VA RNA Genes Research Articles

An Improved Helper Plasmid Containing Deletions Within the E4 and E2a Genes Results in Increased Adeno-Associated Virus Productivity.

The use of a helper plasmid to replace adenovirus infection for adeno-associated virus (AAV) manufacturing has been common practice for decades. Adenovirus E4, E2a, and VA RNA genes are sufficient to support efficient AAV replication. In an effort to ensure that all transfected DNA has a functional role in AAV production, deletions were introduced to the E4 and E2a genes to determine if any portions were dispensable. Although a 900 bp deletion in the E2a intron did not have an impact, the removal of open reading frames (orf) 1-4 from the E4 gene resulted in a doubling of AAV productivity. The E4Δorf1-4 deletion was associated with a reduction in E4orf6 transcripts, along with an increase in Rep and Cap transcripts and protein levels, which corresponded to increased AAV productivity in crude lysate. The final product of these studies was a helper plasmid, termed OXB-Helper_3, that is >3.4 kb smaller than the original control plasmid and resulted in ∼2× improvement in vector genome productivity across multiple capsid serotypes, genome designs, and transfection platforms.

8. Further Characterization of U2 snRNP Mediated Restriction of AAV Vector Transduction

Improving adeno-associated virus (AAV) vector transduction efficiency is central to the development of its continued, widespread use in gene therapy. More effective AAV transduction would reduce vector doses required for efficient gene delivery, minimizing the risks associated with high dose AAV vectors. Several treatments have been reported to increase AAV vector transduction, including adenoviral co-infection. Through screening of a siRNA library, we recently identified U2 snRNP as a host restriction factor for AAV vector transduction. Disruption of U2 snRNP spliceosome and associated proteins, including PHF5A, SF3B1, SF3B2 and U2AF1, potently enhanced AAV vector transduction. Relevant to gene therapy applications of AAV vectors, meayamycin B, a powerful SF3B1 inhibitor, allowed for substantial increases in AAV vector transduction (up to 400-fold). This post-entry restriction appeared to occur after the second-strand synthesis but before transgene expression or accumulation of transgene transcripts and independently of the cellular splicing machinery. No notable changes were found in the cytoplasmic trafficking, nuclear entry, or genome release of AAV vector infection by U2 snRNP inhibition. Here, we further studied the mechanism(s) underlying the U2 snRNP-mediated block of AAV vectors. We first tested another commercially available U2 snRNP inhibitor, pladienolide B (PladB) and verified that PladB treatment also showed substantial dose dependent increases (up to 700-fold) in AAV vector transduction. We next studied the relationship between adenoviral co-infection and U2 snRNP inhibition. Adenoviral co-infection alone enhanced AAV vector transduction up to 30-fold, while adenoviral co-infection, in addition to genetic or pharmacological U2 snRNP inhibition, showed marginal additive effects, suggesting a common pathway targeted by adenoviral co-infection and U2 snRNP inhibition in enhanced AAV vector infection. Using a series of plasmids providing adenoviral helper functions, we have identified Ad5 E4, but not the E2A and VA RNA genes, as partially responsible for the enhanced AAV transduction by adenoviral co-infection through U2 snRNP inhibition. Since the effects of U2 snRNP inhibition are most likely on the regulation of transgene expression, we also assessed the epigenetic modifications of the AAV2 genome in the presence or absence of PladB. Through chromosome immunoprecipitation (ChIP) assays we mapped histone recruitment patterns at different regions of the AAV vector epigenome. PladB treatment reproducibly enhanced recruitment of histone H2b and H3 proteins to the AAV CMV promoter, luciferase transgene, and polyA regions up to 3-fold. Our preliminary study also suggested that U2 snRNP inhibition altered specific histone modification patterns on the AAV vector genome. We are in the process of verifying the influence of U2 snRNP inhibition, as well as AAV capsid variations, on epigenetic modifications of the AAV vector epigenome. Better understanding the underlying mechanism would provide novel insights into host-virus interactions and could lead to the rational design of next generation AAV vectors with improved transduction efficiencies and safety profiles.

Adenovirus-encoding virus-associated RNAs suppress HDGF gene expression to support efficient viral replication.

