Late Phase Of Adenovirus Infection Research Articles

Bovine adenovirus‐3 protein VIII associates with eukaryotic initiation factor‐6 during infection

Adenovirus protein VIII appears to connect core with the inner surface of the adenovirus capsid. Because protein-protein interactions are central to virus replication, identification of proteins interacting with protein VIII may help in understanding their role in adenovirus infection. Our yeast 2-hybrid assay indicated that protein VIII interacts with eukaryotic initiation factor 6 (eIF6). These findings were confirmed by Glutathione S-transferase-pull down assay, bimolecular fluorescent complementation assay, and coimmunoprecipitation assay in plasmid DNA transfected and bovine adenovirus-3 (BAdV-3) infected cells. The C-terminus amino acids 147 to 174 of protein VIII and N-terminus amino acids 44 to 97 of eIF6 are involved in these interactions. Polysome analysis demonstrated increased level of 60S ribosomal subunit and decreased level of 80S complex in protein VIII expressing cells or BAdV-3 infected cells. Our results suggest that formation of functional 80S ribosome appears impaired in the presence of protein VIII at late times post infection. We speculate that this impaired ribosome assembly may be responsible for the inhibition of cellular mRNA translation observed late in adenovirus infected cells. Moreover, analysis of recombinant BAdV-3 expressing mutant protein VIII (deletion of eIF6 interacting domain) suggests that interaction of protein VIII and eIF6 may help in preferential translation of adenovirus genes during late phase of adenovirus infection.

Kinetic Proofreading Scanning Models for Eukaryotic Translational Initiation: the Cap and Poly(A) Tail Dependency of Translation

Two simplified kinetic proofreading scanning (KPS) models were proposed to describe the 5′ cap and 3′ poly(A) tail dependency of eukaryotic translation initiation. In Model I, the initiation factor complex starts scanning and unwinding the secondary structure of the 5′ untranslated region (UTR) from the 5′ terminus of mRNA. In Model II, the initiation factor complex starts scanning from any binding site in the 5′ UTR. In both models, following ATP hydrolysis, the initiation factor complex either dissociates from mRNA or continues to scan and unwind RNA secondary structure in the 5′ UTR. This step repeats n times until the AUG codon is reached. These two models show very different cap and/or poly(A) tail dependency of translation initiation. The models predict that both cap and poly(A) tail dependencies of translation, and translatability of mRNAs are coupled with the structure of 5′ UTR: the translation of mRNA with structured 5′ UTR is strongly cap- and poly(A) tail-dependent; while translation of mRNA with unstructured 5′ UTR is less cap- and poly(A) tail-dependent. We use these two models to explain: (1) the cap and poly(A) tail dependence of translation; (2) the effect of exogenous poly(A) on translation; (3) repression of host mRNA and translation of late adenovirus mRNA in the late phase of adenovirus infection; (4) repression of host mRNA and translation of Vaccinia virus mRNA in virus-infected cell; (5) heat shock repression of translation of normal mRNA and stimulation of translation of hsp mRNA; and (6) the synergistic effect of cap and poly(A) tail on stimulating translation. The kinetic proofreading scanning models provide a coherent interpretation of those phenomena.

The nuclear export signal within the E4orf6 protein of adenovirus type 5 supports virus replication and cytoplasmic accumulation of viral mRNA.

During the late phase of adenovirus infection, viral mRNA is efficiently transported from the nucleus to the cytoplasm while most cellular mRNA species are retained in the nucleus. Two viral proteins, E1B-55 kDa and E4orf6, are both necessary for these effects. The E4orf6 protein of adenovirus type 5 binds and relocalizes E1B-55 kDa, and the complex of the two proteins was previously shown to shuttle continuously between the nucleus and cytoplasm. Nucleocytoplasmic transport of the complex is achieved by a nuclear export signal (NES) within E4orf6. Mutation of this signal sequence severely reduces the ability of the E1B-55 kDa-E4orf6 complex to leave the nucleus. Here, we examined the role of functional domains within E4orf6 during virus infection. E4orf6 or mutants derived from it were transiently expressed, followed by infection with recombinant adenovirus lacking the E4 region and determination of virus yield. An arginine-rich putative alpha helix near the carboxy terminus of E4orf6 contributes to E1B-55 kDa binding and relocalization as well as to the synthesis of viral DNA, mRNA, and proteins. Further mutational analysis revealed that mutation of the NES within E4orf6 considerably reduces its ability to support virus production. The same effect was observed when nuclear export was blocked with a competitor. Further, a functional NES within E4orf6 contributed to the efficiency of late virus protein synthesis and viral DNA replication, as well as total and cytoplasmic accumulation of viral late mRNA. Our data support the view that NES-mediated nucleocytoplasmic shuttling strongly enhances most, if not all, intracellular activities of E4orf6 during the late phase of adenovirus infection.

