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Biomedical subjects

T Shenk

Publications and source records attributed to T Shenk.

At least 127 records · Page 7Linked to original sources
PubMed1986

In vivo identification of sequence elements required for normal function of the adenovirus major late transcriptional control region.

A series of adenovirus type 5 variants were constructed to identify the sequence elements which comprise the major late transcriptional control region in the context of the viral chromosome. The variant chromosomes carried a second copy of DNA sequence derived from the region surrounding the major late mRNA cap site. The reiterated segments replaced the normal transcriptional control region of the E1A gene. By monitoring the rate of E1A transcription subsequent to infection with the variants, it was possible to evaluate the capabilities of the substituted major late elements. A segment derived from -55 to +33 (relative to the major late cap site at +1) functioned for early transcription, in the presence of the E1A enhancer domain, but failed to direct enhanced levels of activity late after infection. A segment from -122 to +33 directed both early and enhanced late transcription. The rate of late E1A transcription directed by this element was about 40% of that displayed by the major late control region at its normal position. Inclusion of additional upstream sequences (to -565) did not increase late transcription rates. Thus, function of the adenovirus major late control region.

Adenoviridae↗
PubMed1986

Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase.

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of host cell and viral mRNAs late after infection. The growth of a viral mutant that is unable to produce the RNA is inhibited by interferon, while wild-type virus is not affected. VAI RNA prevents activation of the interferon-induced P1/eIF-2 alpha kinase. This inhibition can be reproduced in extracts of interferon-treated cells where purified VAI RNA prevents activation of latent kinase by double-stranded RNA.

Adenoviruses, Human↗
PubMed1986

The adenovirus type 5 E1A enhancer contains two functionally distinct domains: one is specific for E1A and the other modulates all early units in cis.

The adenovirus type 5 genome contains two distinct enhancer elements located at the left end of the viral chromosome. The first element is repeated and specifically regulates region E1A transcription within infected cells. One copy of this element is sufficient to fully activate E1A transcription in vivo. The second element is located between these repeated sequences and regulates transcription in cis of all early regions on the chromosome. These enhancer elements function independently of each other, and neither element is required for efficient viral DNA replication. Since mutations within the two E1A enhancer components generate different physiological responses, transcriptional enhancement can be achieved through multiple mechanisms.

Adenovirus Early Proteins↗
PubMed1986

Simian virus 40 agnoprotein facilitates normal nuclear location of the major capsid polypeptide and cell-to-cell spread of virus.

The simian virus 40 agnoprotein is a 61-amino-acid, highly basic polypeptide that is coded within the 5' leader of late 16S mRNAs. To better understand agnoprotein function and to more effectively differentiate cis-from trans-acting effects of an agnogene mutation, we constructed a mutant virus that carries a single-base-pair substitution and fails to produce agnoprotein. pm 1493 contains a T/A to A/T transversion at sequence position 335. This mutation converts the agnoprotein initiation codon from ATG to TTG, preventing synthesis of the protein. The mutant displays only a modest growth defect in CV-1P and AGMK cells and no defect in BSC-1 cells. Early-gene expression, DNA replication, synthesis of late viral products, and the kinetics of virion assembly all appear normal in pm 1493-infected CV-1P cells. Immunofluorescent studies, however, indicate that localization of the major capsid polypeptide VP1 is different in mutant- than wild-type virus-infected cells. Furthermore, the lack of agnoprotein led to inefficient release of mature virus from the infected cell. Agnogene mutants could be severely compromised in their ability to propagate in monkeys given their reduced capacity for cell-to-cell spread.

Capsid↗
PubMed1986

An adenovirus mutant unable to express VAI RNA displays different growth responses and sensitivity to interferon in various host cell lines.

