Biomedical subjects

T Shenk

Publications and source records attributed to T Shenk.

At least 37 records · Page 2Linked to original sources

PubMed1997

Nuclear export of the E1B 55-kDa and E4 34-kDa adenoviral oncoproteins mediated by a rev-like signal sequence.

The E1B 55-kDa and E4 34-kDa oncoproteins of adenovirus type 5 (abbreviated here as E1B-55kD and E4-34kD) promote the export of viral mRNA and inhibit the export of most cellular mRNA species. We show that the intracellular complex containing E1B-55kD and E4-34kD continuously shuttles between the nucleus and the cytoplasm, and may thus serve as a nucleocytoplasmic transporter for viral mRNA. We present evidence that within this complex, it is the E4-34kD protein that directs both nuclear import and nuclear export. E4-34kD contains a functional nuclear export signal similar to corresponding sequences found in the retroviral proteins rev and rex. This sequence element is required for nuclear export of the complex, and it can function autonomously when fused to a carrier protein and microinjected in HeLa cell nuclei. When E4-34kD is expressed alone, a portion of the protein that contains a predicted arginine-rich amphipathic alpha-helical structure mediates nuclear retention of the protein. This retention, however, can be abolished by the association with E1B-55kD or by a specific point mutation within the arginine-rich motif. The export of E4-34kD can be blocked by an HTLV-rex derived competitive inhibitor and overexpressed E4-34kD inhibits rev-mediated transport, suggesting that the export pathways accessed by the adenoviral and retroviral proteins share components. The interplay between two polypeptides as well as the involvement of a dominant nuclear retention domain are novel features that might contribute to the efficiency and regulation of the adenovirus export system.

Adenovirus E1B Proteins↗

PubMed1997

Human cytomagalovirus IE1 and IE2 proteins are mutagenic and mediate "hit-and-run" oncogenic transformation in cooperation with the adenovirus E1A proteins.

Some epidemiological studies have suggested a possible link between human cytomegalovirus (HCMV) infection and various malignancies, and HCMV has been shown to transform cultured cells. However, viral DNA is not detected in most transformants, and the mechanism by which HCMV might contribute to oncogenesis has remained obscure. Here we show that the HCMV immediate early 1 and 2 genes can cooperate with the adenovirus E1A gene to generate transformed foci of primary baby rat kidney cells. HCMV gene expression is transient and viral DNA is not present in clonal cell lines derived from the transformed foci. We find that the HCMV immediate early proteins are mutagenic, and we propose that HCMV has the potential to contribute to oncogenesis through a "hit-and-run" mechanism, by inducing mutations in cellular genes.

Adenovirus E1A Proteins↗

PubMed1997

Activation of the adenovirus major late promoter by transcription factors MAZ and Sp1.

Multiple binding sites for the transcription factors MAZ and Sp1 within the adenovirus type 5 major late promoter have been identified by DNase I protection studies. In the proximal region of the promoter, both MAZ and Sp1 interact with GC-rich sequences flanking the TATA box. Two MAZ binding sites are centered at -18 and -36 relative to the transcriptional initiation site. Sp1 bound only to the -18 GC-rich sequence. Several sites of interaction were also evident in the distal region of the promoter. Both MAZ and Sp1 interacted with a sequence centered at -166, and MAZ bound weakly to an additional site centered at -130. Overexpression of MAZ or Sp1 activated expression from the major late promoter in transient expression assays. Mutational analysis of the GC-rich sequences in the major late promoter suggested that a primary target of MAZ activation is the GC-rich sequences flanking the TATA sequence, whereas Sp1 requires the distal GC-rich sequence elements to stimulate gene expression. This activation is enhanced by the adenovirus E1A protein, and evidence for interaction between E1A and both transcription factors was obtained by using an immunoprecipitation assay. Activation by MAZ and Sp1 also was observed in transfection studies using the complete adenovirus type 5 genome as the target. Increased levels of late mRNA from both the L1 and L5 regions were observed when MAZ or Sp1 expression plasmids were transfected with viral DNA. Unexpectedly, activation of the major late promoter by MAZ and Sp1 was detected irrespective of whether the viral DNA could replicate.