The Role of the L4 33K Gene in Adenovirus Infection

The late phase of adenovirus infection is characterized not only by the synthesis of late proteins and the assembly of new virions, but also by the inhibition of early gene expression and host cell translation. Previous work has demonstrated that both of these inhibitory effects depend upon expression from the major late transcription unit (MLTU), controlled by the major late promoter (MLP). Furthermore, the repression of early gene expression has been shown to be mediated in trans, suggesting a role for one or more MLTU-encoded soluble factor(s). A possible candidate for such a factor is the L4-encoded 33K gene product, a protein conserved throughout the Mastadenoviridae, but of no known function. To test the role of this protein in viral infection, a stop codon was placed at the 20th position of the 33K ORF. Viable virus with genomes containing the mutation were recovered in an overlap recombination assay. Phenotypic analysis revealed that the mutant virus had a significant deficiency in both kinetics of replication and final yield, as compared to the wild-type virus. Detailed analysis of infected cells showed that there was no detectable change in the regulation of expression of several early genes and the pIX gene. This suggests either that 33K is not involved in this late phase phenomenon or that this function is replaceable by another late protein(s). Late protein synthesis and accumulation were similar to those in wild-type-infected cells. However, the reduced yield of infectious mutant virus could be accounted for by a marked deficiency in the accumulation of intermediate particles and completed capsids, suggesting a role for 33K in the process of assembly. In addition there was a small but reproducible deficiency in the shutoff of host cell translation. These results show that the 33K protein plays an important, although apparently not essential, function in the late phase of virus infection.

The tripartite leader sequence of subgroup C adenovirus major late mRNAs can increase the efficiency of mRNA export.

The subgroup C human adenoviruses induce selective export of newly synthesized viral mRNA from the nucleus to the cytoplasm, with concomitant inhibition of export of the majority of cellular mRNA species. Such posttranscriptional regulation of viral and cellular gene expression in infected cells requires viral E1B and E4 proteins. To facilitate the investigation of parameters that govern selective export in adenovirus-infected cells, we constructed a marked human beta-actin minigene under the control of the glucocorticoid-inducible enhancer-promoter of mouse mammary tumor virus and introduced it into the left end of the adenovirus type 5 (Ad5) genome. Transcription of this reporter gene (designated MA) as well as of a sibling, which differed only in the inclusion of a cDNA copy of the Ad2 major late tripartite leader sequence upstream of beta-actin sequences (termed MtplA), in recombinant virus-infected cells was strictly dependent on the addition of dexamethasone to the medium. When transcription of the MA gene was induced during the late phase of infection, newly synthesized MA RNA entered the cytoplasm. These transcripts, which contain no viral sequences, therefore reproduce the behavior of exceptional cellular mRNA species observed when transcription of their genes is activated during the late phase of infection (U.-C. Yang, W. Huang, and S. J. Flint, J. Virol. 70:4071-4080, 1996). Unexpectedly, however, higher concentrations of newly synthesized RNA accumulated in the cytoplasm when the tripartite leader sequence was present in the reporter RNA, despite equal rates of transcription of the two reporter genes. Examination of the partitioning of both newly synthesized and steady-state populations of MA and MtplA RNAs between nuclear and cytoplasmic compartments indicated that the tripartite leader sequence did not increase RNA stability in the cytoplasm. Comparison of nuclear and cytoplasmic reporter RNA species by Northern blotting, primer extension, and reverse transcription-PCR provided no evidence for altered processing induced by the tripartite leader sequence. We therefore conclude that the tripartite leader sequence, long known to facilitate the translation of mRNAs during the late phase of adenovirus infection, can also modulate mRNA export from the nucleus.

Functional Characterization of the Major Late Promoter of Mouse Adenovirus Type 1

