Ad Genome Research Articles

Expression of adenoviral E3 transgenes in beta cells prevents autoimmune diabetes.

The adenovirus (Ad) genome contains immunoregulatory and cytokine inhibitory genes that are presumed to function in facilitating acute infection or in establishing persistence in vivo. Some of these genes are clustered in early region 3 (E3), which contains a 19-kDa glycoprotein (gp19) that inhibits the transport of selected class I major histocompatibility complex (MHC) molecules out of the endoplasmic reticulum. In addition, the E3 region contains three protein inhibitors of the cytolytic function of tumor necrosis factor alpha (TNF-alpha). Because type I autoimmune diabetes destroys islets by mechanisms that involve class I MHC and TNF-alpha, we investigated whether the entire cassette of Ad E3 genes might prevent the onset of diabetes in a well studied lymphocytic choriomeningitis viral (LCMV) murine model of virus-induced autoimmune diabetes. In this model, a LCMV polypeptide (either glycoprotein or nucleoprotein) expressed as a transgene in the islets is a target for autoimmune destruction of beta cells after LCMV infection. In this scenario the LCMV-induced immune response is directed not only against the virus but also against the LCMV transgenes expressed in the beta cells. Our experiments demonstrated a very efficient prevention of this LCMV-triggered diabetes by the Ad E3 genes. This resulted from the inhibition of target cell recognition by a fully competent and LCMV-primed immune system. Unlike the results from the beta-2 microglobulin gene deletion experiments, our approach shows that selective regulation at the level of the target cell is sufficient to prevent autoimmune diabetes without disrupting the function of the systemic immune response. Although the Ad genes in these experiments were provided as transgenes, recent experiments may permit the introduction of such genes through the use of viral vectors. Although the decrease in class I MHC in islets by Ad genes was demonstrated in these in vivo studies, the relative importance of this process and the control of TNF-alpha cytolysis must await further genetic dissection of the introduced Ad genes.

Cytotoxic T lymphocyte responses to proteins encoded by heterologous transgenes transferred in vivo by adenoviral vectors.

Although replication-deficient adenovirus (Ad) vectors are efficient vehicles for in vivo gene transfer, persistence of expression of the Ad genome is limited in immunocompetent hosts by cellular immunity directed against the gene product of the vector. While most attention has been focused on cytotoxic T lymphocytes (CTL) directed against the low-level early and late Ad gene expression in the Ad vector-infected target cells, significant cellular immunity is likely also directed against the product of heterologous transgenes. To evaluate this concept, in vivo generation of CTL was evaluated in C57B1/6 and BALB/c mice with Ad vectors expressing a variety of heterologous transgenes, including Escherichia coli chloramphenicol acetyl transferase (CAT), beta-galactosidase (beta-Gal), cytosine deaminase, and human thrombopoietin (hTPO), with an Ad vector expressing no transgene ("null") as a control. Following intravenous administration of Ad vectors, spleen cells were harvested 2 weeks later, stimulated for 5 days with syngeneic cells infected with various Ad vectors, and then evaluated for CTL activity using 51Cr-release from syngeneic Ad vector-infected targets. In all cases, CTL directed against the heterologous transgene products was observed, although there were differences in the amounts of transgene-specific CTL. CTL directed against the transgene were also observed with other routes of administration, including intratracheal, subcutaneous, and intraperitoneal administration. These observations suggest that inclusion of a heterologous transgene in Ad vectors enhances the elimination of vector-infected cells, a circumstance that will be partially circumvented using autologous genes. For some applications, specific immune responses to products of transgenes delivered by Ad vectors might be exploited for therapeutic purposes.

A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging.