Non-coding small RNAs are involved in many physiological responses including viral life cycles. Adenovirus-encoding small RNAs, known as virus-associated RNAs (VA RNAs), are transcribed throughout the replication process in the host cells, and their transcript levels depend on the copy numbers of the viral genome. Therefore, VA RNAs are abundant in infected cells after genome replication, i.e. during the late phase of viral infection. Their function during the late phase is the inhibition of interferon-inducible protein kinase R (PKR) activity to prevent antiviral responses; recently, mivaRNAs, the microRNAs processed from VA RNAs, have been reported to inhibit cellular gene expression. Although VA RNA transcription starts during the early phase, little is known about its function. The reason may be because much smaller amount of VA RNAs are transcribed during the early phase than the late phase. In this study, we applied replication-deficient adenovirus vectors (AdVs) and novel AdVs lacking VA RNA genes to analyze the expression changes in cellular genes mediated by VA RNAs using microarray analysis. AdVs are suitable to examine the function of VA RNAs during the early phase, since they constitutively express VA RNAs but do not replicate except in 293 cells. We found that the expression level of hepatoma-derived growth factor (HDGF) significantly decreased in response to the VA RNAs under replication-deficient condition, and this suppression was also observed during the early phase under replication-competent conditions. The suppression was independent of mivaRNA-induced downregulation, suggesting that the function of VA RNAs during the early phase differs from that during the late phase. Notably, overexpression of HDGF inhibited AdV growth. This is the first report to show the function, in part, of VA RNAs during the early phase that may be contribute to efficient viral growth.

2-Aminopurine Enhances the Oncolytic Activity of an E1b-Deleted Adenovirus in Hepatocellular Carcinoma Cells

Adenoviruses with deletions of viral genes have been extensively studied as potential cancer therapeutics. Although a high degree of cancer selectivity has been demonstrated with these conditionally replicating adenoviruses, low levels of virus replication can be detected in normal cells. Furthermore, these mutations were also found to reduce the activity of the replicating viruses in certain cancer cells. Recent studies have shown that co-administration of chemotherapeutic drugs may increase the activity of these viruses without affecting their specificity. We constructed an adenovirus with deletions of both the E1b and the VA-RNA genes and found that replication of this virus was selective for human hepatocellular carcinoma (HCC) cell lines when compared to normal cell lines. Furthermore, we show that 2-aminopurine (2′AP) treatment selectively enhanced virus replication and virus-mediated death of HCC cells. 2′AP did not compensate for the loss of VA-RNA activities, but rather the loss of an E1b-55K activity, such as the DNA damage response, suggesting that co-administration of 2′AP derivatives that block host DNA damage response, may increase the oncolytic activity of AdΔE1bΔVA without reducing its selectivity for HCC cells.

Recent progress in adenovirus vectors: focusing on VA-deleted AdV

First-generation adenovirus vectors (FG-AdVs) are widely used because transduction efficiency of the vectors is very high. However, severe immune responses especially to the liver have been a serious problem of this vector. We succeeded to identify a viral protein that cause the immune responses and reported ''low-inflammatory AdVs'' that mostly solve this problem. However, to develop the ultimate form of this vector, it is necessary to remove virus-associated RNA (VA RNA) genes from the AdV vector genome. VA RNAs are transcribed by polymerase III; they are not essential for viral growth but have important roles to make appropriate circumstances for this virus. Large amount of VA RNAs are required in the late phase to support viral growth. Hence it is difficult to establish 293 cell lines that can support replication of AdVs lacking VA RNA genes (VA-deleted AdVs) supplying sufficient amount of VA RNA in trans. Recently we have developed a method for efficient production of VA-deleted AdVs and succeeded to obtain a high titer of VA-deleted AdVs. Then we construct VA-deleted AdVs expressing shRNA that knockdown the replication of hepatitis C virus (HCV). In fact, VA-deleted AdVs expressing these shRNAs suppressed HCV replication more effectively than conventional FG-AdV. Therefore, we showed that VA RNAs expressed from FG-AdVs probably compete with shRNA in the maturation pathway and reduce the effect of shRNAs. We think that VA-deleted AdV may substitute for current FG-AdVs and become a standard AdV.

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively(1). They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans(2). NS1 is the major effector of viral cytotoxicity(3). In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)(4). The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)(4). The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications. We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit(5). The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product(5). In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

The Genome of Human Parvovirus B19 Can Replicate in Nonpermissive Cells with the Help of Adenovirus Genes and Produces Infectious Virus

Human parvovirus B19 (B19V) is a member of the genus Erythrovirus in the family Parvoviridae. In vitro, autonomous B19V replication is limited to human erythroid progenitor cells and in a small number of erythropoietin-dependent human megakaryoblastoid and erythroid leukemic cell lines. Here we report that the failure of B19V DNA replication in nonpermissive 293 cells can be overcome by adenovirus infection. More specifically, the replication of B19V DNA in the 293 cells and the production of infectious progeny virus were made possible by the presence of the adenovirus E2a, E4orf6, and VA RNA genes that emerged during the transfection of the pHelper plasmid. Using this replication system, we identified the terminal resolution site and the nonstructural protein 1 (NS1) binding site on the right terminal palindrome of the viral genome, which is composed of a minimal origin of replication spanning 67 nucleotides. Plasmids or DNA fragments containing an NS1 expression cassette and this minimal origin were able to replicate in both pHelper-transfected 293 cells and B19V-semipermissive UT7/Epo-S1 cells. Our results have important implications for our understanding of native B19V infection.

