Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.

Anderson, Kevin; Klessig, Daniel F.

doi:10.1073/pnas.81.13.4023

Cited by 50 publications

(34 citation statements)

References 42 publications

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“…Studies with DBP hr mutants, which efficiently multiply in monkey cells, indicate that changes in this protein can directly or indirectly alter the patterns of splicing of late viral transcripts-in particular, those processed into fiber mRNA (17,18). Moreover, the translatability of the fiber mRNA in vivo is affected by these hr mutations, which may or may not be a consequence of the altered structure of the message.…”

Section: Discussionmentioning

confidence: 99%

“…The purported negative regulation of early genes mediated by wild-type (wt) DBP appears to operate at the transcription level for E4 (12,13) and at the level of mRNA turnover for regions EIA, EIB, and probably E2A (14). However, studies by Rice and Klessig (15) (16), alterations in the pattern of mRNA splicing for the fiber polypeptide (17,18), and poor utilization of this mRNA in vivo (19). Host-range (hr) mutants of Ad, which overcome these blocks and thus are capable of productive growth in monkey cells, contain alterations in the DBP gene (20)(21)(22).…”

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confidence: 99%

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Association of the adenovirus DNA-binding protein with RNA both in vitro and in vivo.

Cleghon¹,

Klessig²

1986

Proc. Natl. Acad. Sci. U.S.A.

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The multifunctional DNA-binding protein (DBP) encoded by human adenovirus binds RNA. The association of purified DBP with RNA in vitro was demonstrated by using either a gel filtration or a filter binding assay. This association is sensitive to ionic strength and exhibits no apparent sequence specificity. DBP also interacts with RNA in vivo; it can be crosslinked to polyadenylylated RNA by UV-irradiation of intact cells during the late phase of adenovirus infections. The 46-kDa carboxyl-terminal domain ofDBP binds RNA in vitro and was found to be associated with polyadenylylated RNA in vivo. This is the same domain that interacts with DNA. However, the differences in sensitivity of DBP to trypsin when bound to RNA versus DNA suggest that RNA and DNA either bind at different sites within this domain or induce different conformational changes within the protein.The early region 2A gene (E2A) of human adenovirus types 2 and 5 (Ad2 and AdS) encodes a 72-kDa DNA-binding phosphoprotein (DBP) (1, 2), which is synthesized both early and late during an infectious cycle (3,4). This protein has a number of purported functions in infected cells. Analysis of DBP mutants has implicated the protein in viral DNA replication, viral early and late gene expression, and virion assembly.Studies of a DBP temperature-sensitive (ts) mutant, Ad5ts125, provides evidence of the protein's involvement in DNA replication. This mutant encodes a DBP that is thermolabile in its ability to bind to single-stranded (ss) DNA in vitro (5) and, at the nonpermissive temperature, is defective for viral DNA synthesis in vivo (6) and in vitro (7).The role of DBP in the regulation of Ad early gene expression is more controversial. DBP has been implicated in stimulating expression of early region 1B (EIB) (8) and early region 4 (E4) (9), while negatively influencing expression of early genes at intermediate and late times (10, 11). The purported negative regulation of early genes mediated by wild-type (wt) DBP appears to operate at the transcription level for E4 (12,13) and at the level of mRNA turnover for regions EIA, EIB, and probably E2A (14). However, studies by Rice and Klessig (15) (21-24). The ts, DNA replication-minus mutations are located in the 3' two-thirds of the gene, while the hr mutations map in the 5' portion ofthe gene. These functional domains appear to correspond to physical domains because digestion of purified DBP with a variety of proteases generates a C-terminal fragment of =44-46 kDa and an N-terminal fragment of -24-26 kDa (2, 25). The C-terminal fragment binds ss DNA (2) and can substitute for intact DBP in an in vitro DNA replication system (26). The N-terminal region also appears to function independently because DBP encoded by the hr mutants is able to overcome the block to late gene expression in monkey cells, even when its DNA replication function is perturbed by a ts mutation in the C-terminus (27).How the-DBP accomplishes this varied set of purported functions is still poorly understood. No enzymatic functions have...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Association of the adenovirus DNA-binding protein with RNA both in vitro and in vivo.

Cleghon¹,

Klessig²

1986

Proc. Natl. Acad. Sci. U.S.A.

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“…It has been hypothesized over the last two decades that this is a 2-fold defect that is manifested not only in transcription but also as an impediment in translation of the protein due to incomplete splicing or incomplete maturation of the fiber mRNA. This host cell restriction on translation was overcome by cotransfection with SV40 (17)(18)(19)(20). SV40 DNA provided unknown factors necessary to allow the progression of the adenovirus type 2 infection.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional Blocks Limit Adenoviral Replication in Primary Ovarian Tumor

