Genetic identification of adenovirus type 5 genes that influence viral spread

The mechanisms that control cell-to-cell spread of human adenoviruses (Ad) are not well understood. Two early viral proteins, E1B-19K and E3-ADP, appear to have opposing effects since viral mutants that are individually deficient in E1B-19K produce large plaques (G. Chinnadurai, Cell 33:759-766, 1983), while mutants deficient in E3-ADP produce small plaques (A. E. Tollefson et al., J. Virol. 70:2296-2306, 1996) on infected cell monolayers. We have used a genetic strategy to identify different viral genes that influence adenovirus type 5 (Ad5) spread in an epithelial cancer cell line. An Ad5 mutant (dl327; lacking most of the E3 region) with the restricted-spread (small-plaque) phenotype was randomly mutagenized with UV, and 27 large-plaque (lp) mutants were isolated. A combination of analyses of viral proteins and genomic DNA sequences have indicated that 23 mutants contained lesions in the E1B region affecting either 19K or both 19K and 55K proteins. Four other lp mutants contained lesions in early regions E1A and E4, in the early L1 region that codes for the i-leader protein, and in late regions that code for the viral structural proteins, penton base, and fiber. Our results suggest that the requirement of E3-ADP for Ad spread could be readily compensated for by abrogation of the functions of E1B-19K and provide genetic evidence that these two viral proteins influence viral spread in opposing manners. In addition to E1B and E3 proteins, other early and late proteins that regulate viral replication and infectivity also influence lateral viral spread. Our studies have identified novel mutations that could be exploited in designing efficient oncolytic Ad vectors.     

Adeno-Associated Virus Small Rep Proteins Are Modified with at Least Two Types of Polyubiquitination

Adeno-associated virus (AAV) type 2 and 5 proteins Rep52 and Rep40 were polyubiquitinated during AAV-adenovirus type 5 (Ad5) coinfection and during transient transfection in either the presence or absence of Ad5 E4orf6 and E1b-55k. Polyubiquitination of small Rep proteins via lysine 48 (K48) linkages, normally associated with targeting of proteins for proteasomal degradation, was detected only in the presence of E4orf6. The small Rep proteins were ubiquitinated via lysine 63 (K63) following transfection in either the presence or absence of E4orf6 or following coinfection with Ad5. E4orf6/E1b-55k-dependent K48-specific polyubiquitination of small Rep proteins could be inhibited using small interfering RNA (siRNA) to cullin 5.Together, adenovirus type 5 (Ad5) early gene products E1a, E1b-55k, E2a, E4orf6, and virus-associated (VA) RNA can support efficient replication of adeno-associated virus (AAV) (4, 31). E4orf6 and E1b-55k are known to interact with cellular cullin 5 (cul5), elongins B and C, and the ring box protein Rbx1 to form an E3 ubiquitin ligase complex that specifically targets a small population of cellular proteins for degradation by the proteasome (1, 7, 21, 22, 24, 27). This property has been implicated in a number of functions presumed to be required for both Ad and AAV replication (3, 8-10, 17, 23, 24, 34, 35).Previously, only p53, Mre11, DNA ligase IV, and integrin α3 had been shown to be substrates of the Ad5 E3 ubiquitin ligase complex (1, 7, 21, 22, 24, 27); however, we have recently shown (16, 17) that the small Rep proteins and capsid proteins of AAV5 are also degraded in the presence of Ad E4orf6 and E1b-55k in a proteasome-dependent manner. These proteins were restored to levels required during infection by the action of VA RNA (17). The targeting for degradation of AAV5 protein by the E4orf6/E1b-55k E3 ubiquitin ligase complex required functional BC-box motifs in E4orf6 and could be inhibited by depletion of the scaffolding protein cullin 5 using directed small interfering RNA (siRNA) (16). In addition, the degradation of AAV5 protein was partially prevented by overexpression of pUBR7, a plasmid that generates a dominant-negative ubiquitin (16). The role this targeted degradation plays in the life cycle of AAV has not yet been clarified; however, E4orf6 mutants that cannot function in this regard do not support AAV replication as well as wild-type E4orf6 (R. Nayak and D. J. Pintel, unpublished data). Degradation of Mre11 by the Ad5 E3 ligase has also been implicated in allowing efficient Ad5 and AAV replication (24). Ubiquitination of AAV Rep proteins during viral infection, however, has not previously been reported.     