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for the efficient translation of host cell and viral mRNAs late after infection. VAI RNA prevented activation of the interferon-induced P1/eIF-2 alpha kinase. In its absence the kinase was activated, eIF-2 alpha was phosphorylated, and translational initiation was inhibited. H5dl331 (dl331), a mutant which cannot express VAI RNA, grew poorly in 293 cells but generated wild-type yields in KB cells. The growth phenotype of the mutant appeared to correlate with the kinetics of kinase induction and activation. Active kinase appeared more rapidly in cell extracts prepared from infected 293 cells, in which dl331 grew poorly, than in extracts of KB cells, in which the mutant grew well. However, when kinase was induced in KB cells by interferon treatment and then activated subsequent to dl331 infection, viral protein synthesis was less severely inhibited than in interferon-treated 293 cells. Thus, activated kinase per se is insufficient to severely inhibit dl331 protein synthesis in KB cells.

Adenoviruses, Human↗
PubMed1986

The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs.

The adenovirus type 5 mutant H5dl338 lacks 524 base pairs within early region 1B. The mutation removed a portion of the region encoding the related E1B-55K and -17K polypeptides but did not disturb the E1B-21K coding region. The virus can be propagated in 293 cells which contain and express the adenovirus type 5 E1A and E1B regions, but it is defective for growth in HeLa cells, in which its final yield is reduced about 100-fold compared with the wild-type virus. The mutant also fails to transform rat cells at normal efficiency. The site of the dl338 defect was studied in HeLa cells. Early gene expression and DNA replication appeared normal. Late after infection, mRNAs coded by the major late transcription unit accumulated to reduced levels. At a time when transcription rates and steady-state nuclear RNA species were normal, the rate at which late mRNA accumulated in the cytoplasm was markedly reduced. Furthermore, in contrast to the case with the wild type, transport and accumulation of cellular mRNAs continued late after infection with dl338. Thus, the E1B product appears to facilitate transport and accumulation of viral mRNAs late after infection while blocking the same processes for cellular mRNAs.

Adenovirus Early Proteins↗
PubMed1986

A second domain of simian virus 40 T antigen in which mutations can alter the cellular localization of the antigen.

Previous studies have demonstrated that mutations at amino acid position 128 of the simian virus 40 large T antigen can alter the subcellular localization of the antigen. A second domain in which mutations can alter localization of the nuclear antigen has been identified by mutations at amino acid positions 185, 186, and 199. Mutations in this region cause the polypeptide to accumulate in both the nucleus and cytoplasm of monkey cells. These T-antigen variants accumulate to near normal levels, but they don't bind to the simian virus 40 origin of DNA replication and are unable to mediate DNA replication. Furthermore, the altered tumor antigens can no longer transform secondary rat cells at normal efficiency, but they retain the ability to transform established mouse and rat cell lines.

Animals↗
PubMed1985

Selection of sequence elements that substitute for the standard AATAAA motif which signals 3' processing and polyadenylation of late simian virus 40 mRNAs.

A method is described which allows selection of sequences which can substitute for the normal AATAAA hexanucleotide involved in polyadenylation of SV40 late mRNAs. Plaques were generated from viral DNA lacking the motif, forcing acquisition of substitute sequences. Four variants were characterized. All displayed wild-type growth kinetics and produced normal levels of late mRNAs and proteins. Two variants had reacquired AATAAA elements and one acquired an ATTAAA sequence. The last variant carried an ATTTTTTAAA segment, suggesting this novel sequence, or some portion of it, can also signal poly A addition.

Animals↗
PubMed1985

Transcription termination within the E1A gene of adenovirus induced by insertion of the mouse beta-major globin terminator element.

In induced erythroleukemia cells, transcription of the beta-globin gene terminates in a region 600-1500 nucleotides downstream of the poly(A) site. To determine whether this region of the mouse DNA functions to terminate transcription when moved to another genomic site, portions of the putative termination region have been inserted into the E1A transcription unit of the adenovirus (type 5) chromosome. Analysis of RNA labeled either in isolated nuclei or in whole cells early after infection with reconstructed viruses indicated that transcription is terminated if the inserted DNA is oriented in the same direction as in the beta-globin transcription unit and contains the globin poly(A) site plus an additional 1395 nucleotides downstream. In addition to halting transcription within the E1A unit, the insertion of the terminator region had a negative cis effect on the E1B transcription unit, which normally initiates 363 bp downstream of the site of the globin insert. The E1B transcription unit was the only early gene affected, and complementation of the terminator virus with a wild-type E1A gene did not restore transcription of the E1B gene.