The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus (rAAAV), AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein (GFP) gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A,E4, and VA-RNA genes. Chicken embryonic fibroblast (CEF) or chicken embryonic liver (CEL) cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek's disease virus (MDV), avian adenovirus, chicken embryo lethal orphan (CELO) virus or infectious bursal disease virus (IBDV). Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein (gfp) expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV-mediated transgene expression could be enhanced by super infection with the helper viruses.

Deletion of VAI and VAII RNA Genes in the Design of Oncolytic Adenoviruses

Deletion of viral functions that can be complemented by the specific phenotype of tumor cells is a common strategy to design oncolytic viruses. For example, enhanced mRNA cytoplasmic export in tumor cells phenocopies the adenovirus E1B-55K function and renders mutants of this protein tumor selective. Also, an activated RB pathway complements specific E1A functions that can be deleted to produce oncolytic viruses. In this paper we demonstrate that an adenoviral mutant deleted in virus-associated I (VAI) and VAII RNAs (Ad-VAdel) has oncotropism characterized by 100-fold replication deficiency compared with wild-type adenovirus in normal cells and an unaffected ability to replicate and kill different types of tumor cells. This mutant also displays active antitumoral activity in vivo. In contrast, this oncotropism is less evident in a mutant expressing an inactive form of VAI (Adsub719) because VAII RNA expression is upregulated. The mRNA translation promoted by VA RNA genes can be phenocopied in tumor cells with the activation of signal transduction pathways, such as the Ras pathway.

Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components.

Adeno-associated virus (AAV) vector system has several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, in this report, we explored the possibility of generating AAV packaging cell line, 293T R/C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep/Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. We showed that the 293T R/C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep/Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R/C.VA.E2A.E4. cells might be able to tolerate the Rep/Cap and E4 gene products, or have less copy numbers of the Rep/Cap and E4 genes than the traditional method. Moreover, we showed that the AAV vectors derived from 293T R/C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R/C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.

Adeno-associated virus vectors can be efficiently produced without helper virus.

The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M(r), E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-M(r), E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.

Identification of an autonomously initiating RNA polymerase III holoenzyme containing a novel factor that is selectively inactivated during protein synthesis inhibition.

Transcription by RNA polymerase III (Pol III) requires multiple general initiation factors that, in isolated form, assemble onto the promoter in an ordered fashion. Here, it is shown that all components required for transcription of the VA1 and tRNA genes, including TFIIIB, TFIIIC, and RNA Pol III, can be coimmunopurified from a HeLa cell line that constantly expresses a FLAG epitope-tagged subunit of human RNA Pol III. This finding of an RNA Pol III "holoenzyme" suggests similarities between transcription initiation by RNA Pol II and RNA Pol III and has led to the identification of a novel general initiation factor (TDF, translation dependent factor) that is present within the holoenzyme. TDF is selectively inactivated during protein synthesis inhibition by cycloheximide and at a late stage of adenovirus infection, thus accounting for the loss of RNA Pol III-mediated transcription of the tRNA and VA RNA genes under these conditions. On the basis of these observations, possible mechanisms for the global regulation of transcription by RNA Pol III and for disassembly of RNA Pol III initiation complexes are proposed.

Nucleotide sequence of ovine adenovirus tripartite leader sequence and homologues of the IVa2, DNA polymerase and terminal proteins

Ovine adenovirus OAV287 was previously isolated from sheep in Western Australia. Here we describe a portion of its genome between map units 10.3 and 31.7 which includes major ORFs for homologues of the IVa2 polypeptide and the DNA replication proteins, Terminal protein and DNA polymerase, as well as the N-terminal portion of the 52/55-kDa polypeptide. In addition, as a prelude to possible adaptation of this virus as a vector we have mapped the elements which make up the tripartite leader sequence of late mRNAs, thereby defining the probable location of the OAV major late promoter. In other human and animal adenovirus genomes, one or two VA RNA genes are encoded between the ORFs for Terminal protein and 52/55-kDa polypeptides. In OAV, these ORFs overlap, suggesting that if VA RNA genes are present, they may lie elsewhere in the OAV genome.