Adenovirus E3-19K proteins of different serotypes and subgroups have similar, yet distinct, immunomodulatory functions toward major histocompatibility class I molecules

Our understanding of the mechanism by which the E3-19K protein from adenovirus (Ad) targets major histocompatibility complex (MHC) class I molecules for retention in the endoplasmic reticulum is derived largely from studies of Ad serotype 2 (subgroup C). It is not well understood to what extent observations on the Ad2 E3-19K/MHC I association can be generalized to E3-19K proteins of other serotypes and subgroups. The low levels of amino acid sequence homology between E3-19K proteins suggest that these proteins are likely to manifest distinct MHC I binding properties. This information is important as the E3-19K/MHC I interaction is thought to play a critical role in enabling Ads to cause persistent infections. Here, we characterized interaction between E3-19K proteins of serotypes 7 and 35 (subgroup B), 5 (subgroup C), 37 (subgroup D), and 4 (subgroup E) and a panel of HLA-A, -B, and -C molecules using native gel, surface plasmon resonance (SPR), and flow cytometry. Results show that all E3-19K proteins exhibited allele specificity toward HLA-A and -B molecules; this was less evident for Ad37 E3-19K. The allele specificity for HLA-A molecules was remarkably similar for different serotypes of subgroup B as well as subgroup C. Interestingly, all E3-19K proteins characterized also exhibited MHC I locus specificity. Importantly, we show that Lys(91) in the conserved region of Ad2 E3-19K targets the C terminus of the α2-helix (MHC residue 177) on MHC class I molecules. From our data, we propose a model of interaction between E3-19K and MHC class I molecules.     

Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients

First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.     

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

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

Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication.

The human adenovirus type 2 (Ad2) mutant Ad2ts111 has previously been shown to contain two mutations which result in a complex phenotype. Ad2ts111 contains a single base change in the early region 1B (E1B) 19,000-molecular-weight (19K) coding region which yields a cyt deg phenotype and another defect which maps to the E2A 72K DNA-binding protein (DBP) coding region that causes a temperature-sensitive DNA replication phenotype. Here we report that the defect in the Ad2ts111 DBP is due to a single G----T transversion that results in a substitution of valine for glycine at amino acid 280. A temperature-independent revertant, Ad2ts111R10, was isolated, which reverts back to glycine at amino acid 280 yet retains the cyt and deg phenotypes caused by the 19K mutation. We physically separated the two mutations of Ad2ts111 by constructing a recombinant virus, Ad2ts111A, which contained a wild-type Ad2 E1B 19K gene and the gly----val mutation in the 72K gene. Ad2ts111A was cyt+ deg+, yet it was still defective for DNA replication at the nonpermissive temperature. The Ad2ts111 DBP mutation is located only two amino acids away from the site of the mutation in Ad2+ND1ts23, a previously sequenced DBP mutant. Biochemical studies of purified Ad2+ND1ts23 DBP showed that this protein was defective for elongation but not initiation of replication in a cell-free replication system consisting of purified Ad polymerase, terminal protein precursor, and nuclear factor I. Ad2+ND1ts23 DBP bound less tightly to single-strand DNA than did Ad2 DBP, as shown by salt gradient elution of purified DBPs from denatured DNA cellulose columns. This decreased binding to DNA was probably due to local conformational changes in the protein at a site that is critical for DNA binding rather than to global changes in protein structure, since both the Ad2+ND1ts23 and Ad2 DBPs showed identical cleavage patterns by the protease thermolysin at various temperatures.     

