Oncogenic transformation by the human polyomaviruses.
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Cell Transformation, Neoplastic↗
Biomedical subjects
M J Imperiale
Publications and source records attributed to M J Imperiale.
RNA polymerase II-dependent positional effects on mRNA 3' end processing in the adenovirus major late transcription unit.
During the early phase of adenovirus infection, the promoter-proximal L1 poly(A) site in the major late transcription unit is used preferentially despite the fact that the distal L3 poly(A) site is stronger (i.e. it competes better for processing factors and is cleaved at a faster rate, in vitro). Previous work had established that this was due at least in part to the stable binding of the processing factor, cleavage and polyadenylation specificity factor, to the L1 poly(A) site as mediated by specific regulatory sequences. It is now demonstrated that in addition, the L1 poly(A) site has a positional advantage because of its 5' location in the transcription unit. We also show that preferential processing of a particular poly(A) site in a complex transcription unit is dependent on RNA polymerase II. Our results are consistent with recent reports demonstrating that the processing factors cleavage and polyadenylation specificity factor and cleavage stimulatory factor are associated with the RNA polymerase II holoenzyme; thus, processing at a weak poly(A) site like L1 can be enhanced by virtue of its being the first site to be transcribed.
Adenoviridae↗
Prospects for an SV40 vaccine.
The identification of SV40 as a possible cause of human cancer leads to the question of whether the unique properties of the virus can be exploited to treat patients with SV40-positive mesotheliomas, which are otherwise refractory to successful intervention. A modified SV40 T antigen, from which the transforming domains have been removed, has been cloned into a vaccinia virus vector and tested in animal tumor model systems. It has been shown to be effective against both subsequent tumor challenge and pre-existing tumors. Thus, the potential exists for use of such a vaccine in mesothelioma patients.
Humans↗
Role for the adenovirus IVa2 protein in packaging of viral DNA.
Although it has been demonstrated that the adenovirus IVa2 protein binds to the packaging domains on the viral chromosome and interacts with the viral L1 52/55-kDa protein, which is required for viral DNA packaging, there has been no direct evidence demonstrating that the IVa2 protein is involved in DNA packaging. To understand in greater detail the DNA packaging mechanisms of adenovirus, we have asked whether DNA packaging is serotype or subgroup specific. We found that Ad7 (subgroup B), Ad12 (subgroup A), and Ad17 (subgroup D) cannot complement the defect of an Ad5 (subgroup C) mutant, pm8001, which does not package its DNA due to a mutation in the L1 52/55-kDa gene. This indicates that the DNA packaging systems of different serotypes cannot interact productively with Ad5 DNA. Based on this, a chimeric virus containing the Ad7 genome except for the inverted terminal repeats and packaging sequence from Ad5 was constructed. This chimeric virus replicates its DNA and synthesizes Ad7 proteins, but it cannot package its DNA in 293 cells or 293 cells expressing the Ad5 L1 52/55-kDa protein. However, this chimeric virus packages its DNA in 293 cells expressing the Ad5 IVa2 protein. These results indicate that the IVa2 protein plays a role in viral DNA packaging and that its function is serotype specific. Since this chimeric virus cannot package its own DNA, but produces all the components for packaging Ad7 DNA, it may be a more suitable helper virus for the growth of Ad7 gutted vectors for gene transfer.
Adenoviridae↗
The human polyomaviruses, BKV and JCV: molecular pathogenesis of acute disease and potential role in cancer.
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Acute Disease↗
Production of first generation adenovirus vectors: a review.
In the past decade, adenovirus vectors have generated tremendous interest, especially in gene therapy applications. In the so-called 'first generation' adenovirus vectors, the transgenes are inserted in place of the E1 region, or less often the E3 region. Although second-generation and helper-dependent adenovirus vectors will probably prevail in the future in applications that require long-term gene expression, first generation adenovirus vectors will remain very useful in other settings, such as cancer and vaccination, or simply to transfect cell lines that are refractory to other transfection methods. Until a few years ago, the construction of first generation adenovirus vectors was a labor-intensive and time-consuming process. More than 20 methods have appeared that facilitate their construction and are reviewed below.
