Adenovirus type 40 and 41 growth in vitro: host range diversity reflected by differences in patterns of DNA replication.

Subgroup F adenoviruses adapt poorly to cell culture, but the reasons for their fastidious nature are as yet ill defined. In an attempt to gain an overview of the differences in replication between adenovirus type 2 (Ad2) and representative strains of Ad40 and Ad41, cell lines which show different degrees of permissiveness to Ad40 and Ad41 were infected and examined with respect to three key functions in the Ad2 life cycle: host protein shutoff, DNA synthesis, and late antigen synthesis. The complexity of growth patterns exhibited by the subgroup F adenoviruses suggests that defectiveness is a multifactorial phenomenon not easily explainable by a single aberrant function. Furthermore, results suggest that there may be replicative defects in subgroup F adenoviruses which are not shared by both serotypes or by all strains.     

Stability of the structure and antigenic determinants of adenovirus type 1 native hexon to proteases

Hexon capsomers of human adenovirus type 1 (h1) labeled by iodine 125 were digested in a native state (trimers) by trypsin, chymotrypsin or papain, and the resulting hydrolysates were analyzed by SDS-PAGE. In each case, a discrete and temporally stable pattern of relatively large fragments was revealed. The degree of hexon polypeptide hydrolysis was maximal for papain, intermediate for chymotrypsin and minimal for trypsin, the largest fragments in the digest being 32, 40 and 80 kD, respectively. At room temperature, all the electrophoretically discernible hexon proteolytical fragments were held together in structures resembling intact hexon trimers and could be regarded as "hexon cores", of which papain hexon cores were the most stable during SDS-PAGE. Radioimmunoprecipitation analysis revealed a complete absence of native hexon antigenicity in thermodenaturated fragments of hexon protease digests, while native trypsin, chymotrypsin and papain hexon cores could be precipitated by hexon-specific antibodies. The immunoprecipitated material contained all of the hexon fragments found in appropriate hexon cores and retained the structure of the original cores. Trypsin, chymotrypsin and papain hexon cores were shown to possess at least part of native Ad h1 hexon antigenic determinants of each of the following specificities: species-specific (epsilon), cross-reactive with hexon of human adenoviruses (h3 and h6), simian adenovirus (sim 16), bovine adenoviruses (bos 3 and bos 7) and avian adenovirus (Aviadenovirus gal 1 or CELO). Thus, the full spectrum of known hexon antigenic determinants (species-specific to intergenus-crossreactive) is at least portly stable against protease attack of native hexon capsomers.     

Physical organization of subgroup B human adenovirus genomes.

Cleavage sites of nine bacterial restriction endonucleases were mapped in the DNA of adenovirus type 3 (Ad3) and Ad7, representative serotypes of the "weakly oncogenic" subgroup B human adenoviruses. Of 94 sites mapped, 82 were common to both serotypes, in accord with the high overall sequence homology of DNA among members of the same subgroups. Of the sites in Ad3 and Ad7 DNA, fewer than 20% corresponded to mapped restriction sites in the DNA of Ad2 or Ad5. The latter serotypes represent the "nononcogenic" subgroup C, having only 10 to 20% overall sequence homology with the DNA of subgroup B adenoviruses. Hybridization mapping of viral mRNA from Ad7-infected cells resulted in a complex physical map that was nearly identical to the map of early and late gene clusters in Ad2 DNA. Thus the DNA sequences of human adenoviruses of subgroups B and C have significantly diverged in the course of viral evolution, but the complex organization of the adenovirus genome has been rigidly conserved.     