Integration and transcription of group C human adenovirus sequences in the DNA of five lines of transformed rat cells

We have used the Southern blotting technique to analyze the integration patterns of human adenovirus sequences in the DNA of four rat cell lines, F17, 8617, T2C4 and F4, which were transformed by Ad-2 virus, and 5RK clone 6, which was transformed by Ad-5 HindIII-G fragment. We have also analyzed the Ad-specific messenger RNAs synthesized in these cell lines, in 293 cells (an Ad-5 transformed human cell line), and in Ad-2 early infected human KB cells, using the RNA geltransfer hybridization technique. We were interested in whether the Ad sequences are integrated, what the integration patterns are, whether the transforming region is present in an intact form, and whether the transforming region and other early regions are expressed at the mRNA level.Our results show that the integration patterns of Ad sequences range from simple to quite complex. Cells from line 8617 contain a single copy of right-end sequences flanked by left-end sequences. T2C4 cells have four different left-end sequences and two different right-end sequences. 5RK cells contain multiple different pieces of left-end sequences. In agreement with the results of Sambrook et al. (1979a,b), F17 cells contain a single copy of the left 17% of the genome, and F4 cells contain multiple copies of the right 5% of the genome fused to the left ~ 68% of the genome. The complete Ad genome is not present in any of the cell lines, and different regions may not be equimolar. There are no specific sites on the cellular or viral genome at which integration occurs. In 8617, F17 and F4 cells the Ad-2 sequences appear to be located close together on a single chromosome, suggesting that the Ad sequences in these cells arose from a single integration event. F17, 8617, T2C4, F4, and probably 5RK, cells all have an intact early region E1a (map position 1·3–4·6); F17, 8617, T2C4 and F4 cells also have E1b (m.p. 4·6–11·2) intact. E1a and E1b are the regions responsible for transformation. 8617 cells also have an intact early region E4 (m.p. 99-91·5) and T2C4 cells have an intact early region E3 (m.p. 76–86).Ad-2 early infected KB cells were shown to synthesize major E1a-specific mRNAs of 13 S, 12 S and 9 S, and major E1b-specific mRNAs of 22 S and 13 S. All the transformed cells synthesize the E1a 13 S and E1a 12 S mRNAs, and all cells except 5RK synthesize the E1b 22 S and E1b 13 S mRNAs. Early infected KB cells synthesize E3-specific mRNAs of 26 S, 24 S, 22 S, 19 S, 12 S and 9 S: T2C4 cells synthesize the major 22 S and 19 S RNA species, and possibly the less pronounced E3 mRNAs. Early infected cells and 8617 cells synthesize E4-specific mRNAs of 19 S, 17 S, 14 S, 12 S, 11 S, 9 S and 8 S. 8617 cells also synthesize E4 mRNAs of about 23 to 24 S and 21 S. F4 cells synthesize 24 S and 19 S hybrid mRNAs that contain both E4 and E1a sequences: these RNAs arise because F4 cells contain a portion of the E4 region fused to the left end (m.p. 0) of the genome.Our results, as well as those from other laboratories, are consistent with the idea that the transformed phenotype of Ad transformed cells is maintained by expression of Ad genes in E1a and E1b.     

Association of adenovirus type 2 early proteins with a soluble complex that synthesizes adenovirus DNA in vitro

A soluble Ad2 DNA synthesizing complex was prepared from Ad2-infected KB cell nuclei and purified by exclusion chromatography on a BioGel A-50m column. The purified complex was able to synthesize DNA from all regions of the virus genome, as indicated by $\text{Eco}$RI restriction endonuclease analysis of $\text{in vitro}$ labeled DNA. Experiments were performed to identify Ad2-induced early polypeptides present in the complex. Ad2-infected and mock-infected cells were labeled with [³⁵S]methionine 7–10 h postinfection, then incubated for 8 h to allow the ³⁵S-labeled early polypeptides to become associated with the complex. The polypeptides in the purified complex and each of the cell fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major components of the purified complex were the 73K DNA binding phosphoprotein and 11K, two adenovirus 2-induced early polypeptides. The 11K has a preferred nuclear location. Small quantities of other Ad2-induced early proteins, 21K, 15K, and possibly 8.3K were also associated with the complex.     

Absence of an essential regulatory influence of the adenovirus E1B 19-kilodalton protein on viral growth and early gene expression in human diploid WI38, HeLa, and A549 cells.