Cloning and characterization of a TFIIIC2 subunit (TFIIIC beta) whose presence correlates with activation of RNA polymerase III-mediated transcription by adenovirus E1A expression and serum factors.

TFIIIC2 is a general factor essential for transcription of 5S RNA, tRNA, and VA RNA genes by mammalian RNA polymerase III and consists of two forms designated TFIIIC2a and TFIIIC2b. TFIIIC2a and TFIIIC2b share common subunits of 220, 102, 90, and 63 kD but differ with respect to transcription activity and the presence of a presumptive 110-kD subunit in the active form (TFIIIC2a). Because both forms can bind the promoter directly, a selective role for the 110-kD subunit in the regulation of RNA polymerase III activity has been suggested. To investigate this possibility, we have cloned and expressed a cDNA encoding the 110-kD subunit (TFIIIC beta). Immunoprecipitation studies with anti-TFIIIC beta antibodies have confirmed that TFIIIC beta is a bona fide subunit present only in TFIIIC2a, that TFIIIC2a and the general factor TFIIIC1 are associated in unfractionated extracts, and that previously undetected polypeptides (potential TFIIIC1 subunits) can be isolated in association with TFIIIC2a. Previous studies have shown that increases in RNA polymerase III activity during infection of cells by adenovirus (with concomitant E1A expression) or during cell growth at high serum concentration results from an increased activity in the TFIIIC fraction. Studies with antibodies to TFIIIC beta have shown that this is strongly correlated with a selective increase in the cellular concentration of the TFIIIC beta 110-kD subunit and a concomitant rise in the ratio of the active-to-inactive forms of TFIIIC2.

Human and Simian Adenoviruses: Phylogenetic Inferences from Analysis of VA RNA Genes

Adenovirus VA RNA genes have primary sequence constraints due to internal promoter regions and a high degree of secondary structure in the RNA product. To determine the relationships between human and simian adenoviruses, the VA RNA genes of several primate adenoviruses were characterized and compared to those sequences already published. Human adenoviruses of subgenera A, B:2, and F have only one VA RNA gene, whereas human adenoviruses of subgenera 8:1, C, D, and E have two. The genomes of 12 monkey adenoviruses were found to have only one VA RNA gene, whereas the genomes of six representative chimpanzee adenoviruses were each found to have two VA RNA genes. Phylogenetic analysis of representative VA RNA gene sequences individually, irrespective of their strain of origin or partnering VA RNA gene, gave the following inferences. (1) The single VA RNA genes of human adenovirus subgenera A and F are most closely related to these of monkey adenoviruses. (2) The VA RNA I genes of human adenoviruses in subgenera B:1, D, and E, and also the single VA RNA genes of subgenus B:2 probably diverged from a common ancestral VA RNA gene. (3) This ancestral gene most likely reduplicated to give the precursor of all VA RNA II genes, the evidence for which has been almost totally lost in subgenus B:2 adenoviruses. (4) The two VA RNA genes of human subgenus C adenoviruses are relatively distant from each other phylogenetically. Since the Ad2 and Ad5 VA RNA I genes have a higher identity to the single VA RNA gene of SAV13 (SV36) than to those of any of the other human adenoviruses, these genes may have entered the human subgenus C adenovirus genome by substitution involving recombination with a simian adenovirus. The results of this study suggest that a renewed appraisal of VA RNA function in adenoviruses other than Ad2 and Ad5 may be necessary.

Cloning and characterization of an evolutionarily divergent DNA-binding subunit of mammalian TFIIIC.

Transcription factor IIIC (TFIIIC) is required for the assembly of a preinitiation complex on 5S RNA, tRNA, and adenovirus VA RNA genes and contains two separable components, TFIIIC1 and TFIIIC2. TFIIIC2 binds to the 3' end of the internal control region of the VAI RNA gene and contains five polypeptides ranging in size from 63 to 220 kDa; the largest of these directly contacts DNA. Here we describe the cloning of cDNAs encoding all (rat) or part (human) of the 220-kDa subunit (TFIIIC alpha). Surprisingly, TFIIIC alpha has no homology to any of the yeast TFIIIC subunits already cloned, suggesting a significant degree of evolutionary divergence for RNA polymerase III factors. Antibodies raised against the N terminus of recombinant human TFIIIC alpha specifically inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepletion assays indicate that TFIIIC alpha is absolutely required for RNA polymerase III transcription of 5S RNA, tRNA, and VAI RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VAI RNA genes in TFIIIC-depleted nuclear extracts can be restored by addition of purified TFIIIC. In contrast, restoration of 5S RNA gene transcription requires readdition of both TFIIIC and TFIIIA, indicating a promoter-independent interaction between these factors. Immunoprecipitation experiments demonstrate a tight association of all five polypeptides previously identified in the TFIIIC2 fraction, confirming the multisubunit structure of the human factor.