Adenoviruses (Ads) are intermediate-sized mammalian DNA viruses with a double-stranded linear genome of 36 kb. The icosohedral virion has been shown to accommodate up to 105% of the wild-type genome length, and genomes larger than this size are either unpackageable or extremely unstable, frequently undergoing DNA rearrangement. Here we show that the Ad virion also has a lower packaging limit of approximately 75% of the wild-type genome length. We have constructed a series of vectors with sizes ranging from 15.1 to 33.6 kb and used these to show that in our Cre/loxP helper-dependent system (R. J. Parks, L. Chen, M. Anton, U. Sankar, M. A. Rudnicki, and F. L. Graham, Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996), vectors with genomes greater than or equal to 27.7 kb are packaged with equal efficiencies, whereas vectors with smaller genomes are inefficiently packaged. A 15.1-kb vector, approximately half the size of the wild-type adenovirus genome, was packaged with an efficiency intermediate between that of the small (21.3- to 25.7-kb) and large (27.7- to 33.5-kb) vectors. Analysis of vector DNA after amplification in helper virus-infected cells showed that vectors below 75% of the Ad genome had undergone DNA rearrangements, whereas larger vectors were unaltered. The 15.1-kb vector was recovered primarily as a mix of head-to-tail and tail-to-tail covalent dimers, with a size of 30 kb. We conclude that the Ad virion can efficiently accommodate viral DNA of greater than 75% of the viral genome but that smaller viral genomes tend to undergo rearrangement, resulting in a final size of greater than approximately 27 kb before they can be efficiently packaged. Knowledge of the lower limit to Ad DNA packaging should allow for the design of better and more stable vectors.

Insertion of the adenoviral E3 region into a recombinant viral vector prevents antiviral humoral and cellular immune responses and permits long-term gene expression.

Recombinant adenoviruses (Ads) are highly efficient at transferring foreign genes to the liver in vivo; however, the duration of gene expression is limited by the host antiviral immune response, which prevents expression upon readministration of the virus. To test whether overexpression of the immunomodulatory products of the early Ad genome region 3 (E3) could prevent the antiviral immune response and prolong expression of foreign genes delivered by Ad vectors, we injected a recombinant Ad (Ad-E3-hBUGT), containing both E3 and the human bilirubin-uridine-diphosphoglucuronate-glucuronosyltransferase (BUGT) genes, into BUGT-deficient hyperbilirubinemic Gunn rats. Control Gunn rats received Ad-hBUGT, which expresses human BUGT alone. An initial injection of either virus resulted in hepatic expression of human BUGT as evidenced by excretion of bilirubin glucuronides in bile and a reduction of mean serum bilirubin levels from 7.0 mg/dl to 1.9-2.7 mg/dl within 7 days. In Ad-E3-hBUGT-injected rats, serum bilirubin levels increased to 4.5 mg/dl by 84 days after infection, but a second administration of the virus on that day resulted in a hypobilirubinemic response similar to that seen with the first injection. In contrast, rats receiving Ad-hBUGT had serum bilirubin levels of 7 mg/dl on day 84 after infection, but showed no reduction of serum bilirubin by reinjection of the virus on that day. In the rats injected with Ad-E3-hBUGT, but not in the ones injected with Ad-hBUGT, there was a marked inhibition of the antiviral antibody and Ad-specific cytotoxic T lymphocyte responses. This is the first demonstration that insertion of E3 genes in recombinant Ads facilitates readministration of a functional vector for long-term correction of an inherited metabolic disorder.

Circumvention of anti-adenovirus neutralizing immunity by administration of an adenoviral vector of an alternate serotype.

Effective gene transfer and expression following repetitive administration of adenoviral (Ad) vectors in experimental animals is limited by anti-Ad neutralizing antibodies. Knowing that anti-Ad humoral immunity is serotype-specific, we hypothesized that anti-Ad neutralizing immunity could be circumvented using Ad vectors of different serotypes (Ad2, Ad5) within the same subgroup (C) to transfer and express beta-glucuronidase (beta glu) in the lung. Sprague-Dawley rats received an intratracheal administration of either Ad2 beta glu or Ad5 beta glu, and, 14 days later, repeat administration of either the same vector or a vector of a different serotype. Analysis of serum and bronchoalveolar lavage fluid following initial vector administration demonstrated systemic and local serotype-specific neutralizing antibodies. For both the Ad2 and Ad5 vectors, beta glu expression 24 hr following the second administration of the same serotype was < 30% of that of naive animals. In contrast, beta glu expression 24 hr following second administration of a different serotype Ad vector was similar to expression at 24 hr of naive animals receiving a single administration (Ad5 beta glu followed by Ad2 beta glu, as well as Ad2 beta glu followed by Ad5 beta glu; p > 0.2 both comparisons). Although the alternative serotype bypassed anti-Ad neutralizing immunity, persistence of expression was reduced compared to that following administration to naive animals. Compatible with this observation, systemic administration of the same vectors to C57B1/6 mice demonstrated induction of cytotoxic T lymphocytes directed against the beta glu transgene, as well as products of the Ad genome. Interestingly, intratracheal administration of vectors with different serotypes and different transgenes to rats resulted in longer expression (but still not normalized) compared to that achieved with vectors of different serotypes but the same transgene. These observations demonstrate that alternate use of Ad vectors from different serotypes within the same subgroup can circumvent anti-Ad humoral immunity to permit effective gene transfer after repeat administration, although the chronicity of expression is limited, likely by cellular immune process directed against both the transgene and viral gene products expressed by the vector.