Mutations in the gene encoding the adenovirus (Ad) early region 1B 19-kDa protein (the 19K gene) result in multiple phenotypic effects upon infection of permissive human cells. It has been reported, for example, that Ad type 2 (Ad2) and Ad5 with mutations in the 19K gene (19K-defective mutants) have a marked growth advantage compared with wild-type virus in human diploid WI38 cells (E. White, B. Faha, and B. Stillman, Mol. Cell. Biol. 6:3763-3773, 1986), and it was proposed that this host range phenotype stems from the large increase in viral early gene expression reported to occur in the mutant-infected cells. These observations gave rise to the hypothesis that the 19-kDa protein (the 19K protein) normally functions as a negative regulator of Ad early gene expression and growth. We have tested this hypothesis and find that Ad5 and Ad12 wild-type viruses grow as efficiently as their respective 19K-defective mutants, in1 and dl337 and pm700 and in700, in WI38 and other human cell types. Neither the accumulation of E1A cytoplasmic mRNAs nor the synthesis of E1A and other viral early proteins in these cells is altered as a result of these mutations in the 19K gene, and we conclude that the 19K protein does not play an essential role in regulating viral early gene expression or viral growth in human cells.     

Mouse anti-adenovirus cytotoxic T lymphocytes. Inhibition of lysis by E3 gp19K but not E3 14.7K

Early region E3 of adenovirus (Ad) appears to encode proteins involved in the interaction of the virus with the host immune system. The E3 region 19-kDa glycoprotein (gp19K) binds to class I MHC Ag in the endoplasmic reticulum and inhibits their transport to the cell surface; it has been proposed that this protects virus infected cells from lysis by CTL. We have found that the E3 14.7-kDa protein (14.7K) inhibits lysis of infected cells by TNF, and here we show that it also protects cells from lysis by lymphotoxin, which has been implicated as a mediator of CTL lysis. We have developed a method for producing CTL specific for human Ad2 and Ad5 in mice, in order to test directly which of the genes in the E3 region protect infected cells from lysis by virus specific CTL. The presence of the E3 region inhibits both the induction of Ad-specific CTL in culture and the lysis of infected target cells by these CTL. The inhibition varies between different mouse strains, with almost complete inhibition in C57BL/10 (H-2b) mice, partial inhibition with BALB/c (H-2d) and little or no inhibition with C3H (H-2k); results were similar for Ad2 and Ad5. By using a panel of E3 deletion mutants, inhibition of target cell lysis by Ad5 specific CTL was mapped exclusively to the gp19K gene. The 14.7K gene had no effect on CTL lysis despite its ability to protect cells against lysis by lymphotoxin. gp19K was synthesized abundantly in mouse cells by mutants retaining the gp19K gene; some mutant forms of the protein were synthesized but were nonfunctional. These data support the hypothesis that gp19K can protect Ad infected cells against lysis by virus specific CTL.     

The 19-kDa glycoprotein coded by region E3 of adenovirus. Purification, characterization, and structural analysis

The 19-kDa glycoprotein (gp 19K) coded by early region E3 of adenovirus is of interest as a model for glycoprotein processing and sorting, as well as for the interaction between viral antigens and class I transplantation antigens. In this paper, we show that gp 19K is a major protein synthesized during early stages of infection of human KB cells. We report the purification of gp 19K to near homogeneity, the preparation of a gp 19K antiserum, and structural analyses on the protein. We have determined the DNA sequence of the gp 19K gene in adenovirus type 5 (Ad5) for comparison with the published sequence (Hérissé, J., Courtois, G., and Galibert, F. (1980) Nucleic Acids Res. 8, 2173-2192) of adenovirus type 2 (Ad2). Fragments produced by cyanogen bromide cleavage of Ad2 gp 19K are in accord with the DNA sequence, as are synthetic peptide antibodies targeted to the NH2 terminus of Ad2 gp 19K and the COOH terminus of Ad5 gp 19K. The Ad2 and Ad5 proteins are quite homologous. Conserved features include an NH2-terminal signal sequence, two potential Asn-linked glycosylation sites, and a 20-residue putative transmembrane hydrophobic domain followed by a 15-residue polar domain at the COOH terminus. We show that cleavage of the signal peptide occurs between the 17th and 18th amino acids on both the Ad2 and Ad5 versions of gp 19K and that both potential sites are glycosylated with exclusively high-mannose (as opposed to complex) oligosaccharides. Secondary structure predictions suggest six alpha-helix regions including the signal peptide and transmembrane domain, two or three beta-sheet regions, and about eight beta-turns including the two glycosylation sites and the regions flanking the transmembrane domain.     