Subgroup B and F fiber chimeras eliminate normal adenovirus type 5 vector transduction in vitro and in vivo

Altering adenovirus vector (Ad vector) targeting is an important goal for a variety of gene therapy applications and involves eliminating or reducing the normal tropism of a vector and retargeting through a distinct receptor-ligand pathway. The first step of Ad vector infection is high-affinity binding to a target cellular receptor. For the majority of adenoviruses and Ad vectors, the fiber capsid protein serves this purpose, binding to the coxsackievirus and adenovirus receptor (CAR) present on a variety of cell types. In this study we have explored a novel approach to altering Ad type 5 (Ad5) vector targeting based on serotypic differences in fiber function. The subgroup B viruses bind to an unidentified receptor that is distinct from CAR. The subgroup F viruses are the only adenoviruses that express two distinct terminal exons encoding fiber open reading frames. We have constructed chimeric fiber adenoviruses that utilize the tandem fiber arrangement of the subgroup F genome configuration. By taking advantage of serotypic differences in fiber expression, fiber shaft length, and fiber binding efficiency, we have developed a tandem fiber vector that has low binding efficiency for the known fiber binding sites, does not rely on an Ad5-based fiber, and can be grown to high titer using conventional cell lines. Importantly, when characterizing these vectors in vivo, we find the subgroup B system and our optimal tandem fiber system demonstrate reduced liver transduction by over 2 logs compared to an Ad5 fiber vector. These attributes make the tandem fiber vector a useful alternative to conventional strategies for fiber manipulation of adenovirus vectors.     

The 100K-chaperone protein from adenovirus serotype 2 (Subgroup C) assists in trimerization and nuclear localization of hexons from subgroups C and B adenoviruses

Recombinant hexons from subgroup C adenoviruses (Ad2 and Ad5) and from a member of subgroup B (Ad3) adenoviruses have been expressed in insect cells. When expressed alone, all three hexons were found to be insoluble and accumulated as inclusion bodies in the cytoplasm. However, co-expression of recombinant Ad2, Ad5 or Ad3 hexon with Ad2 L4-100K protein resulted in the formation of soluble trimeric hexons. EM analysis of hexons revealed that they were indistinguishable from native hexon capsomers isolated from Ad2-infected human cells, or released from partially disrupted adenovirions. This suggests that 100K acts as a chaperone for hexon folding and self-assembly into capsomer in insect cells. Since 100K protein assists in the trimerization of subgroup C hexon, and of subgroup B hexon protein, it implies that it functions in a manner that is both homo- and heterotypic. During the course of recombinant protein expression, the 100K protein was found in association with hexon monomers and trimers within the cytoplasm. In the nucleus, however, 100K was found in complexes with hexon trimers exclusively. EM observation of purified 100K protein samples showed a dumb-bell-shaped molecule compatible with a monomeric protein. EM analysis of hexon-100K protein complexes showed that interaction of hexon with the 100K protein occurred via one of the globular domains of the 100K protein molecule. Our data confirm the role of the 100K protein as a scaffold protein for hexon, and provide evidence suggesting its function in hexon nuclear import in insect cells.     

Genetic analysis of adenovirus type 2. VIII. Physical locations of temperature-sensitive mutations.

Temperature-sensitive mutations of human adenoviruses can be physically located on the viral genome by determining the DNA structures of recombinants formed in genetic crosses between different members of the same subgroup. We have analyzed the DNA structures of many interserotypic recombinants formed in crosses between temperature-sensitive (ts) mutants of adenovirus type 2 and adenovirus type 5 with the restriction endonucleases BamHI, EcoRI, HindIII, and and Sma I. In this way, we have mapped the physical coordinates of adenovirus type 2 (Ad2) ts1, Ad2 ts3, Ad2 ts4, and Ad2 ts48, and refined the mapping of Ad5 ts1.     

Pseudopackaging of adenovirus type 5 genomes into capsids containing the hexon proteins of adenovirus serotypes B, D, or E

Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major challenge in the development of Ad vectors is the circumvention of the host immune responses to Ad infection, including both the host cytotoxic T-cell response and the humoral response resulting in neutralizing antibodies. One method to circumvent the effect of neutralizing antibodies against an Ad vector is to use different Ad serotypes to deliver the transgene of interest. This approach has been demonstrated with Ad genomes of highly related members of subgroup C. However, it is not known whether an Ad5-based vector DNA molecule can be packaged into capsids of evolutionarily more divergent adenoviruses. The aim of these studies was to determine if capsids containing hexon proteins from other Ad subgroups could package the Ad5 genome. A genetic approach utilizing an Ad5 temperature-sensitive (ts) mutant with a mutation in the hexon protein was used. When grown at the nonpermissive temperature, Ad5 ts147 replicates normally, providing a source of Ad5 DNA for virus assembly, but does not produce virus particles due to the hexon protein mutation. Coinfection of Ad5 ts147 with a wild-type virus of other Ad serotypes (Ad3, Ad4, or Ad9), which supply functional hexon proteins, resulted in the pseudopackaging of the Ad5 DNA genome. Furthermore, the pseudopackaged Ad5 DNA virions obtained in the coinfections were infectious. Therefore, switching hexons did not impair the infectivity or uncoating process of the pseudopackaged virion. Since hexon protein is a major antigenic determinant of the Ad capsid, this approach may prove useful to reduce the antigenicity of therapeutic Ad vectors and allow repeated vector administration.     