Adenoviridae↗
PubMed1985

Adenovirus VAI RNA prevents phosphorylation of the eukaryotic initiation factor 2 alpha subunit subsequent to infection.

The virus-associated VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of mRNAs late after infection. Deletion mutant dl331 fails to produce this RNA and, as a result, grows poorly. Three lines of evidence suggest that VAI RNA facilitates translation by preventing inactivation of the function of eukaryotic initiation factor 2 (eIF-2). First, the mutant's translational defect can be relieved by addition of eIF-2 or eIF-2B (GTP recycling factor). Second, extracts of mutant-infected cells exhibit enhanced protein P1/eIF-2 alpha subunit kinase activity. Third, dl331 can grow with nearly normal kinetics in cells that do not express the kinase.

Adenoviridae Infections↗
PubMed1985

Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff.

To delineate the function of adenovirus early region 4 (E4) gene products, we constructed a set of mutant viruses which carry defined lesions within this coding region. Deletion and insertion mutations within six of seven known E4 coding regions had no measurable effect on virus growth in cultured cells. A variant carrying a deletion within the last coding region (encoding a 34,000-molecular-weight polypeptide) was modestly defective, and a mutant lacking the majority of the E4 region was severely defective for growth. The phenotypes of the two defective mutants are similar and complex. Both display perturbations in DNA replication, translation of the E2A mRNA, accumulation of late viral mRNAs, and host cell shutoff.

Adenoviruses, Human↗
PubMed1985

Sequence-independent autoregulation of the adenovirus type 5 E1A transcription unit.

The adenovirus E1A gene is known to be autoregulated at the level of transcription. Autoregulation was found to be mediated by products of the E1A 13S mRNA, which induced a fivefold increase in E1A transcription rate. Deletion analysis suggested that the autoregulation did not require any specific sequence in the E1A transcriptional control region. This conclusion was reinforced by the demonstration that a cellular alpha-globin gene substituted for the E1A gene on the adenovirus chromosome was also positively regulated by E1A gene products.

Adenovirus Early Proteins↗
PubMed1984

Dissection of overlapping functions within the adenovirus type 5 E1A gene.

The adenovirus E1A gene encodes multiple, overlapping mRNAs whose products function both to regulate mRNA levels during the lytic cycle of the virus and to facilitate transformation of non-permissive cells. To assign specific functions to the E1A gene products, two adenovirus type 5 variants have been constructed. Mutants dl347 and 348 carry cloned segments corresponding to the E1A 12 and 13S mRNAs, respectively, in place of the normal E1A gene. The variants produced the predicted E1A-specific mRNAs and polypeptides. Both viruses grew efficiently in HeLa cells. Although the 13S mRNA products were more effective, the products of either mRNA species could stimulate the accumulation of mRNAs from additional transcription units. Both viruses could induce the formation of transformed foci in an established rat cell line. Neither virus could transform primary rat embryo cells at normal frequency, and the dl347 foci which were induced were incomplete or abortive transformants. Thus, functions encoded by both 12S and 13S mRNAs are required for efficient and complete transformation of primary rat cells.

Adenoviridae↗
PubMed1984

Adenovirus VAI RNA facilitates the initiation of translation in virus-infected cells.

The adenovirus VAI RNA is a small polymerase III-transcribed species that is required for optimal translation of mRNAs late after infection. Mutant dl331 fails to produce this RNA species and, as a result, grows poorly. Mutant-infected cells contain normal levels of late mRNAs, but reduced levels of polypeptides are synthesized late after infection. Translational elongation occurs at normal rates in mutant, as compared to wild-type, virus-infected cells. Initiation of translation occurs with reduced efficiency in dl331 -infected cells. VAI RNA is required for formation of a stable 48S preinitiation complex and very likely functions to facilitate the interaction between 43S preinitiation complex and mRNA to form the 48S species.