Adenoviridae↗

PubMed1997

Characterization of the human cytomegalovirus irs1 and trs1 genes: a second immediate-early transcription unit within irs1 whose product antagonizes transcriptional activation.

We have characterized the irs1 and trs1 genes of human cytomegalovirus. The previously identified mRNAs as well as their corresponding protein products pIRS1 and pTRS1 could be detected during all phases of the viral replication cycle. The proteins were present in the nucleus and cytoplasm during the immediate-early and early phases of the viral growth cycle but were predominantly cytoplasmic late after infection. Although pIRS1 and pTRS1 exhibited little transcriptional activation potential on their own, both cooperated with the IE1 and IE2 proteins to enhance expression from a variety of viral promoters. We have also identified a previously undescribed immediate-early gene product encoded within the irs1 gene that we have termed pIRS1(263). This new protein is encoded within the 3' end of the irs1 gene and is in the same reading frame as the large pIRS1 protein. Expression of the irs1(263) gene is controlled by a promoter that resides within the irs1 open reading frame in the unique short region of the viral genome. pIRS1(263) resides in the nucleus and antagonizes transcriptional activation by cytomegalovirus immediate-early proteins. We propose that pIRS1(263), whose promoter responds to immediate-early transcriptional activators, serves as part of a regulatory loop, antagonizing the function of the viral proteins that are responsible for its synthesis.

Base Sequence↗

PubMed1997

Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle.

Three tegument proteins of human cytomegalovirus (HCMV), ppUL82 (pp71), pUL69, and ppUL83 (pp65), were examined for the ability to stimulate the production of infectious virus from human diploid fibroblasts transfected with viral DNA. Although viral DNA alone had a low intrinsic infectivity of 3 to 8 plaques/microg of viral DNA, cotransfection of a plasmid expressing pp71 increased the infectivity of HCMV DNA 30- to 80-fold. The increase in infectivity produced by pp71 was reflected in an increased number of nuclei observed to express high levels of the major immediate-early proteins IE1 and IE2. Cotransfection of viral DNA with plasmids directing expression of IE1 and IE2 also resulted in extensive IE1 and IE2 expression in the transfected cells; however, the infectivity of viral DNA was only marginally increased. pp71 also facilitated late gene expression, virus transmission to adjacent cells, and plaque formation. In contrast, expression of pUL69 reduced the pp71- and IE1/IE2-mediated enhancement of HCMV DNA infectivity and also failed to produce any increase in the number of cells expressing IE1 and IE2 over that seen with viral DNA alone. Expression of pp65 did not alter the infectivity of HCMV DNA, nor did it modify the effects of pp71 or pUL69. These results imply that pp71 plays a critical role in the initiation of infection apart from its function as a transactivator of IE1 and IE2.

Cells, Cultured↗

PubMed1997

pIRS1 and pTRS1 are present in human cytomegalovirus virions.

The virus-coded proteins pIRS1 and pTRS1 were found associated with purified human cytomegalovirus virions. The proteins were not degraded when intact virions were treated with trypsin, which suggests that they are localized inside the viral particle. In transfection experiments pIRS1 and pTRS1 modestly activated expression from a reporter plasmid containing the viral major immediate-early promoter but did not influence the activity of a reporter carrying the irs1/trs1 immediate-early promoter. Both reporters were activated by the combination of pIRS1 or pTRS1 and pUL69, which is also present in virions.

Cytomegalovirus↗

PubMed1997

Viral transactivating proteins.

Many viruses utilize the cellular transcription apparatus to express their genomes, and they encode transcriptional regulatory proteins that modulate the process. Here we review the current understanding of three viral regulatory proteins. The adenovirus E1A protein acts within the nucleus to regulate transcription through its ability to bind to other proteins. The herpes simplex type 1 virus VP16 protein acts within the nucleus to control transcription by binding to DNA in conjunction with cellular proteins. The human T-cell leukemia virus Tax protein influences transcription through interactions with cellular proteins in the nucleus as well as the cytoplasm.