Adenoviridae↗
Interaction of the adenovirus IVa2 protein with viral packaging sequences.
We have demonstrated previously that the adenovirus L1 52/55-kDa protein binds to the viral IVa2 protein in infected cells. The significance of this interaction was unclear, however, based on the known functions of these two proteins: the 52/55-kDa protein is required for viral DNA packaging, while the IVa2 protein is a transactivator of the major late promoter (MLP). In this report, we have attempted to elucidate a role for each of the two proteins in the other's known function. There is no apparent effect of the 52/55-kDa protein on the interaction of the IVa2 protein with the MLP. Surprisingly, however, we found that the IVa2 protein can interact with the adenoviral packaging signal and that this interaction involves DNA sequences that have previously been demonstrated to be required for packaging.
Adenoviruses, Human↗
Recombinant adenovirus vector mediated expression of lipoprotein (a) [Lp(a)] in rabbit plasma.
Lipoprotein (a) [Lp(a)] is a heterodimer of apolipoprotein (a) [apo(a)] and apolipoprotein B-100 (apoB-100) of low density lipoprotein linked by a disulfide bond. Apo(a) and apoB-100 are synthesized by the liver and covalently associate or couple to form Lp(a) extracellularly. Elevated plasma Lp(a) is an independent risk factor for vascular injury disorders such as restenosis after balloon angioplasty and accelerated graft atherosclerosis following heart transplantation. Lp(a) is not expressed in laboratory animals making studies of its pathophysiology difficult. To overcome this problem, we explored the possibility of generating Lp(a) in rabbit plasma using replication-deficient adenovirus vector mediated gene delivery. Rabbits were chosen because of their large vessels and unlike mouse or rat, rabbit apoB-100 could interact with apo(a) to generate Lp(a). The recombinant (r) adenovirus vector construct used encoded a 200 kDa apo(a) [Ad-apo(a)]. Ad-apo(a) injection into the rabbit marginal vein caused the appearance of plasma rLp(a). Injection of a r adenovirus vector expressing the bacterial LacZ gene (Ad-LacZ) or PBS (vehicle) did not result in detectable plasma rLp(a). These are the first results to demonstrate plasma expression of rLp(a) in rabbits using adenovirus vector mediated gene transfer. Therefore, this system may be suitable for investigating Lp(a)'s role in the development of vascular injury diseases in a rabbit model.
Adenoviridae↗
Induction of tumor antigen-specific immunity in vivo by a novel vaccinia vector encoding safety-modified simian virus 40 T antigen.
BACKGROUND: Evidence that simian virus 40 (SV40) is associated with human mesotheliomas, osteosarcomas, and brain tumors suggests that a recombinant vaccine directed against lethal cancers expressing SV40 T antigen (Tag) could have clinical utility. To address this potential need, we designed a novel vaccinia virus construct that encodes an SV40 Tag in which oncogenic domains were excluded and immunogenic domains were preserved. We named this recombinant construct vaccinia-encoding safety-modified SV40 Tag (vac-mTag). METHODS: Purified vac-mTag was characterized by DNA sequencing, reverse transcription-coupled polymerase chain reaction, western blot analysis, and immunocytochemical techniques. Induction of Tag-specific immunity was examined by cytolytic T-cell assays, and the efficacy of vac-mTag in protecting animals against Tag-expressing tumors and in treating pre-established microscopic tumors was evaluated in vac-mTag-immunized BALB/c mice. RESULTS: The immune response elicited by vac-mTag in C57BL/6 and BALB/c mice included an SV40 Tag-specific cytolytic T-lymphocyte activity against syngeneic (identical genetic background) SV40 Tag-expressing tumor targets. Immunization of mice with a single dose of vac-mTag resulted in potent protection against subsequent challenge with a lethal mouse cancer expressing SV40 Tag. In addition, single-dose vac-mTag immunization coadministered with interleukin 2 produced a possible therapeutic effect against a preadministered microscopic (but lethal) burden of Tag-expressing tumor cells in vivo. CONCLUSION: vac-mTag induces an effective immune response in mice that is specific for a tumor-associated antigen. This response protects against a lethal tumor challenge and results in a possible therapeutic effect against Tag-expressing tumors in vivo. Thus, vac-mTag provides a new avenue for the development of therapies for human cancers thought to be associated with SV40.