Cloning and Characterization of an Evolutionarily Divergent DNA-Binding Subunit of Mammalian TFIIIC

Transcription factor IIIC (TFIIIC) is required for the assembly of a preinitiation complex on 5S RNA, tRNA, and adenovirus VA RNA genes and contains two separable components, TFIIIC1 and TFIIIC2. TFIIIC2 binds to the 3' end of the internal control region of the VAI RNA gene and contains five polypeptides ranging in size from 63 to 220 kDa; the largest of these directly contacts DNA. Here we describe the cloning of cDNAs encoding all (rat) or part (human) of the 220-kDa subunit (TFIIIC alpha). Surprisingly, TFIIIC alpha has no homology to any of the yeast TFIIIC subunits already cloned, suggesting a significant degree of evolutionary divergence for RNA polymerase III factors. Antibodies raised against the N terminus of recombinant human TFIIIC alpha specifically inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepletion assays indicate that TFIIIC alpha is absolutely required for RNA polymerase III transcription of 5S RNA, tRNA, and VAI RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VAI RNA genes in TFIIIC-depleted nuclear extracts can be restored by addition of purified TFIIIC. In contrast, restoration of 5S RNA gene transcription requires readdition of both TFIIIC and TFIIIA, indicating a promoter-independent interaction between these factors. Immunoprecipitation experiments demonstrate a tight association of all five polypeptides previously identified in the TFIIIC2 fraction, confirming the multisubunit structure of the human factor.

Isolation and characterization of adenovirus-associated VA RNAs of human adenovirus type 7

Two genes for virus-associated VA RNAs in the human adenovirus type 7 (Ad7) genome are compared to Ad2, Ad4, Ad5, simian VA RNAs, and pol III transcripts of Epstein-Barr virus. The newly identified VA RNA-encoding genes of Ad7 contain two relatively conserved intragenic promoter (elements A and B), the double-stranded RNA-dependent protein kinase (PK) active site-binding domain and transcriptional terminators. The conserved features extend to the VA RNA genes of simian and avian origin.

Formation of large, sedimentable transcription complexes with VARNA genes and other related genes

We have used an Eppendorf centrifuge for isolation of transcription complexes assembled on VARNA genes and other related genes with NTP-depleted cell-free extracts. Similar to the 5 S rRNA gene, sedimentable, stable transcription preinitiation complexes could be assembled from two VARNA genes, two EB virus-specific EBER genes, four human tRNA genes, and one human Alu-family RNA gene, suggesting that the 5 S rRNA-specific transcription factor, TFIIIA, was not required for formation of these sedimentable, stable preinitiation complexes. Parameters affecting assembly of these complexes were sequences in circular DNA templates, sizes and sequences of linear DNA templates, temperature and incubation time. These complexes were stable at from 4 to 37 degrees C, and somewhat stable to salt wash. From results of effects of various mutations on assembly of these sedimentable complexes, we concluded that they were transcription machineries. Addition of the supernatant and partially purified factors to salt-washed complexes stimulated their transcription, we concluded that these sedimentable complexes were minimal transcription machineries containing suboptimal quantities of loosely bound transcription factors, TFIIIB, and RNA polymerase III. DNase 1 footprints of these sedimentable preinitiation complexes showed that two regions were protected, from +34 to +80 including the B block promoter element, and from +98 to +105. Similar DNase 1 footprints were also obtained from salt-washed complexes and stable preinitiation complexes isolated by molecular sieve column chromatography.

Cloning of the Human Thyrotropin β-Subunit Gene and Transient Expression of Biologically Active Human Thyrotropin after Gene Transfection

A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) beta-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG alpha-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSH beta containing both coding exons, we constructed hCG alpha and hTSH beta expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-associated virus vectors, each containing one subunit of hTSH, together with a plasmid containing the adenovirus VA RNA genes produced hTSH as well as free human alpha- and TSH beta-subunits in an adenovirus transformed human embryonal kidney cell line (293). The levels of protein expression in this system were 10- to 100-fold greater than that found in a simian virus transformed monkey kidney cell line (COS) using vectors containing the early promoter of simian virus 40. The hTSH synthesized in 293 cells was glycosylated as indicated by complete binding to concanavalin A-Sepharose but was larger in apparent molecular weight than a standard hTSH preparation on gel chromatography suggesting an altered glycosylation pattern. However, it was immunologically and biologically indistinguishable from two pituitary hTSH standards in an immunoradiometric and in vitro iodide trapping assay, respectively.