Adenoviruses, Human↗
PubMed1984

Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection.

A series of adenovirus type 5 variants was constructed to probe the function of the tripartite leader sequence, a 200-nucleotide, 5' noncoding sequence carried on the majority of late viral mRNAs. Recombinant plasmids were constructed that carried the major late transcriptional control region followed by portions of the tripartite leader sequence fused to the E1A coding region. These modified E1A genes were then rebuilt into intact viral chromosomes, replacing the corresponding wild-type region. The leader segments had no effect on the translation of E1A mRNAs early after infection, but the tripartite leader significantly enhanced (5-fold) the efficiency with which the mRNAs were translated late after infection.

Adenoviruses, Human↗
PubMed1984

A viable simian virus 40 variant that carries a newly generated sequence reiteration in place of the normal duplicated enhancer element.

A segment comprising the transcriptional enhancer elements was deleted from a recombinant plasmid carrying the simian virus 40 genome. The mutated viral chromosome was excised from the plasmid and propagated through several cycles of growth in monkey kidney cells. A variant was obtained that carried reiterations of sequences that span both sides of the deleted enhancer region. The mutant virus, dup1495, displays a lag in its growth kinetics as compared to its parent, but it ultimately generates wild-type yields. The mutant virus expresses early mRNAs at near-normal levels, and the reiterated sequence functioned in cis to enhance transformation of mouse cells by the herpesvirus thymidine kinase gene. Thus dup1495 reiterated segments encode enhancer activity even though their primary sequence is radically different from that of the normal simian virus 40 enhancer.

Base Sequence↗
PubMed1984

Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells.

In soluble protein extracts obtained from adenovirus productively infected cells, monoclonal antibodies directed against the early region 1B 58,000-dalton (E1B-58K) protein immunoprecipitated, in addition to this protein, a polypeptide of 25,000 molecular weight. An analysis of tryptic peptides derived from this 25K protein demonstrated that it was unrelated to the E1B-58K protein. The tryptic peptide maps of the 25K protein produced in adenovirus 5 (Ad5)-infected HeLa cells and BHK cells were identical, whereas Ad3-infected HeLa cells produced a different 25K protein. The viral origin of this 25K protein was confirmed by an amino acid sequence determination of five methionine residues in two Ad2 tryptic peptides derived from the 25K protein. The positions of these methionine residues in the 25K protein were compared with the nucleotide sequence of Ad2 and uniquely mapped the gene for this protein to early region 4, subregion 6 of the viral genome. A mutant of Ad5 was obtained (Ad5 dl342) which failed to produce detectable levels of the E1B-58K protein. In HeLa cells infected with this mutant, monoclonal antibodies directed against the E1B-58K protein failed to detect the associated 25K protein. In 293 cells infected with Ad5 dl342, which contain an E1B-58K protein encoded by the integrated adenovirus genome, the mutant produced an E4-25K protein which associated with the E1B-58K protein derived from the integrated genome. Extracts of labeled Ad5 dl342-infected HeLa cells (E1B-58K-) were mixed in vitro with extracts of unlabeled Ad5 wild type-infected HeLa cells or 293 cells (E1B-58K+). When the mixed extracts were incubated with the E1B-58K monoclonal antibody, a labeled E4-25K protein was coimmunoprecipitated. When extracts of Ad5 dl342-infected HeLa cells and uninfected HeLa cells (both E1B-58K-) were mixed, the E1B-58K monoclonal antibody failed to immunoselect the E4-25K protein. These data provide evidence that the E1B-58K antigen is physically associated with an E4-25K protein in productively infected cells. This is the same E1B-58K protein that was previously shown to be associated with the cellular p53 antigen in adenovirus-transformed cells.

Adenoviridae↗