Adenovirus E1A Proteins↗

PubMed1996

YY1 transcriptional initiator: protein interactions and association with a DNA site containing unpaired strands.

The Ying-Yang 1 protein (YY1) DNA-binding site functions as an initiator element at which YY1, transcription factor IIB (TFIIB), and RNA polymerase II sponsor basal transcription from a supercoiled DNA template. We show that TFIIB binds to YY1, stabilizing its interaction with DNA, and YY1 contacts the large subunit of polymerase II, directing it to the initiation site. YY1 directs initiation from linear DNA containing mismatched sequences within its binding site, leading us to infer that supercoiling facilitates the separation of DNA strands and to suggest that YY1 likely remains bound to the start site as DNA strands separate during initiation. These results provide a mechanistic basis for transcriptional initiation directed by YY1 in the absence of the TATA box-binding protein.

Base Sequence↗

PubMed1996

Cocrystal structure of YY1 bound to the adeno-associated virus P5 initiator.

Ying-Yang 1 protein (YY1) supports specific, unidirectional initiation of messenger RNA production by RNA polymerase II from two adjacent start sites in the adeno-associated virus P5 promoter, a process which is independent of the TATA box-binding protein (TBP). The 2.5-A resolution YY1-initiator element cocrystal structure reveals four zinc fingers recognizing a YY1-binding consensus sequence. Upstream of the transcription start sites protein-DNA contacts involve both strands and downstream they are virtually restricted to the template strand, permitting access to the active center of RNA polymerase II and ensuring specificity and directionality. The observed pattern of protein-DNA contacts also explains YY1 binding to a preformed transcription bubble, and YY1 binding to a DNA/RNA hybrid analog of the P5 promoter region containing a nascent RNA transcript. A model is proposed for YY1-directed, TBP-independent transcription initiation.

Amino Acid Sequence↗

PubMed1996

Oncogenic potential of the adenovirus E4orf6 protein.

The group C adenovirus E4orf6 protein has previously been shown to bind to the p53 cellular tumor suppressor protein and block its ability to activate transcription. Here we show that the E4orf6 protein blocks the induction of p53-mediated apoptosis when AT6 cells, which harbor a temperature-sensitive p53, are shifted to the permissive temperature. The E4orf6 protein does not, however, prevent the induction of apoptosis in p53-deficient H1299 cells by treatment with tumor necrosis factor alpha and cycloheximide. The E4orf6 protein also cooperates with the adenovirus E1A protein to transform primary baby rat kidney cells, and it cooperates with the adenovirus E1A plus E1B 19-kDa and E1B 55-kDa proteins to increase the number of baby rat kidney cell transformants and enhance the rate at which they arise. The level of p53 is substantially reduced in transformed cells expressing the E4orf6 protein in comparison to adenovirus transformants lacking it. The E4orf6 gene also accelerates tumor formation when transformed baby rat kidney cells are injected subcutaneously into the nude mouse, and it converts human 293 cells from nontumorigenic to tumorigenic in nude mice. In addition to the well-studied E1A and E1B oncogenes, group C adenoviruses harbor a third oncogene, E4orf6, which functions in some respects similarly to the E1B oncogene.

Adenoviridae↗

PubMed1996

Molecular cloning of the cDNA and chromosome localization of the gene for human ubiquitin-conjugating enzyme 9.