NAP-I is a functional homologue of TAF-I that is required for replication and transcription of the adenovirus genome in a chromatin-like structure.

For the activation of replication and transcription from DNA in a chromatin structure, a variety of factors are thought to be needed that alter the chromatin structure. Template activating factor-I (TAF-I) has been identified as such a host factor required for replication of the adenovirus (Ad) genome complexed with viral basic core proteins (Ad core). TAF-I also stimulates transcription from the Ad core DNA. Using mutant TAF-I proteins, we have demonstrated that the acidic stretch present in the carboxyl terminal region is essential for the stimulation of transcription from the Ad core. A genomic footprinting experiment with restriction endonuclease has revealed that TAF-I causes a structural change in the Ad core. TAF-I has been shown to have significant amino acid similarity to nucleosome assembly protein-I (NAP-I), which is involved in the formation of the chromatin structure. We have shown that TAF-I can be substituted by NAP-I in the activation of the cell-free Ad core transcription system. Two of the tripartite acidic regions and the region homologous to TAF-I in NAP-I are required for the maximal TAF-I activity of NAP-I. Furthermore, TAF-I has been shown to have NAP-I activity, and the acidic region of TAF-I is required for this activity. Since TAF-I causes the structural change of the Ad core and thereby activates transcription, TAF-I is thought to be one of the proteins which is involved in chromatin remodeling. NAP-I is structurally related to TAF-I and functionally substitutes for TAF-I. Furthermore, TAF-I has NAP-I activity. These observations suggest that this type of molecule has dual functions, possibly by participating in facilitating the assembly of the chromatin structure as well as perturbing the chromatin structure to allow transcription to proceed.

Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors.

Adenovirus (Ad) vectors have been extensively used to deliver recombinant genes to a great variety of cell types in vitro and in vivo. Ad-based vectors are available that replace the Ad early region 1 (E1) with recombinant foreign genes. The resultant E1-deleted vectors can then be propagated on 293 cells, a human embryonal kidney cell line that constitutively expresses the E1 genes. Unfortunately, infection of cells and tissues in vivo results in low-level expression of Ad early and late proteins (despite the absence of E1 activity) resulting in immune recognition of virally infected cells. The infected cells are subsequently eliminated, resulting in only a transient expression of foreign genes in vivo. We hypothesize that a second-generation Ad vector with a deletion of viral genes necessary for Ad genome replication should block viral DNA replication and decrease viral protein production, resulting in a diminished immune response and extended duration of foreign gene expression in vivo. As a first step toward the generation of such a modified vector, we report the construction of cell lines that not only express the E1 genes but also constitutively express the Ad serotype 2 140-kDa DNA polymerase protein, one of three virally encoded proteins essential for Ad genome replication. The Ad polymerase-expressing cell lines support the replication and growth of H5ts36, an Ad with a temperature-sensitive mutation of the Ad polymerase protein. These packaging cell lines can be used to prepare Ad vectors deleted for the E1 and polymerase functions, which should facilitate development of viral vectors for gene therapy of human diseases.

Adenovirus vector technology: an efficient method for constructing recombinant adenovirus and on/off switching of gene expression.

An efficient method of constructing recombinant adenoviruses (Ad) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique SwaI site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected to 293 cells together with the Ad DNA-terminal protein complex digested at several sites with EcoT22I. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the restriction digestion drastically reduced regeneration of the parent virus. This method may facilitate the application of recombinant Ad and should be useful for further improvement of Ad vectors. Also a recombinant adenovirus expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ad showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1 cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in future gene therapy.

Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.