Expression of the chloramphenicol acetyl transferase gene in human cells under the control of early adenovirus subgroup C promoters: effect of E1A gene products from other subgroups on gene expression

A hierarchy of dominance has been observed in HeLa cells co-infected with two serotypes of adenovirus belonging to different subgroups. DNA replication and late protein synthesis of one serotype are inhibited by those of the other. The degree of inhibitory effect has the following decreasing order: adenovirus type 3 (Ad3) and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad12 (A), Ad2 and Ad5 (C) [Delsert and D'Halluin, Virus Res. 1 (1984) 365-380]. HeLa cells were first transfected with recombinant plasmids carrying Ad5 E2A or E3 promoters fused to the chloramphenicol acetyl transferase gene (cat), and then infected with human Ad belonging to different subgroups. All the serotypes tested were found to be able to stimulate both E2A and E3 promoters. When HeLa cells were co-transfected with either of the previous plasmids, plus a second plasmid carrying the Ad3 E1A region, the same stimulatory effect was observed. However, an inhibitory effect on Ad5 E2A and E3 promoters seemed to occur when both Ad2 E1A (subgroup C) and Ad3 E1A (subgroup B) genes were present together. To determine which one of the early products was responsible for the observed repression effect, and to assign the target on the genome of subgroup C Ad, a plasmid was constructed in which the sequences at the 5' end of the Ad2 E1A region were fused to the structural sequences of the cat gene. In HeLa cells transfected with this plasmid, CAT activity was significantly increased after co-transfection with a plasmid carrying the Ad2 E1A region, but decreased with a plasmid carrying the Ad3 E1A region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tropism modification of adenovirus vectors by peptide ligand insertion into various positions of the adenovirus serotype 41 short-fiber knob domain

Recombinant adenoviruses have emerged as promising agents in therapeutic gene transfer, genetic vaccination, and viral oncolysis. Therapeutic applications of adenoviruses, however, would benefit substantially from targeted virus cell entry, for example, into cancer or immune cells, as opposed to the broad tropism that adenoviruses naturally possess. Such tropism modification of adenoviruses requires the deletion of their natural cell binding properties and the incorporation of cell binding ligands. The short fibers of subgroup F adenoviruses have recently been suggested as a tool for genetic adenovirus detargeting based on the reduced infectivity of corresponding adenovectors with chimeric fibers in vitro and in vivo. The goal of our study was to determine functional insertion sites for peptide ligands in the adenovirus serotype 41 (Ad41) short fiber knob. With a model peptide, CDCRGDCFC, we could demonstrate that ligand incorporation into three of five analyzed loops of the knob, namely, EG, HI, and IJ, is feasible without a loss of fiber trimerization. The resulting adenovectors showed enhanced infectivity for various cell types, which was superior to that of viruses with the same peptide fused to the fiber C terminus. Strategies to further augment gene transfer efficacy by extension of the fiber shaft, insertion of tandem copies of the ligand peptide, or extension of the ligand-flanking linkers failed, indicating that precise ligand positioning is pivotal. Our study establishes that internal ligand incorporation into a short-shafted adenovirus fiber is feasible and suggests the Ad41 short fiber with ligand insertion into the top (IJ loop) or side (EG and HI loops) of the knob domain as a novel platform for genetic targeting of therapeutic adenoviruses.     