Further mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments

RNA isolated from the cytoplasm of human cells at late times after infection by adenovirus type 2 (Ad2) has been fractionated by hybridization to fragments of Ad2 DNA which were produced by digestion with the restriction endonucleases Hpa I, Eco RI, Bam HI and Hind III. Cell-free translation of these partially purified mRNAs indicates that the genes for the late Ad2 proteins lie within the following intervals on the conventional Ad2 map: 15K (4.4–17.0 map units), IX and IVa₂ (7.5–17.0), IIIa (29.1–40.9), III and V (29.1–57.0), pVIII (40.9–57.0), pVI and II (40.9–70.7), 100K (59.0–83.4), pVIII (70.7–83.4) and IV (85.0–100). In addition to the primary hybridization of the late Ad2 mRNAs to the regions indicated above, most late Ad2 mRNAs (except those for 15K, IX and IVa₂) exhibited some hybridization to a secondary site between 17.0 and 29.1 map units.     

Expression of adenovirus type 12 E1b 58-kDa protein in Escherichia coli and production of antibodies raised against a 58-kDa::beta-galactosidase fusion protein

DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector. The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal. Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein. Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells. Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression. We have not been able to detect substantial amounts of the 58-kDa protein in these cells. However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E. coli.     

Adenovirus genes that modulate the sensitivity of virus-infected cells to lysis by TNF

TNF is a key inflammatory cytokine with antiviral properties. Human adenoviruses encode several intracellular proteins that mediate the effects of TNF. Expression of the adenovirus immediate early E1A proteins induces viral genes and a host of cellular genes, drives G₀ cells into S-phase, and induces apoptosis and susceptibility to TNF-induced apoptosis. The adenovirus E1B-19K protein inhibits both E1A- and TNF-induced apoptosis. The E3-14.7K protein and the E3-10.4K/14.5K complex of proteins inhibit TNF- but not E1A-induced apoptosis. The E3 14.7K and 10.4K/14.5K proteins inhibit TNF activation of cytosolic phospholipase A₂ (cPLA₂), which may explain how they inhibit TNF cytolysis. Since eicosinoids produced from arachidonic acid (the product of cPLA₂) are potent mediators of inflammation, the E3 proteins may block the inflammatory response to adenovirus infection. These adenovirus proteins should be novel tools to understand adenovirus pathogenesis, TNF signal transduction, and TNF cytolysis.     

The adenovirus E1b55K/E4orf6 complex induces degradation of the Bloom helicase during infection

The adenovirus (Ad) E1b55K and E4orf6 gene products assemble an E3 ubiquitin ligase complex that promotes degradation of cellular proteins. Among the known substrates are p53 and the Mre11-Rad50-Nbs1 (MRN) complex. Since members of the RecQ helicase family function together with MRN in genome maintenance, we investigated whether adenovirus affects RecQ proteins. We show that Bloom helicase (BLM) is degraded during adenovirus type 5 (Ad5) infection. BLM degradation is mediated by E1b55K/E4orf6 but is independent of MRN. We detected BLM localized at discrete foci around viral replication centers. These studies identify BLM as a new substrate for degradation by the adenovirus E1b55K/E4orf6 complex.     

Sequence of the immunoregulatory early region 3 and flanking sequences of adenovirus type 35