Subgroup B Adenovirus Type 35 Early Region 3 mRNAs Differ from Those of the Subgroup C Adenoviruses

Adenovirus type 35 (Ad35) is a member of Ad subgroup B, DNA homology cluster B2. The B2 Ads are unique in that they are isolated most frequently from immunosupressed individuals such as AIDS patients and bone marrow transplant recipients and in that they have a tropism for the urinary tract. One region of the Ad genome which may influence serotype specific pathology is early region 3 (E3). E3 of subgroup C Ad2 and Ad5 has been shown to encode proteins which counteract the immune response to Ad infection. While a great deal is known about gene expression of the subgroup C Ad E3s, little is known about the E3 gene expression from the subgroup B Ads. Although some E3 open reading frames (ORFs) are shared between subgroups B and C, there are additional ORFs that appear in subgroup B. This paper demonstrates the results of an analysis of gene expression from the Ad35 E3 and describes differences in splicing and polyadenylation between the Ad35 and Ad2 E3s. RT–PCR, cDNA sequencing, RNase protection, 3′ RACE, and Northern blotting techniques were utilized to identify, quantify, and determine the structure of six Ad35 E3 mRNAs predicted to encode at least seven proteins. A common intron that is removed during splicing of the subgroup C E3 mRNAs is not removed from Ad35 E3 mRNAs, and only one E3 polyadenylation signal is present in the Ad35 E3 while two polyadenylation signals are used in the formation of subgroup C E3 mRNAs. The quantity of individual mRNAs encoding homologous proteins for Ad35 and Ad2 also differ substantially, presumably because of the absence in Ad35 ofcis-acting signals which have been shown to be important for regulation of Ad2 E3 pre-mRNA processing. Such information should contribute to an understanding of the role the E3 plays in determining subgroup B Ad pathogenesis in general and Ad35 pathogenesis in particular.

Role of adenovirus type 5 early region 3 in the pathogenesis of ocular disease and cell culture infection.

Experimental animal virus replication models make it possible to study the role of viral gene products in adenovirus (Ad)-induced ocular disease. This study tested the hypothesis that the early region 3 (E3) of the human Ad genome plays an important role in the pathogenesis of Ad-induced ocular disease. Both Ad5 wt300, a genetically defined E3+ parent, and Ad5 dl327, a deletion mutant without E3 (E3-), replicated in ocular-derived cell cultures. Ad5 E3+ and Ad8 replicated more efficiently than did Ad5 E3- in cornea and conjunctival cell cultures. Lacrimal gland-derived cell cultures supported human Ad8 replication significantly more efficiently than did either Ad5 E3+ or Ad5 E3-. After intrastromal and topical inoculation of rabbits with either Ad wt300 or Ad dl327, a specific immune response was elicited that coincided with the appearance of subepithelial opacities that mimicked human disease both clinically and histologically. The clinical features (i.e., conjunctivitis, iritis, and corneal edema) were not significantly different for Ad5 E3(+)- and Ad5 E3(-)-induced ocular infection. Ad5 E3(+)- and Ad5 E3(-)-inoculated eyes shed virus for up to 7 and 5 days, respectively, and occasionally established persistent and/or latent infections in corneal, conjunctival, and, infrequently, lacrimal gland cells. Both Ad5 E3+ and Ad5 E3- spread from virus-inoculated animals to the cornea and conjunctiva of normal animals. Under current experimental conditions, expression of the E3 gene does not appear to affect the degree of virulence in ocular disease induced by Ad5 in the rabbit eye model. Deletion of the E3 gene from Ad5 does not make the model more like human disease.

Stimulation of DNA Transcription by the Replication Factor from the Adenovirus Genome in a Chromatin-like Structure

Adenovirus (Ad) genome DNA is complexed with viral core proteins in the virus particle and in host cells during the early stages of infection. This DNA protein complex, called Ad core, is thought to be the template for transcription and DNA replication in infected cells. The Ad core functioned as template for DNA replication in the cell-free system consisting of viral replication proteins, uninfected HeLa nuclear extracts, and a novel factor, template activating factor-I (TAF-I) that we have isolated from uninfected HeLa cytoplasmic fractions. The Ad core did not function as an efficient template in the cell-free transcription system with nuclear extracts of uninfected HeLa cells. The addition of TAF-I resulted in the stimulation of transcription from E1A and ML promoters on the Ad core. TAF-I was required, at least, for the formation of preinitiation complexes. These observations suggest that, in addition to factors essential for transcription on naked DNA template, the factor such as TAF-I needed for replication of the Ad core is also required for transcription from the Ad genome in a chromatin-like structure.