Antineoplastic Agents↗
Immortalized cementoblasts and periodontal ligament cells in culture.
Cementum, a mineralized tissue lining the surface of the tooth root, is required for formation of a functional periodontal ligament attachment during development. Additionally, during regeneration of tissues after disease, cementum is thought to play a critical role in the reparative process. Research efforts aimed toward understanding mechanisms involved in periodontal development and regeneration, and in particular the formation of root cementum, have been hampered by an inability to isolate and culture cells involved in cementum production, i.e., cementoblasts. Using classical techniques for osteoblast isolation, immortalized, heterogeneous cementoblast/periodontal ligament cell (CM/PDL) populations were established from cells lining the tooth root surface of: 1) CD-1 mice, where cells were immortalized using SV40, or 2) H-2KbtsA58 "immorto" mice, where cells containing an immortalizing transgene were removed and cultured. CM/PDL populations were derived from tissues adherent to developing tooth root surfaces, while tissues adherent to the surrounding alveolar bone were specifically excluded from the population. Immortalized CM/PDL cells were characterized to ensure their phenotype reflected that previously demonstrated in situ and in primary, nonimmortalized cultures. Proteins/mRNAs associated with bone/cementum and known to be expressed by root lining cementoblasts, but not by PDL cells, in situ, e.g., bone sialoprotein, osteopontin, and osteocalcin, were expressed by cells within the immortalized populations. Furthermore, CM/PDL cells, in vitro, attached to bone sialoprotein in an arginine-glycineaspartic acid (RGD)-dependent manner, promoted mineral nodule formation and exhibited a PTH/PTHrP-mediated cAMP response. These immortalized heterogeneous populations, containing both CM and PDL cells, provide a unique opportunity to study cells involved in cementogenesis and to enhance our knowledge of the mechanisms controlling development, maintenance, and regeneration of periodontal tissues.
Animals↗
Processing mRNA 3' ends in vitro.
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Bacteriophages↗
Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro.
BACKGROUND: Although recombinant adenovirus vectors are attractive for use in gene expression studies and therapeutic applications, the construction of these vectors remains relatively time-consuming. We report here a strategy that simplifies the production of adenoviruses using the Cre-loxP system. MATERIALS AND METHODS: Full-length recombinant adenovirus DNA was generated in vitro by Cre-mediated recombination between loxP sites in a linearized shuttle plasmid containing a transgene and adenovirus genomic DNA. RESULTS: After transfection of Cre-treated DNA into 293 cells, replication-defective viral vectors were rapidly obtained without detectable wild-type virus. CONCLUSION: This system facilitates the development of recombinant adenoviral vectors for basic and clinical research.
Adenoviridae↗
Encapsidation of viral DNA requires the adenovirus L1 52/55-kilodalton protein.