Comparative transduction efficiencies of human and nonhuman adenoviral vectors in human, murine, bovine, and porcine cells in culture

Clinical usefulness of human Ad serotype 5 (HAd5) based vectors is limited primarily because of preexisting Ad immunity and lack of targeting to specific cell types. Alternative vectors based on less prevalent HAd serotypes as well as nonhuman adenoviruses such as porcine Ad serotype 3 (PAd3) and bovine Ad serotype 3 (BAd3) are being developed to overcome these shortcomings. Using virus neutralization assay, we examined whether preexisting Ad immunity in humans would cross-neutralize PAd3 or BAd3. To further evaluate the potential of PAd3 and BAd3 vectors as gene delivery vehicles, we compared their transduction efficiencies in a panel of human, murine, bovine, and porcine cell lines to those obtained with a HAd5 vector. Transduction by the HAd5 vector in the majority of human cell lines correlated with the expression levels of coxsackievirus-adenovirus receptor (CAR), the primary HAd5 receptor; while transduction by PAd3 and BAd3 vectors was CAR-independent. The results suggest that PAd3 and BAd3 vectors are promising gene delivery vehicles for human gene therapy as well as for recombinant vaccines for human and animal use.     

Specific properties of two enteric adenovirus 41 clones mapped within early region 1A.

Enteric adenovirus type 40 (Ad40) and Ad41 form the sixth subgenus of human adenoviruses. They are associated with infantile diarrhea but cannot be isolated in conventional cell cultures. The genome of the fastidious enteric Ad41 has been cloned, and the cleavage sites of the genome produced by restriction endonucleases BamHI, EcoRI, HpaI, NruI, PvuI, and SalI have been mapped. To develop useful hybridization methods for direct detection of adenoviruses, a restriction fragment library containing Ad41 DNA, with plasmid pBR322 as vector, has been constructed. Clones have been isolated which contain 8 of 10 possible BamHI fragments of Ad41, inserted into the BamHI cleavage site of the vector. Two of these clones are particularly useful for the detection of adenoviruses. One clone detects members of all human adenovirus subgenera, and the second clone is specific for enteric adenoviruses, in particular Ad41. A conspicuous absence of detectable homology was noted at 1.5 to 3.3 map units of the Ad41 genome in hybridizations against other serotypes of adenoviruses, including the closely related enteric Ad40. This sequence corresponds to the 5' portion of early region Ia.     

Adenovirus type 11 uses CD46 as a cellular receptor

The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type 11 (Ad11; of species B) binds to Chinese hamster ovary (CHO) cells transfected with CD46 (membrane cofactor protein)-cDNA at least 10 times more strongly than to CHO cells transfected with cDNAs encoding CAR or CD55 (decay accelerating factor). Nonpermissive CHO cells were rendered permissive to Ad11 infection upon transfection with CD46-cDNA. Soluble Ad11 fiber knob but not Ad7 or Ad5 knob inhibited binding of Ad11 virions to CD46-transfected cells, and anti-CD46 antibodies inhibited both binding of and infection by Ad11. From these results we conclude that CD46 is a cellular receptor for Ad11.     

Detection of bovine and porcine adenoviruses for tracing the source of fecal contamination

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.     

Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.     

Transforming genes among three different oncogenic subgroups of human adenoviruses have similar replicative functions.

We have examined the functional similarity of the transforming genes for replicative functions among three different subgroups of human adenoviruses (A, B, and C), using mutant complementation as an assay. A host range deletion mutant (dl201.2) of Ad2 (nononcogenic subgroup C) lacking about 5% of the viral DNA covering two early gene blocks (E1a and E1b) involved in cellular transformation was isolated and tested for its ability to replicate in nonpermissive KB cells in the presence of Ad7 (weakly oncogenic group B) or ad12 (highly oncogenic group A). The complementation of the mutant defect was demonstrated by cleaving the viral DNA extracted from mixed infected cells or the DNA extracted from purified virions from mixed infected cells with restriction endonuclease BamHI, which produces a different cleavage pattern with the DNA of each serotype. It was found that the defects in E1a plus E1b of dl201.2 could be complemented by Ad7 and Ad12, indicating that these genes in Ad2, Ad7, and Ad12 have similar functions during productive infection.     

