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

Abstract

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.

FIG. 1

Organization of glucocorticoid-inducible β-actin minigenes and recombinant viruses that contain them. (A) The organization of the human β-actin gene is shown schematically at the top, with exons and introns represented by black boxes and lines, respectively. As illustrated, the β-actin minigene used in these experiments comprised part of exon 5 and exon 6 and was marked by insertion of a synthetic DNA fragment (white box). The MA and MtplA reporter genes, whose organization is shown, were constructed by ligation of appropriate DNA fragments as described in Materials and Methods. Rescue of these genes into the Ad5 genome generated recombinant viruses with the organization shown for Ad5/MtplA at the bottom of panel A; Ad5/MA differs only in the absence of tripartite leader sequences (tpl) from the reporter gene. (B) The unspliced RNAs predicted for the initiation of transcription from the initiation site of the mouse mammary tumor virus (MMTV) long terminal repeat promoter are shown by arrows. RNA lengths are listed in nucleotides, and intron 5 of the β-actin gene is indicated by broken lines.

FIG. 2

Transcription of MMTV β-actin minigenes in Ad5/MA- and Ad5/MtplA-infected cells. 293 cells were infected with 20 PFU of Ad5/MA or Ad5/MtplA per cell and exposed to 3 mM dexamethasone for 3 h at 11 h after infection (+) or mock treated (−). RNA labeled in run-on transcription reaction mixtures (see Materials and Methods) in the absence (A) or presence (B) of 2 μg of α-amanitin per ml, used to inhibit RNA polymerase II, was hybridized to the SpeI oligonucleotide probe specific for the β-actin reporter minigene and to major late (ML) and E2E probes as described in Materials and Methods. For each probe, 0.5, 2.5, or 5.0 μg of DNA (left to right) was loaded on the filter as described in Materials and Methods. Because the E2E probe used in these experiments detects the products of both RNA polymerase II and RNA polymerase III transcription (42), α-amanitin-resistant E2E transcription was observed (E2E probe in panel B).

FIG. 3

Expression of the inducible β-actin minigenes in Ad5/MA- and Ad5/MtplA-infected 293 cells. Equal numbers of human 293 cells infected with 20 PFU of Ad5/MA or Ad5/MtplA per cell or mock infected (M) were exposed to dexamethasone for 1 h at 10 h after infection (I+) as described in Materials and Methods or mock treated (I−). At 14 h after infection, one set of cells was harvested for run-on transcription assays (A), while a duplicate set was used for [³H]uridine pulse-labeling (B). The labeled RNAs were purified and quantified by hybridization to filter-bound DNA as described in Materials and Methods. To control for variations in the number of cells recovered under each condition or in the MOIs of the two viruses, DNA was purified from the nuclear fractions of the [³H]uridine-labeled cells and hybridized to saturating quantities of ³²P-labeled human Alu repeat DNA and viral E2E DNA, respectively. ML, major late.

FIG. 4

Northern blotting of reporter RNAs. (A) Poly(A)-containing cytoplasmic (C) or nuclear (N) RNAs isolated from equal numbers of Ad5/MA (lanes 3 and 5)- and Ad5/MtplA (lanes 2 and 4)-infected 293 cells exposed to dexamethasone for 1 h during the late phase of infection and from Ad5-infected cells (lane 6) were examined by Northern blotting with the reporter gene-specific SpeI oligonucleotide probe as described in Materials and Methods. Glyoxylated DNA markers, whose lengths (in kilobase pairs) are listed on the left, were run in lane 1. (B) The concentrations of the processed and unprocessed reporter RNA species shown in panel A, determined with a Molecular Dynamics PhosphorImager, are expressed relative to those of cytoplasmic processed MA RNA in Ad5/MA-infected cells.

FIG. 5

Partitioning of newly synthesized MA and MtplA RNAs between nuclear and cytoplasmic fractions of recombinant virus-infected cells. (A and B) The concentrations of the newly synthesized RNA species in cytoplasmic (A) and nuclear (B) fractions of Ad5/MA- and Ad5/MtplA-infected 293 cells treated with dexamethasone (Dex.) for 1 h at 14 h after infection were determined as described in Materials and Methods. U, uninfected; ML Tpl, major late tripartite leader sequence. (C) Total concentrations of newly synthesized MA and MtplA reporter RNAs and their distribution between nuclear and cytoplasmic fractions. In these experiments, the concentrations of cellular and viral DNAs were also examined by blotting with a human Alu repeat sequence and the viral L2 probe, respectively, to monitor variations in the number of cells under each condition and differences in the efficiencies of infection. No major variation was detected.

FIG. 6

Characterization of MA and MtplA RNA species by RT-PCR. Cytoplasmic poly(A)-containing (lanes 2, 4, 6, 8, 10, and 12) or poly(A)-lacking (lanes 3, 5, 7, 9, 11, and 13) RNAs isolated from Ad5/MtplA-, Ad5/MA-, or Ad5-infected cells following exposure to dexamethasone for 1 h at 11 h after infection were analyzed by RT-PCR with a 3′ primer complementary to sequences near the β-actin poly(A) addition site and a 5′ primer corresponding to positions +21 to +44 of mouse mammary tumor virus (MMTV) transcription unit A or to the 5′ end of the tripartite leader sequence (Tpl). The lengths (in kilobase pairs) of DNA markers (lane 1) are indicated on the left.