Adenovirus type 35 (Ad35) is an important pathogen in immunosuppressed individuals such as AIDS patients and bone marrow transplant recipients. Ad35, a member of Ad subgroup B, differs with respect to pathogenic properties from the more fully characterized subgroup C Ad, such as Ad2 and Ad5. One region of human Ad which varies between subgroups and which may influence Ad pathogenesis is early region 3 (E3), a region which appears to modulate the immune response to Ad infection. In order to begin to characterize the differences between the Ad35 E3 and the E3 of other Ad, the complete DNA sequence of the Ad35 E3 promoter and coding sequence along with two flanking structural proteins, pVIII and fiber, has been determined. Ad35 contains open reading frames which are unique to the subgroup B Ad in addition to the four characterized immunoregulatory proteins encoded by the subgroup C Ad. Further evaluation of the sequence of one of these proteins, 18.5K, which is the class-I major histocompatibility complex (MHC) binding protein of 18.5 kDa, demonstrates that the amino acid sequence of this Ad2 gp19K homologue fits a proposed model of gp19K-MHC interaction. Analysis of promoter sequences demonstrates that an NF-κB site found in the subgroup C E3 promoter is absent from the Ad35 E3 promoter. In addition, the fiber genes of Ad35 and other subgroup B Ad have been shown to diverge in an unexpected way, yielding three clusters of fiber homology.     

Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA

The adenovirus mutant Ad2ts111 has been previously shown to contain a mutation in the early region 2A gene encoding the single-stranded-DNA-binding protein that results in thermolabile replication of virus DNA and a mutation in early region 1 that causes degradation of intracellular DNA. A recombinant virus, Ad2cyt106, has been constructed which contains the Ad2ts111 early region 1 mutation and the wild-type early region 2A gene from adenovirus 5. This virus, like its parent Ad2ts111, has two temperature-independent phenotypes; first, it has the ability to cause an enhanced and unusual cytopathic effect on the host cell (cytocidal [cyt] phenotype) and second, it induces degradation of cell DNA (DNA degradation [deg] phenotype). The mutation responsible for these phenotypes is a single point mutation in the gene encoding the adenovirus early region 1B (E1B) 19,000-molecular-weight (19K) tumor antigen. This mutation causes a change from a serine to an asparagine in the 20th amino acid from the amino terminus of the protein. Three other mutants that affect the E1B 19K protein function have been examined. The mutants Ad2lp5 and Ad5dl337 have both the cytocidal and DNA degradation phenotypes (cyt deg), whereas Ad2lp3 has only the cytocidal phenotype and does not induce degradation of cell DNA (cyt deg+). Thus, the DNA degradation is not caused by the altered cell morphology. Furthermore, the mutant Ad5dl337 does not make any detectable E1B 19K protein product, suggesting that the absence of E1B 19K protein function is responsible for the mutant phenotypes. A fully functional E1B 19K protein is not absolutely required for lytic growth of adenovirus 2 in HeLa cells, and its involvement in transformation of nonpermissive cells to morphological variants is discussed.     

Anti-tumor immunotherapy despite immunity to adenovirus using a novel adenoviral vector Ad5 [E1-, E2b-]-CEA

Adenovirus serotype 5 (Ad5) has been widely used in clinical trials because it expresses inserted transgenes robustly and augments the innate immune response. Strategies to improve Ad5 vectors that can circumvent Ad5 immunity have become a critical issue, especially for use as a cancer immunotherapeutic in which repeated immunization is required. In this study, we constructed a novel Ad5 vector with unique deletions of the viral DNA polymerase and the pre-terminal protein region (Ad5 [E1-, E2b-]). This vector contains the carcinoembryonic antigen (CEA) gene insert and is designed to induce cell-mediated immunity (CMI) against the tumor-associated target. The CEA immunogenicity and in vivo anti-tumor effects of repeated immunizations with Ad5 [E1-, E2b-]-CEA compared with those observed with current generation Ad5 [E1-]-CEA were tested in Ad5 pre-immunized mice. We report that Ad5-immune mice immunized multiple times with Ad5 [E1-, E2b-]-CEA induced CEA-specific CMI responses that were significantly increased over those detected in Ad5-immune mice immunized multiple times with a current generation Ad5 [E1-]-CEA. Ad5 immune mice bearing CEA-expressing tumors that were treated with Ad5 [E1-, E2b-]-CEA had increased anti-tumor response as compared with Ad5 [E1-]-CEA treated mice. These results demonstrate that Ad5 [E1-, E2b-]-CEA can induce CMI immune responses which result in tumor growth inhibition despite the presence of pre-existing Ad5 immunity. Multiple re-immunizations using the same vector platform are now possible with the novel Ad5 [E1-, E2b-] platform.