Template activating factor I, a novel host factor required to stimulate the adenovirus core DNA replication

The adenovirus (Ad) genome in virion exists in the form of the Ad core, which is a complex of Ad DNA and viral basic proteins. The Ad core supported only initiation and limited elongation in DNA replication in a cell-free system which was developed for DNA replication of naked Ad DNA. We found that addition of a cytoplasmic fraction from uninfected HeLa cells on the top of the system stimulated Ad core DNA replication, although it had no effect on DNA replication of naked Ad DNA. The factor stimulating Ad core DNA replication, designated as template activating factor I (TAF-I), was purified by successive steps of ammonium sulfate precipitation, column chromatographies on phosphocellulose and Q-Sepharose, and glycerol density gradient centrifugation. SDS-polyacrylamide gel electrophoresis of the purified fraction revealed the presence of proteins with molecular masses of 41 and 39 kDa. TAF-I stimulated both initiation and elongation in Ad core DNA replication. Judging from its behavior in purification steps, TAF-I could be acidic. These findings suggest that TAF-I stimulates Ad core DNA replication by interaction with viral basic core proteins.

Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector.

A defective adenovirus (Ad) type 5 E1- vector has been combined with the powerful constitutive cytomegalovirus (CMV) major immediate early (IE) promoter to produce a novel eukaryotic expression system. The Ad vector can replicate to high titres in 293 cells and then be used to infect a wide variety of non-permissive cell types. The Escherichia coli lacZ and CMV IE1 genes have been cloned to generate the Ad recombinants RAd35 and RAd31 respectively. In human fibroblasts infected with RAd35 beta-galactosidase (beta-gal) expression could be detected in virtually 100% of target cells, there was no detectable transcription from the Ad genome and extremely high levels of expression could be achieved with beta-gal representing the predominant cytoplasmic cellular protein. Additionally, a number of agents, including the CMV IE1 gene product (in RAd31) and forskolin, significantly enhanced expression from RAd35-infected human fibroblasts. Lower levels of constitutive beta-gal expression were obtained in RAd35-infected HeLa cells but again expression could be enhanced (up to 60 fold) by chemical inducing agents. Expression from the IE promoter in the Ad vector could be repressed by coinfection with CMV.

Terpenoids in Foliar Pigment Glands of A, D, and AD Genome Cottons: Introgression Potential for Pest Resistance

High-performance liquid chromatographic analyses showed that leaves of 58 accessions of 18 species of Gossypium exhibited relatively consistent differences in terpenoids among species. Cotton terpenoids, such as gossypol, are economically important secondary metabolites that accumulate primarily in gland structures throughout the plant. Color and other morphological characters associated with foliar pigment glands also were distinctive in this germplasm and could be important for taxonomic and evolutionary studies. Upland cotton (G. hirsutum) was crossed with G. raimondii, a species that produces the unique terpenoid, raimondal. In the inter-specific hybrids, we detected raimondal concentrations in leaves and flower buds that averaged 9% and 12.3%, respectively, of the level in G. raimondil. Large reductions occurred in the quantity of para-oxidation terpenoids in the hybrids in comparison to the cultivated cotton parents; these reductions were not associated with major changes in the number or size of the glands. Thus, terpenoid introgression for profile shifts are possible but will require careful progeny selection to maximize expression.

Transcription in the reverse orientation at either terminus of the adenovirus type 5 genome.

The right terminus of the adenovirus type 5 (Ad5) genome, which contained the early-region 4 (E4) promoter, was capable of initiating transcription in the reverse orientation in in vitro and in vivo assays. Multiple cis-acting elements, required for original-oriented transcription, were also important for reverse-oriented transcription except for the original TATA box. We constructed a plasmid in which the E4 promoter region was linked to the chloramphenicol acetyltransferase gene in the original orientation and linked to the lacZ gene in the reverse orientation and tested the ability of a co-transfected E1A gene to stimulate transcription in both orientations. Activities of transcription in both orientations were stimulated 6- to 10-fold in this assay. More than one cis-acting element was necessary for the stimulation. The region between -39 and -177 was necessary for the E1A-mediated trans-activation of transcription in both orientations, indicating that the region functioned in a bidirectional manner. The activity of transcription in the reverse orientation was similarly detected at the left terminus of the Ad genome and the activity was stimulated several-fold by E1A gene products.

The precise structure and coding capacity of mrnas from early region 2B of human adenovirus serotype 2

Replication of human adenovirus (Ad) DNA requires three virus-encoded proteins that are coordinately transcribed from a single promoter at early times after infection. The mRNAs for two of these proteins, the precursor to the terminal protein (pTP) and the Ad DNA polymerase (Ad Poll, share several exons, including one encoded near Ad genome coordinate 39. The positions of the splice points of these mRNAs have been mapped by S1 nuclease mapping, by RNA sequencing, and by cDNA cloning. As a result of RNA splicing events, a short open reading frame (ORF) encoded at genome coordinate 39 is connected to the beginning of both the pTP and Ad Pol coding sequences; inclusion of this upstream ORF is essential for expression of functional pTP and Ad Pol proteins.