Preuss

Lam

Wang

et al. 2004

Clinical Cancer Research

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Purpose: Despite the success of conditionally replicating adenoviruses in tumor models, clinical success has been limited when they are used as a single modality agent. Overcoming the disparity in efficacy between in vivo animal models and human use is a key hurdle for better conditionally replicating adenovirus therapy in humans. We endeavored to identify biological blocks to adenoviral infection and replication in tumor cells.Experimental Design: We hypothesized that the differences in adenoviral replication between ovarian cancer cell lines and patient tumor samples are the result of a block in viral RNA transcription. To test this hypothesis, established ovarian cancer cell lines and purified patient ovarian cancer cells were infected with wild-type adenovirus. RNA for early adenoviral genes E1A and E1B as well as the late transcripts for fiber and hexon were measured using real-time PCR.Results: Established ovarian cancer cell lines treated with wild-type virus had a lower E1A:E1B ratio than the patient samples. Additionally, the levels of fiber and hexon relative to E1A were also decreased in the patient samples compared with the established cell lines. These findings were consistent with an early-to late-phase block in the adenovirus replication cycle.Conclusions: These data suggest that the biology of abortive infection in the patient samples may be linked to a defect in the production of early and late viral transcripts.Identification of factors leading to abortive infection will be crucial to understanding the low viral replication in patient samples.

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“…Defective splicing of the Li transcripts (little or no Illa mRNA) and fiber transcripts (altered patterns of ancillary leaders on the mRNA) in abortively infected monkey cells is overcome by host range mutations in the adenovirus DBP or the presence of the SV40 T antigen (3,4,27 Li transcripts from pK3, a vector containing large deletions both 5' and 3' of the Illa splice junction, were spliced to the Illa acceptor in the presence of T antigen. This result suggests that a cis-acting element(s) essential for T-antigen induction of Illa acceptor usage is located within a region of 132 nt 5' and 130 nt 3' of the Illa splice junction.…”

Section: Discussionmentioning

confidence: 99%

“…For example, Forbes et al (17) reported cell cycle-dependent variations in small nuclear ribonucleoprotein concentration, which could affect splicing. i Our earlier studies indicated that T antigen, as well as DBP, could affect RNA splicing in monkey cells (4,27). Here we demonstrate that an SV40 early gene product (presumably large T antigen) alters Li transcript splicing in human cells as well as in monkey cells.…”

Section: Discussionmentioning

confidence: 99%

cis-acting elements and a trans-acting factor affecting alternative splicing of adenovirus L1 transcripts.

Delsert¹,

Morin²,

Klessig³

1989

Mol. Cell. Biol.

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Expression of the Li region of adenovirus is temporally regulated by alternative splicing to yield two major RNAs encoding the 52-to 55-kilodalton (52-55K) and lIa polypeptides. The distal acceptor site (IHa) is utilized only during the late phase of infection, whereas the proximal site (52-55K) is used at both early and late times. Several parameters that might affect this alternative splicing were tested by using expression vectors carrying the Li region or mutated versions of it. In the absence of a virus-encoded or -induced factor(s), only the 52-55K acceptor was used. Decreasing the distance between the donor and the IlIa acceptor had no effect. Removal of the 52-55K acceptor induced IIIa splicing slightly, implying competition between the two acceptors. Fusion of the Hla exon to the 52-55K intron greatly enhanced splicing of the HIa junction, suggesting that the Ila exon does not contain sequences that inhibit splicing. Thus, the lack of splicing to the Ila acceptor in the absence of a virus-encoded or -induced factor(s) is probably due to the absence of a favorable sequence and/or the presence of a negative element 5' of the Hla splice junction, or both. The presence of several adenovirus gene products, including VA RNAs, the E2A DNA-binding protein, and the products of ElA and ElB genes, did not facilitate use of the lIla acceptor. In contrast, the simian virus 40 early proteins, probably large T antigen, induced IlIa splicing. This result, together with those of earlier studies, suggest that T antigen plays a role in modulation of alternative RNA splicing.Most eucaryotic genes contain introns that are removed from the transcript during mRNA maturation. During processing of the precursor RNA of some of these genes, different combinations of splice donor and acceptor sites are joined to form different mRNAs. It is now clear that this alternative RNA splicing not only increases the complexity of products that are encoded by a given gene but also serves as an important regulatory mechanism. For example, both the rat fibroblast tropomyosin 1 and the skeletal muscle ,-tropomyosin are expressed from a single rat gene via alternative RNA splicing (25). Many other examples of tissue-specific (9,32) or developmentally regulated alternative splicing are known (7, 12; for reviews, see references 11 and 33).Although many studies are being conducted to characterize alternative splicing, the mechanisms that govern this process are still ill defined. Specifically, it is not known how alternative splicing sites are chosen or even whether different signals are used during processing of transcripts that undergo simple versus complex patterns of splicing. Both simple and complex transcriptional units use more or less conserved consensus sequences identified at the donor and acceptor splice sites and the branch point (24, 36, 39). Moreover, sequence comparisons between splice junctions of constitutive and alternative exons have not revealed significant differences (11). Therefore, it is likely that regulation of alternative...

show abstract

Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.

Cited by 50 publications

References 42 publications

Association of the adenovirus DNA-binding protein with RNA both in vitro and in vivo.

Association of the adenovirus DNA-binding protein with RNA both in vitro and in vivo.

Transcriptional Blocks Limit Adenoviral Replication in Primary Ovarian Tumor

cis-acting elements and a trans-acting factor affecting alternative splicing of adenovirus L1 transcripts.

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