Role of cellular heparan sulfate proteoglycans in infection of human adenovirus serotype 3 and 35

Species B human adenoviruses (Ads) are increasingly associated with outbreaks of acute respiratory disease in U.S. military personnel and civil population. The initial interaction of Ads with cellular attachment receptors on host cells is via Ad fiber knob protein. Our previous studies showed that one species B Ad receptor is the complement receptor CD46 that is used by serotypes 11, 16, 21, 35, and 50 but not by serotypes 3, 7, and 14. In this study, we attempted to identify yet-unknown species B cellular receptors. For this purpose we used recombinant Ad3 and Ad35 fiber knobs in high-throughput receptor screening methods including mass spectrometry analysis and glycan arrays. Surprisingly, we found that the main interacting surface molecules of Ad3 fiber knob are cellular heparan sulfate proteoglycans (HSPGs). We subsequently found that HSPGs acted as low-affinity co-receptors for Ad3 but did not represent the main receptor of this serotype. Our study also revealed a new CD46-independent infection pathway of Ad35. This Ad35 infection mechanism is mediated by cellular HSPGs. The interaction of Ad35 with HSPGs is not via fiber knob, whereas Ad3 interacts with HSPGs via fiber knob. Both Ad3 and Ad35 interacted specifically with the sulfated regions within HSPGs that have also been implicated in binding physiologic ligands. In conclusion, our findings show that Ad3 and Ad35 directly utilize HSPGs as co-receptors for infection. Our data suggest that adenoviruses evolved to simulate the presence of physiologic HSPG ligands in order to increase infection.     

Phylogenetic relationships between adenoviruses as inferred from nucleotide sequences of inverted terminal repeats

The nucleotide (nt) sequences of inverted terminal repeats (ITR) from human adenovirus (Ad) 19, bovine Ad1 (BAd1), bovine Ad3 (BAd3), canine Ad2 (CAd2) and an avian Ad, EDS-76, were determined. The length of the ITR sequence was 160 bp in Ad19, 159 bp in BAd1, 195 bp in BAd3, 196 bp in CAd2 and 52 bp in EDS-76. CAd2 had the longest ITR among the examined Ads, BAd3 the second longest, and EDS-76 had the shortest ITR. A TAAT sequence located between the 10th and 13th nt counted from the ends was conserved in all Ads examined so far. To determine phylogenetic relationships among human and animal Ads, sequences of their ITRs were compared, and a phylogenetic tree was constructed by using the maximum-likelihood method. It is the method involving statistical analysis of computing the probability of a particular set of sequences on a given tree and maximizing this probability over all evolutionary trees [Felsenstein, J. Mol. Evol. 17 (1981) 368-376]. From these analyses, it was found that members belonging to the same human Ad subgenus are related closely to each other, whereas representatives of different human subgenera are distributed rather divergently among animal Ads.     

CD/TK单、双自杀基因重组腺病毒的制备及初步应用

为制备表达大肠杆菌胞嘧啶脱氨酶 (cytosinedeaminase ,CD)和单纯疱疹病毒 1 胸苷激酶(herpessimplexvirustype 1thymidinekinase ,TK)双自杀基因的重组腺病毒载体 ,为再狭窄基因治疗的应用奠定基础 ,首先构建了含单、双自杀基因的腺病毒穿梭载体 ,细菌内同源重组法获得重组腺病毒质粒 .脂质体介导下转染 2 93细胞进行包装并大量扩增得到Ad TK、Ad CD、Ad TK CD、Ad LacZ 4种重组腺病毒 .氯化铯 (CsCl)梯度离心法纯化后利用报告基因绿色荧光蛋白 (GFP)测定病毒滴度 ,可达 10 9pfu ml .PCR和Western印迹法对外源基因进行鉴定 ,证明单、双自杀基因均已整合入腺病毒基因组并表达 .重组腺病毒感染血管平滑肌细胞后 ,用MTT法比较了单、双自杀基因的杀伤作用 ,发现在感染复数≤ 2 0时 ,双自杀基因对细胞的抑制作用明显高于单基因 .成功地构建腺病毒双自杀基因共表达载体 ,为进一步的体内外实验奠定了基础 .