We report a novel human gene whose product specifically associates with the negative regulatory domain of the Wilms' tumor gene product (WT1) in a yeast two-hybrid screen and with WT1 in immunoprecipitation and glutathione S-transferase (GST) capture assays. The gene encodes a 17-kDa protein that has 56% amino acid sequence identity with yeast ubiquitin-conjugating enzyme (yUBC) 9, a protein required for cell cycle progression in yeast, and significant identity with other subfamilies of ubiquitin-conjugating enzymes. The human gene fully complements yeast that have a temperature-sensitive yUBC9 gene mutation to fully restore normal growth, indicating that we have cloned a functionally conserved human (h) homolog of yUBC9. Transcripts of hUBC9 of 4.4 kilobases (kb), 2.8 kb, and 1.3 kb were found in all human tissues tested. A single copy of the hUBC9 gene was found and localized to human chromosome 16p13.3. We conclude that hUBC9 retains striking structural and functional conservation with yUBC9 and suggest a possible link of the ubiquitin/proteosome proteolytic pathway and the WT1 transcriptional repressor system.

Amino Acid Sequence↗

PubMed1996

Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor.

The adenovirus E4orf6 protein is shown here to interact with the cellular tumor suppressor protein p53 and to block p53-mediated transcriptional activation. The adenovirus protein inhibited the ability of p53 to bind to human TAFII31, a component of transcription factor IID (TFIID). Earlier work demonstrated that the interaction of p53 with TAFII31 involves a sequence near the NH2-terminus of p53, whereas the E4orf6-p53 interaction occurs within amino acids 318 to 360 of p53. Thus, the E4orf6 protein interacts at a site on p53 distinct from the domain that binds to TAFII31 but nevertheless inhibits the p53-TAFII31 interaction.

Adenoviridae↗

PubMed1996

The serotonin 1a receptor gene contains a TATA-less promoter that responds to MAZ and Sp1.

The structure and function of the 5'-flanking region of the mouse and human serotonin 1a receptor gene have been analyzed by RNA 5' end mapping, DNA-protein interaction, and transient expression assays. A large number of mRNA 5' termini, detected by mapping 5' ends from mouse brain RNA, were found dispersed over a region of about 700 base pairs flanking the receptor coding sequence. Consistent with the apparently heterogeneous pattern of transcription initiation, the flanking DNA sequence lacked typical TATA box elements and was rich in guanine and cytosine. The mouse and human 5'-flanking sequences were 63% homologus and similarly organized. A guanine-cytosine-rich DNA sequence motif related to the sequence 5'-GGGG(C/A)GGGG-3' was repeated within the 5'-flanking region and located at or near several mRNA 5' ends. This DNA sequence motif bound to proteins in a crude HeLa cell nuclear extract. A cDNA encoding a protein that interacts with this sequence was cloned and found to be the MAZ (Pur-1, Zif87) protein. The interaction between MAZ and the receptor gene 5'-flanking region proximal to the protein coding sequence was examined by DNase I footprinting, and four sites of MAZ interaction were identified. Three of the four MAZ binding sites also were shown to interact with transcription factor Sp1. Overproduction of MAZ or Sp1 in transient transfection assays increased expression directed by the human 5'-flanking sequence, although MAZ was substantially more effective. This result suggests that MAZ and Sp1 both participate in regulating expression from the serotonin 1a receptor gene promoter, and it raises the possibility that MAZ may act at a variety of promoters through the guanosine-cytosine-rich sequences generally thought to serve as binding sites for the Sp1 family of transcription factors. Analysis of one of the guanosine-cytosine-rich DNA sequences also revealed that it can serve as a transcription initiator sequence in vitro. This initiator sequence differs from previously characterized initiators and may represent a new class of this transcriptional control sequence.

Animals↗

PubMed1996

Mammary tumors induced by human adenovirus type 9: a role for the viral early region 4 gene.

Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors when injected into female rats. Three different histological types of mammary tumor (benign fibroadenomas, phyllodes tumors, and malignant solid sarcomas) have been described in Ad9-infected animals, with benign fibroadenomas being seen most frequently. Interestingly, in contrast to other adenoviruses, in which oncogenic viral functions are entirely encoded within the E1 region, Ad9 requires an E4 region transforming protein (ORF1) for its unique mammary oncogenicity. Studies of Ad9-induced rat mammary tumors may lead to a detailed molecular understanding for the development of fibroadenoma, a common human breast tumor.