Previous work demonstrated that the adenovirus L1 52/55-kDa protein is required for assembly of viral particles, although its exact role in the assembly process is unclear. The 52/55-kDa protein's early expression, however, suggests that it might have other roles at earlier times during infection. To uncover any role the 52/55-kDa protein might have at early times and to better characterize its role in assembly, a mutant adenovirus incapable of expressing the 52/55-kDa protein was constructed (H5pm8001). Analysis of the onset and extent of DNA replication and late protein synthesis revealed that H5pm8001-infected 293 cells entered the late stage of infection at the same time as did adenovirus type 5 (Ad5)-infected cells. Interestingly, H5pm8001-infected cells displayed slightly lower levels of replicated viral DNA and late proteins, suggesting that although not required, the 52/55-kDa protein does augment these activities during infection. Analysis of transcripts produced from the major late and IVa2 promoters indicated a slight reduction in H5pm8001-infected compared to Ad5-infected cells at 18 h postinfection that was not apparent at later times. Analysis of particles formed in H5pm8001 cells revealed that empty capsids could form, suggesting that the 52/55-kDa protein does not function as a scaffolding protein. Subsequent characterization of these particles demonstrated that they lacked any associated viral DNA. These findings indicate that the 52/55 kDa-protein is required to mediate stable association between the viral DNA and empty capsid and suggest that it functions in the DNA encapsidation process.
Adenoviridae↗
Novel mechanisms of E2F induction by BK virus large-T antigen: requirement of both the pRb-binding and the J domains.
E2F activity is regulated in part by the retinoblastoma family of tumor suppressor proteins. Viral oncoproteins, such as simian virus 40 (SV40) large-T antigen (TAg), adenovirus E1A, and human papillomavirus E7, can disrupt the regulation of cellular proliferation by binding to pRb family members and dissociating E2F-pRb family protein complexes. BK virus (BKV), which infects a large percentage of the human population and has been associated with a variety of human tumors, encodes a TAg homologous to SV40 TAg. It has been shown that BKV TAg, when expressed at low levels, does not detectably bind to pRb family members, yet it induces a serum-independent phenotype and causes a decrease in the overall levels of pRb family proteins. The experiments presented in this report show that, despite the lack of TAg-pRb interactions, BKV TAg can induce transcriptionally active E2F and that this induction does in fact require an intact pRb-binding domain as well as an intact J domain. In addition, E2F-pRb family member complexes can be detected in both BKV and SV40 TAg-expressing cells. These results suggest the presence of alternate cellular mechanisms for the release of E2F in addition to the well-established model for TAg-pRb interactions. These results also emphasize a role for BKV TAg in the deregulation of cellular proliferation, which may ultimately contribute to neoplasia.
Animals↗
BK virus as a potential co-factor in human cancer.
BK virus (BKV) is a member of the polyomavirus family that persistently infects 75-80% of the human population. BKV encodes a large T antigen which is responsible for the transforming functions of the virus. Recent studies have shown an association of BKV DNA with a variety of human tumours including pancreatic islet, brain, urinary tract and Kaposi's sarcoma. Despite the detection of BKV DNA in several human tumours, there is no clear evidence for a causative role in tumour formation. We have sought to characterize the interactions of BKV TAg with cellular tumour suppressor proteins including p53, pRb, p107, and p130 in an attempt to further understand the molecular mechanisms of transformation by BKV. We have shown that BKV TAg can bind to and functionally inhibit p53 and the p53-mediated response to DNA damage. Additionally, we have shown that low levels of BKV TAg are sufficient to induce free E2F and a serum-independent phenotype despite the absence of detectable interactions with pRb family members. Taken together, these results suggest that BKV TAg can both inhibit the cellular response to DNA damage and induce proliferation, allowing for potential accumulation of mutations in cellular growth control genes. These results suggest a possible role for BKV TAg in cellular transformation and tumour formation in the human host.
Antigens, Polyomavirus Transforming↗
Promoter attenuation in gene therapy: interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression.
