Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts

Abstract

The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. During the late phase of human adenovirus type 5 (Ad5) infection, cellular protein synthesis is severely inhibited, while viral late proteins are made in large quantities (3, 6). Such preferential expression of viral genes is the result of posttranscriptional regulatory mechanisms: several viral gene products, including VA-RNA1 and the L4 100-kDa protein, contribute to selective translation of viral late mRNAs (see references 18, 64, and 85 for reviews), while the E1B 55-kDa and E4 Orf 6 proteins induce selective export of these mRNAs from the nucleus (reviewed in references 25, 33, and 39). In infected cells, these last two early proteins form a complex (84) that has been implicated in regulation of mRNA export (12, 19). Indeed, the E1B 55-kDa and E4 Orf 6 proteins colocalize to specific sites within infected cell nuclei, the peripheral zones of replication centers (70). Transcription of viral late genes and at least initial processing of viral pre-mRNAs take place at these same sites (4,14,74,75). Mutations that prevent synthesis of the E4 Orf 6 protein or reduce binding of this to the E1B 55-kDa protein (83) result in both mislocalization of the E1B 55-kDa protein and defects in export of viral late mRNAs (39, 70). These properties indicate that E4 Orf 6 protein-dependent recruitment of the E1B protein to the specialized nuclear sites at which viral late pre-mRNAs are synthesized promotes selective export of the processed mRNAs. The observation that the accumulation of viral mRNAs at enlarged interchromatin granules, which form in infected cells as the late phase progresses, correlates with efficient late mRNA export (4, 13) provides further support for the view that efficient mRNA export is intimately coupled to the organization of infected cell nuclei. However, the molecular basis of such coupling remains unknown, nor has the cellular export pathway by which viral late mRNAs are transported from the nucleus to the cytoplasm been identified.AThe E1B 55-kDa protein contains a leucine-rich nuclear export signal (NES) that is recognized by the cellular exportin 1 export receptor and mediates shuttling of the viral protein when it is synthesized either alone or in Ad5-infected cells (26,58). It has also been reported that the E4 Orf 6 protein contains a similar NES nece...

The Repression Domain of the E1B 55-Kilodalton Protein Participates in Countering Interferon-Induced Inhibition of Adenovirus Replication

“…1). The 2-to 3-order-of-magnitude decreases in yield of this null mutant observed when cells were not exposed to IFN are also consistent with the requirement for the E1B 55-kDa protein to allow maximally efficient viral DNA synthesis in HFFs (47,62). In the absence of IFN, no significant impact of the E1B Sub19 substitutions on virus yield was observed (Fig.…”

“…As these initial observations indicated that the enhanced sensitivity of replication of this mutant to IFN is not a trivial consequence of failure to synthesize stable E1B protein, we examined the properties of this mutant in more detail. We first compared its sensitivity to IFN to that of the corresponding E1B 55-kDa protein null mutant, AdEasyE1⌬2347 (48), following infection of HFFs at a high multiplicity (50 PFU/cell) for 36 h or at a low multiplicity (3 PFU/cell) for 88 h. The former condition corresponds to a single infectious cycle with the great majority of cells initially infected (62). In contrast, the low-multiplicity protocol allowed spread of infection from the small number of cells initially infected and is therefore likely to be more representative of infection and the impact of host antiviral defenses in vivo.…”

Timely Synthesis of the Adenovirus Type 5 E1B 55-Kilodalton Protein Is Required for Efficient Genome Replication in Normal Human Cells

“…We therefore initiated investigations of the roles played by the E1B 55-kDa protein during Ad5 replication in normal human cells. One unexpected observation was that, in the absence of this protein, viral DNA synthesis was impaired in proliferating human fibroblasts (32), although it is not in HeLa and other lines of transformed human cells (5,34,38,66,100). Furthermore, McCormick and colleagues had previously reported that no differences in viral DNA synthesis were observed in quiescent small airway epithelial cells infected by wild-type virus or the E1B 55-kDa null mutant ONYX-015 (also known as dl1520) (63).…”

Impact of the Adenoviral E4 Orf3 Protein on the Activity and Posttranslational Modification of p53

“…To assess the effect, if any, of the E4 Orf3 protein on p53 stabilization and accumulation, HFFs were infected with the E1B 55-kDa-null mutant and the ⌬E1B/⌬EOrf3 double mutant for increasing periods, and p53 was examined by immunoblotting, as described in Materials and Methods. By 24 h after infection, the initial period of the late phase in these cells (102), p53 was readily detected in cells infected by the mutant viruses, but not in cells infected by the phenotypically wild-type parent of ⌬E1B, AdEasyE1 ( Fig. 2A).…”