Adenoviruses, Human↗

PubMed1996

Human cytomegalovirus infection inhibits cell cycle progression at multiple points, including the transition from G1 to S.

Human cytomegalovirus inhibits the growth of human foreskin fibroblast cells by 12 h after infection. Analysis of the cellular DNA content of infected cells by flow cytometry demonstrated that cytomegalovirus does not arrest cell cycle progression at a single point. At least two blockages occur, one of which is in the G1 phase of the cell cycle. The G1 arrest introduced by cytomegalovirus infection blocks S-phase entry after serum stimulation.

Cell Cycle↗

PubMed1996

Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors.

The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. Using various adenovirus mutants, we were able to map this function to early-region E4 open reading frame 6. Plasmid expression of open reading frame 6 protein in cells infected with recombinant AAV increased transduction between 100- and 1,000-fold. The increase in transduction was not dependent on the recombinant AAV gene cassette but instead appeared to involve an immediate early step of the AAV life cycle. Chemical and physical agents that have been shown to induce helper-free replication of wild-type AAV were also able to stimulate recombinant AAV transduction, suggesting that the phenomenon might affect AAV DNA replication. Further experiments showed that viral uncoating was not affected and that the rate-limiting step involved synthesis of a second strand on the single-stranded genomic AAV DNA. These data suggest that the adenovirus E4 region, as well as chemical and physical agents, can play an essential role in an immediate-early step of the AAV life cycle, specifically in second-strand synthesis, and have important implications for the use of AAV vectors in gene therapy protocols.

Adenoviridae↗

PubMed1996

Proteins associated with purified human cytomegalovirus particles.

Virion-associated proteins isolated from purified human cytomegalovirus particles (strain AD169) were used as substrates for chemical sequence analysis. Extracellular virions, noninfectious enveloped particles, and dense bodies were purified by negative viscosity-positive density gradient centrifugation, and their component proteins were separated by denaturing polyacrylamide gel electrophoresis. The deduced amino acid sequence of individual protein bands was used to identify six corresponding viral genes whose products have not previously been identified as virion constituents: UL47, UL25, UL88, UL85, UL26, and UL48.5. In addition, a 45-kDa cellular protein was identified, and the protein fragments sequenced have a high degree of amino acid identity with actin. However, antiactin monoclonal and polyclonal antibodies did not react with a specific protein in the virus preparations, suggesting that this 45-kDa protein is an immunologically distinct isoform of actin. The newly identified viral and cellular proteins were resistant to protease treatment of purified virions, suggesting that they are unlikely to be contaminants of the viral preparations.

Actins↗

PubMed1996

Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells.

The localization of the adenovirus type 5 34-kDa E4 and 55-kDa E1B proteins was determined in the absence of other adenovirus proteins. When expressed by transfection in human, monkey, hamster, rat, and mouse cell lines, the E1B protein was predominantly cytoplasmic and typically was excluded from the nucleus. When expressed by transfection, the E4 protein accumulated in the nucleus. Strikingly, when coexpressed by transfection in human, monkey, or baby hamster kidney cells, the E1B protein colocalized in the nucleus with the E4 protein. A complex of the E4 and E1B proteins was identified by coimmunoprecipitation in transfected HeLa cells. By contrast to the interaction observed in primate and baby hamster kidney cells, the E4 protein failed to direct the E1B protein to the nucleus in rat and mouse cell lines as well as CHO and V79 hamster cell lines. This failure of the E4 protein to direct the nuclear localization of the E1B protein in REF-52 rat cells was overcome by fusion with HeLa cells. Within 4 h of heterokaryon formation and with protein synthesis inhibited, a portion of the E4 protein present in the REF-52 nuclei migrated to the HeLa nuclei. Simultaneously, the previously cytoplasmic E1B protein colocalized with the E4 protein in both human and rat cell nuclei. These results suggest that a primate cell-specific factor mediates the functional interaction of the E1B and E4 proteins of adenovirus.

3T3 Cells↗