One of the major limitations to current gene therapy is the low-level and transient vector gene expression due to poorly defined mechanisms, possibly including promoter attenuation or extinction. Because the application of gene therapy vectors in vivo induces cytokine production through specific or nonspecific immune responses, we hypothesized that cytokine-mediated signals may alter vector gene expression. Our data indicate that the cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) inhibit transgene expression from certain widely used viral promoters/enhancers (cytomegalovirus, Rous sarcoma virus, simian virus 40, Moloney murine leukemia virus long terminal repeat) delivered by adenoviral, retroviral or plasmid vectors in vitro. A constitutive cellular promoter (beta-actin) is less sensitive to these cytokine effects. Inhibition is at the mRNA level and cytokines do not cause vector DNA degradation, inhibit total cellular protein synthesis, or kill infected/transfected cells. Administration of neutralizing anti-IFN-gamma monoclonal antibody results in enhanced transgene expression in vivo. Thus, standard gene therapy vectors in current use may be improved by altering cytokine-responsive regulatory elements. Determination of the mechanisms involved in cytokine-regulated vector gene expression may improve the understanding of the cellular disposition of vectors for gene transfer and gene therapy.
Adenoviruses, Human↗
Distinct domains of adenovirus E1A interact with specific cellular factors to differentially modulate human immunodeficiency virus transcription.
Transcription of human immunodeficiency virus (HIV) type 1 and other viruses is regulated by the transcription factor NF-kappaB, which interacts with the multifunctional cellular protein p300. p300, originally identified by its ability to bind adenovirus early region 1A (E1A), has also been shown to regulate HIV transcription through its interaction with NF-kappaB. The 13S form of E1A activates HIV gene expression, while the 12S form represses its transcription. In this report, we have investigated whether these divergent effects of E1A are dependent upon common or distinct cellular cofactors, including p300, pRb, and the TATA box-binding protein (TBP). Unlike activation in the absence of E1A, cooperative stimulation of HIV gene expression by 13S E1A and RelA was independent of the ability of E1A to bind p300 but was critically dependent on the E1A CR3 region which associates with TBP. In contrast, inhibition of basal HIV gene expression by the 12S form of E1A was dependent on p300 but not pRb or TBP. Interestingly, mutation of the CR2 region of 12S E1A responsible for pRb binding abolished the repression of HIV transcription stimulated by tumor necrosis factor alpha, suggesting that repression of cytokine-activated transcription involves cofactors different from those used in unstimulated cells. Repression and activation of HIV transcription by different forms of E1A are mediated by distinct sets of cellular cofactors. These findings suggest that E1A has evolved to interact by alternative mechanisms with a transcriptional coactivator and its associated cofactors to differentially modulate cellular and viral gene expression.
Adenovirus E1A Proteins↗
Promoter-proximal poly(A) sites are processed efficiently, but the RNA products are unstable in the nucleus.
The presence of two polyadenylation signals in the primary transcript of the human immunodeficiency virus type 1 (HIV-1) provirus leads to a requirement for regulation of 3'-end processing. To ensure that viral genome replication and gene expression occur, polyadenylation must occur at the poly(A) site transcribed from the 3' long terminal repeat (LTR) but not the 5' LTR. Models that have been proposed to explain this regulation include (i) inhibition of the 5' site as a result of proximity to the promoter and (ii) enhancement of the 3' site by U3 sequences that are transcribed upstream of only the 3' poly(A) site. In previous studies designed to investigate these models, a reduction in the levels of steady-state RNA was observed when the HIV-1 poly(A) site was placed within 500 nucleotides of the cap site. Although these findings were interpreted to be the result of promoter proximity effects on 3'-end processing, in vitro studies demonstrated that the HIV-1 poly(A) site was equally functional in promoter-proximal and promoter-distal positions. These results led to the hypothesis that, in vivo, the poly(A) site is fully active at this close distance but the short transcripts produced are highly unstable in the nucleus and undergo rapid degradation, precluding their appearance as abundant mRNAs in the steady-state pool. To investigate the biogenesis of these short RNAs in vivo, experiments were performed to examine directly the nuclear processing rates of the HIV-1 poly(A) site in intact cells. By using recombinant adenoviruses as expression vectors, it is now demonstrated conclusively that the HIV-1 poly(A) site is indeed processed at equivalent levels independent of its distance from the promoter. Although transcripts containing the promoter-proximal poly(A) site are processed efficiently, most undergo degradation in the nucleus instead of nucleocytoplasmic transport.
Adenoviridae↗