Expression of enzymatically active adenovirus DNA POLYMERASE FROM CLONED DNA requires sequences upstream of the main open reading frame

Replication of human adenovirus (Ad) DNA requires three virus-encoded proteins that are coordinately transcribed from a single promoter at early times after infection. The mRNAs for two of these proteins, the preterminal protein (pTP) and the Ad DNA polymerase (Ad Pol), share several exons, including one encoded near Ad genome coordinate 39. Plasmids containing the putative exons that encode Ad Pol mRNA were constructed to determine if enzymatically active Ad Pol protein could be synthesized. An Ad Pol of 140 kDa was detected by immunoprecipitation with specific antibody and its enzymatic activity was confirmed by complementation of Ad DNA replication in vitro. In addition to an Ad2 DNA fragment from 24.7 to 9.2 map units which contains an open reading frame for a protein of 120 kDa, the HindIII-J fragment that encodes the axon at genome coordinate 39 can be shown to be essential for production of full-length (140 kDa), enzymatically active Ad Pol.

Inhibition of protein synthesis enhances transformation of primary cells by viral DNA.

Inhibition of protein synthesis by cycloheximide after transfection and subsequent removal of the drug increased the transformation efficiency of primary cells by plasmids containing the left 4.5, 6.7, or 16% of the adenovirus (Ad) genome. The enhancement factor ranged from 2 to as much as 70 depending on the size of the viral DNA fragments used. Addition of cycloheximide before or at the time of transfection inhibited transformation, suggesting that viral protein synthesis is important during the early phase of transformation. Transient expression assays showed that cells treated with cycloheximide post-transfection contained as much as three times the amount of viral RNA transcribed from regions E1A and E1B. Conversion of a rat cell line lacking thymidine kinase activity (TK-) to the TK+ phenotype by a plasmid containing the herpes TK gene was severely inhibited by the drug treatment, suggesting that the enhancement effects of cycloheximide on transformation may be specific for Ad DNA. Cycloheximide treatment also increased the number of transformants induced by a transformation defective E1B mutant of Ad12 (cyt mutant). Plasmid containing only the E1A region of Ad12 transformed primary rat kidney cells with very low efficiency. The inclusion of E1B in the transfecting DNA fragments increased the transformation frequency by more than 400-fold, much higher than that achieved by cycloheximide. Thus, cycloheximide cannot replace E1B functions in transformation efficiency.

DNA sequences required for the in vitro replication of adenovirus DNA.

Initiation of adenovirus (Ad) DNA replication occurs on viral DNA containing a 55-kilodalton (kDa) protein at the 5' terminus of each viral DNA strand and on plasmid DNAs containing the origin of Ad replication but lacking the 55-kDa terminal protein (TP). Initiation of replication proceeds via the synthesis of a covalent complex between an 80-kDa precursor to the TP (pTP) and the 5'-terminal deoxynucleotide, dCMP. Formation of the covalent pTP-dCMP initiation complex with Ad DNA as the template requires the viral-encoded pTP and DNA polymerase and, in the presence of the Ad DNA binding protein, is dependent upon a 47-kDa host protein, nuclear factor I. Initiation of replication with recombinant plasmid templates requires the aforementioned proteins and an additional host protein, factor pL. Deletion mutants of the Ad DNA replication origin contained within the 6.6-kilobase plasmid pLA1 were used to analyze the nucleotide sequences required for the formation and subsequent elongation of the pTP-dCMP initiation complex. The existence of two domains within the first 50 base pairs of the Ad genome, both of which are required for the efficient use of recombinant DNA molecules as templates in an in vitro DNA replication system, was demonstrated. The first domain, consisting of a 10-base-pair "core" sequence located at nucleotide positions 9-18, has been identified tentatively as a binding site for the pTP [ Rijinders , A. W. M., van Bergen, B. G. M., van der Vliet , P. C. & Sussenbach , J. S. (1983) Nucleic Acids Res. 11, 8777-8789]. The second domain, consisting of a 32-base-pair region spanning nucleotides 17-48, was shown to be essential for the binding of nuclear factor I.