During the late phase of adenovirus infection, the major late promoter (MLP) controls the regulated expression of the genes that encode most viral structural proteins. Recently, the region of the genome of mouse adenovirus type 1 (MAV-1), predicted to contain the MLP, was sequenced and compared to that of the human virus MLP. The general organization of the transcriptional elements of the putative MAV-1 MLP is similar to that of the human virus counterpart, with some interesting differences. We wished to investigate the function of the predicted MLP of MAV-1 and to determine the significance of the differences found in the MAV-1 MLP. To test the activity of the predicted MLP of MAV-1, both Northern blot and primer extension analyses were performed on intracellular RNA isolated from cells infected with MAV-1. The results show that late RNA can be detected 48 hr postinfection and increases up to 6 days p.i. Primer extension analysis revealed that the major start sites of transcription are 28 and 31 nt downstream of the first T residue of the predicted TATA box. To analyze the functional significance of the predicted transcriptional elements, a transient transfection system, using the firefly luciferase gene controlled by the MAV-1 MLP sequence, was established. The predicted MLP sequence was capable of directing luciferase gene expression, to a level some 60% of that of the human virus MLP. Mutations were created in the inverted CAAT box, the SP1 site, and the TATA box, either singly or in combination. Each single-element mutation causes a marked reduction in luciferase gene expression, with the SP1 mutation showing the greatest effect. Double mutations were even more deficient, suggesting a level of functional redundancy among the various transcriptional elements. Finally, the putative SP1-binding site was examined by gel mobility shift assay and shown to interact with purified SP1 protein specifically, supporting the functional significance of this transcriptional element. These findings contribute to a better understanding of gene expression in MAV-1 and to its development as an appropriate model for the study of the molecular basis of pathogenesis in a natural host animal.

MRNA export correlates with activation of transcription in human subgroup C adenovirus-infected cells

To investigate the mechanisms by which viral mRNA species are distinguished from their cellular counterparts for export to the cytoplasm during the late phase of subgroup C adenovirus infection, we have examined the metabolism of several cellular and viral mRNAs in human cells productively infected by adenovirus type 5 (Ad5). Several cellular mRNAs that were refractory to, or could escape from, adenovirus-induced inhibition of export of mRNA from the nucleus have been identified. This group includes Hsp70 mRNAs synthesized upon heat shock of Ad5-infected 293 or HeLa cells during the late phase of infection. However, successful export in Ad5-infected cells is not a specific response to heat shock, for beta-tubulin and interferon-inducible mRNAs were also refractory to virus-induced export inhibition. The export of these cellular mRNAs, like that of viral late mRNAs, required the E1B 55-kDa protein. Export to the cytoplasm during the late phase of Ad5 infection of several cellular mRNAs, including members of the Hsp70 family whose export was inhibited under some, but not other, conditions, indicates that viral mRNA species cannot be selectively exported by virtue of specific sequence or structural features. Cellular and viral late mRNAs that can be exported from the nucleus to the cytoplasm were expressed from genes whose transcription was induced or activated during the late phase of Ad5 infection. Consistent with the possibility that successful export is governed by transcriptional activation in the late phase of adenovirus infection, newly synthesized viral early E1A mRNA was subject to export inhibition during the late phase of infection.

Modification of eukaryotic initiation factor 4F during infection by influenza virus.

Influenza virus infection of cells is accompanied by a striking shutoff of cellular protein synthesis, resulting in the exclusive translation of viral mRNAs. The mechanism for control of cellular protein synthesis by influenza virus is poorly understood, but several translation properties of influenza virus mRNAs which are potentially involved have been described. Influenza virus mRNAs possess the surprising ability to translate in the presence of inhibitory levels of inactive (phosphorylated) eukaryotic initiation factor 2 (eIF-2). In addition, influenza virus mRNAs were shown to be capable of translating in cells during the late phase of adenovirus infection but not in cells infected by poliovirus. Since both adenovirus and poliovirus facilitate virus-specific translation by impairing the activity of initiation factor eIF-4F (cap-binding protein complex) but through different mechanisms, we investigated the translation properties of influenza virus mRNAs in more detail. We show that influenza virus infection is associated with the significant dephosphorylation and inactivation of eIF-4E (cap-binding protein), a component of eIF-4F, and accordingly that influenza virus mRNAs possess a moderate ability to translate by using low levels of eIF-4F. We also confirm the ability of influenza virus mRNAs to translate in the presence of high levels of inactive (phosphorylated) eIF-2 but to a more limited extent than reported previously. We suggest a potential mechanism for the regulation of protein synthesis by influenza virus involving a decreased requirement for large pools of active eIF-4F and eIF-2.

Phosphoprotein phosphatase 2A dephosphorylates eIF-4E and does not alter binding to the mRNA cap

The phosphorylation and dephosphorylation of the 25 kDa mRNA cap binding protein eukaryotic initiation factor-4E (eIF-4E) is regulated during different physiologic and pathophysiologic states that include cell growth and the late phase of adenovirus infection. We have found that okadaic acid is much more effective in increasing the phosphorylated fraction of eIF-4E than phorbol 12-myristate 13-acetate in Hep G2 cells. Phosphoprotein phosphatase 2A dephosphorylated eIF-4E isolated from both phorbol 12-myristate 13-acetate- or okadaic acid-treated cells, whereas alkaline and acid phosphatase were relatively ineffective. The ability of purified [ 35S]eIF-4E isolated from okadaic acid-treated cells to bind mRNA caps was compared to phosphoprotein phosphatase 2A-treated [ 35S]eIF-4E and found to be no different. This suggests that alternative explanations for the previously observed effects of eIF-4E phosphorylation on protein synthesis must be considered. In addition, our results indicate that the in vivo phosphorylation eIF-4E is not catalyzed solely by protein kinase C.

Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination.

We have studied the adenovirus-induced inhibition of host cell protein synthesis and the effect of infection on the overall metabolism of host cell mRNA during the late phase of adenovirus infection by following the fate of a number of cellular mRNAs complementary to specific cloned DNA segments. At a time in infection when the rate of total cellular protein synthesis is drastically (greater than 90%) reduced, transcription of specific cellular genes is undiminished. However, the transport of newly synthesized cellular mRNA to the cytoplasm is greatly decreased. This decreased appearance of new mRNA in the cytoplasm cannot account for the observed cessation of cell specific protein synthesis, however, since the concentration of several preexisting cellular mRNAs, including the mRNA for actin, remains unchanged throughout the course of infection. The preexisting mRNA is intact, capped, and functional as judged by its ability to direct protein synthesis in vitro in a cap-dependent fashion. The interruption in host translation appears to operate at the level of initiation directly, since we find that fewer ribosomes are associated with a given cellular mRNA after infection than before infection. Furthermore, the in vivo inhibition of cellular protein synthesis does not appear to be the result of competition with viral mRNA, since conditions which prevent the efficient initiation of translation of viral mRNA (infection with a viral mutant) do not result in the recovery of cell translation. Thus, it appears that a late adenovirus gene product directly mediates a shutoff of host protein synthesis.

Synthesis and processing of simian virus 40-specific RNA in adenovirus-infected, simian virus 40-transformed human cells

Human simian virus 80 (SV80) cells transformed by simian virus 40 (SV40) synthesize substantial quantities of the SV40 large T-antigen (Henderson & Livingston, 1974; Tjian, 1978) and cytoplasmic, poly(A)-containing RNA species that exhibit spliced structures characteristic of the SV40, early messenger RNA species that encode both large and small T-antigens (Flint & Beltz, 1979). When SV80 cells were infected with type C adenovirus, both the synthesis of SV40 large T-antigen and the appearance in the cytoplasm of newly synthesized, SV40-specific RNA sequences were inhibited during the late phase of infection. The results of hybridization to SV40 DNA of SV80 nuclear RNA, prepared from mock- or adenovirus-infected cells after labeling for short periods in vivo or in vitro, indicated that transcription of integrated SV40 was, by contrast, not disrupted during the late phase of adenovirus infection. Poly(A)-containing, nuclear RNA species that hybridized to SV40 DNA sequences and exhibited the sizes of spliced, large and small T-antigen mRNA species were also synthesized in infected cells at a time when the corresponding mRNA sequences did not leave the nucleus. These results suggest that the failure of non-adenoviral mRNA sequences to enter the cytoplasm of adenovirus-infected cells does not reflect inhibition of either their transcription or the normal enzymatic processing reactions to which pre-mRNA species are subject. Several lines of evidence do, however, establish that nuclear, SV40-specific RNA sequences are less stable in adenovirus-infected compared to mock-infected SV80 cells.

Nucleotide sequence complementarity between adenovirus 2-coded VA RNA and host cell pre-mRNA. A possible regulatory mechanism of cellular RNA splicing by VA RNA.

Using a computer program, complementary of nucleotide sequences was assessed between adenovirus 2-coded VA RNA and presumptive cellular and viral 'pre-mRNAs'. In this paper, the possibility is considered that the splicing of cellular 'pre-mRNA' can be regulated in such a way that the formation of the proper intramolecular double-stranded hairpin structures, key elements for RNA splicing, is prevented by the binding of VA RNAI or RNAII to the nucleotide sequences around the exon-intron and intron-exon joint sites of cellular 'pre-mRNA' molecules. Complementarity assessment showed that VA RNAI can bind to the joint sites in such a way as to form an omega shape at two separate regions around the joint sites of cellular 'pre-mRNA'. Whereas VA RNAI is not capable of binding to viral hexon 'pre-mRNA' in the same manner as it does to cellular 'pre-mRNA', the binding may occur in a different way. Such differential binding is discussed in relation to the post-transcriptional sequence selection which takes place during the late phase of adenovirus infection.