify these proposed primate-specific cellular factors, the interaction of the E1B-55K and E4orf6 proteins was studied in a panel of stable human-rodent monochromosomal somatic cell hybrids. Analysis of this panel of cell lines has demonstrated the existence of an activity associated with human chromosome 21 that permits the E1B-55K and E4orf6 proteins to colocalize in the nucleus of a rodent cell. Additional hybrid cells bearing portions of human chromosome 21 were used to map this activity to a 10-megabase-pair segment of the chromosome, extending from 21q22.12 to a region near the q terminus. Strikingly, this region also facilitates the expression of adenovirus late genes in a rodent cell background while having little impact on the expression of early viral genes.     

The E3 ubiquitin ligase activity associated with the adenoviral E1B-55K-E4orf6 complex does not require CRM1-dependent export

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via leucine-rich nuclear export signals (NES). However, the role of their NES-dependent export in viral replication has not been established. It was initially shown that mutations in the E4orf6 NES negatively affect viral late gene expression in transfection/infection complementation assays, suggesting that E1B-55K/E4orf6-dependent viral late mRNA export involves a CRM1 export pathway. However, a different conclusion was drawn from similar studies showing that E1B-55K/E4orf6 promote late gene expression without active CRM1 or functional NES. To evaluate the role of the E1B-55K/E4orf6 NES in viral replication in the context of Ad-infected cells and in the presence of functional CRM1, we generated virus mutants carrying amino acid exchanges in the NES of either or both proteins. Phenotypic analyses revealed that mutations in the NES of E1B-55K and/or E4orf6 had no or only moderate effects on viral DNA replication, viral late protein synthesis, or viral late mRNA export. Significantly, such mutations also did not interfere with the degradation of cellular substrates, indicating that the NES of E1B-55K or E4orf6 is dispensable both for late gene expression and for the activity associated with the E3 ubiquitin ligase.     

The adenovirus type 5 E1B-55K oncoprotein actively shuttles in virus-infected cells, whereas transport of E4orf6 is mediated by a CRM1-independent mechanism

The E1B-55K and E4orf6 proteins of adenovirus type 5 are involved in viral mRNA export. Here we demonstrate that adenovirus infection does not inhibit the function of the E1B-55K nuclear export signal and that E1B-55K also shuttles in infected cells. Even during virus infection, E1B-55K was exported by the leptomycin B-sensitive CRM1 pathway, whereas E4orf6 transport appeared to be mediated by an alternative mechanism. Our results strengthen the potential role of E1B-55K as the "driving force" for adenoviral late mRNA export.     

Diverse roles for E4orf3 at late times of infection revealed in an E1B 55-kilodalton protein mutant background

Species C human adenovirus mutants that fail to express open reading frame 3 of early region 4 (E4orf3) are phenotypically indistinguishable from the wild-type virus when evaluated in cells cultured in vitro. However, E4orf3 gene function has been productively studied in the context of additional viral mutations. This study identifies diverse roles for the E4orf3 protein that are evident in the absence of early region 1B 55-kDa protein (E1B-55K) function. In an E1B-55K-deficient background, the E4orf3 protein promotes viral replication by increasing both the burst size and the probability that an infected cell will produce virus. Early viral gene expression is not impaired in E1B-55K/E4orf3 double mutant virus-infected cells. Cells infected with the double mutant virus accumulated concatemers of viral DNA. However, the E1B-55K/E4orf3 double mutant virus did not replicate any better in MO59J cells, in which viral DNA concatemers did not accumulate, than in MO59K cells, in which viral DNA concatemers were produced, suggesting that viral DNA concatenation is not the primary growth defect of the E1B-55K/E4orf3 double mutant virus. Accumulation of viral mRNA in the nucleus and cytoplasm of E1B-55K/E4orf3 double mutant virus-infected cells was severely reduced compared to that on wild-type virus-infected cells. Thus, in an E1B-55K mutant background, the E4orf3 protein promotes the accumulation of late viral RNA and enhances late gene expression. Finally, within the context of an E1B-55K mutant virus, the E4orf3 protein acts to suppress host cell translation and preserve the viability of cells at moderately late times of infection.     

Analysis of the adenovirus E1B-55K-anchored proteome reveals its link to ubiquitination machinery

During the early phase of infection, the E1B-55K protein of adenovirus type 5 (Ad5) counters the E1A-induced stabilization of p53, whereas in the late phase, E1B-55K modulates the preferential nucleocytoplasmic transport and translation of the late viral mRNAs. The mechanism(s) by which E1B-55K performs these functions has not yet been clearly elucidated. In this study, we have taken a proteomics-based approach to identify and characterize novel E1B-55K-associated proteins. A multiprotein E1B-55K-containing complex was immunopurified from Ad5-infected HeLa cells and found to contain E4-orf6, as well as several cellular factors previously implicated in the ubiquitin-proteasome-mediated destruction of proteins, including Cullin-5, Rbx1/ROC1/Hrt1, and Elongins B and C. We further demonstrate that a complex containing these as well as other proteins is capable of directing the polyubiquitination of p53 in vitro. These ubiquitin ligase components were found in a high-molecular-mass complex of 800 to 900 kDa. We propose that these newly identified binding partners (Cullin-5, Elongins B and C, and Rbx1) complex with E1B-55K and E4-orf6 during Ad infection to form part of an E3 ubiquitin ligase that targets specific protein substrates for degradation. We further suggest that E1B-55K functions as the principal substrate recognition component of this SCF-type ubiquitin ligase, whereas E4-orf6 may serve to nucleate the assembly of the complex. Lastly, we describe the identification and characterization of two novel E1B-55K interacting factors, importin-alpha 1 and pp32, that may also participate in the functions previously ascribed to E1B-55K and E4-orf6.     

Adenovirus ubiquitin-protein ligase stimulates viral late mRNA nuclear export

Theadenovirus 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 dominant-negative cullin 5 protein stabilized p53 and the MRN complex, indicating that it inhibited the viral ubiquitin-protein 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.     

Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex.

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa EIB protein can exert its multiple activities in lytically infected cells and in adenovirus transformation.     

Adenovirus exploits the cellular aggresome response to accelerate inactivation of the MRN complex

Results reported here indicate that adenovirus 5 exploits the cellular aggresome response to accelerate inactivation of MRE11-RAD50-NBS1 (MRN) complexes that otherwise inhibit viral DNA replication and packaging. Aggresomes are cytoplasmic inclusion bodies, observed in many degenerative diseases, that are formed from aggregated proteins by dynein-dependent retrograde transport on microtubules to the microtubule organizing center. Viral E1B-55K protein forms aggresomes that sequester p53 and MRN in transformed cells and in cells transfected with an E1B-55K expression vector. During adenovirus infection, the viral protein E4orf3 associates with MRN in promyelocytic leukemia protein nuclear bodies before MRN is bound by E1B-55K. Either E4orf3 or E4orf6 is required in addition to E1B-55K for E1B-55K aggresome formation and MRE11 export to aggresomes in adenovirus-infected cells. Aggresome formation contributes to the protection of viral DNA from MRN activity by sequestering MRN in the cytoplasm and greatly accelerating its degradation by proteosomes following its ubiquitination by the E1B-55K/E4orf6/elongin BC/Cullin5/Rbx1 ubiquitin ligase. Our results show that aggresomes significantly accelerate protein degradation by the ubiquitin-proteosome system. The observation that a normal cellular protein is inactivated when sequestered into an aggresome through association with an aggresome-inducing protein has implications for the potential cytotoxicity of aggresome-like inclusion bodies in degenerative diseases.