Nucleotide sequence of the adenovirus type 40 inverted terminal repeat: close relation to that of adenovirus type 5

Human adenovirus type 40 (Ad40) is a pathogen that causes acute infantile gastroenteritis. Ad40 has the distinct characteristic of being difficult to propagate in conventional cultured human cells. The nucleotide sequence of the inverted terminal repeat (ITR) of Ad40, which includes the origin of adenoviral DNA replication, was determined using recombinant plasmid DNA. By using our newly developed program to express the ITR homologies simply, we found that the ITR of Ad40, which is 163 nucleotides long, was related most closely to that of adenovirus type 5, which replicates efficiently.     

Replication of an adenovirus type 34 mutant DNA containing tandem reiterations of the inverted terminal repeat

A mutant of human adenovirus type 34 (Ad34) has been isolated which contains DNA molecules with tandem reiterations of from two to eight copies of a 131-bp sequence within the right-sided inverted terminal repetition. Terminal heterogeneity was not eliminated by repeated plaque purifications indicating that the population of DNA molecules with various numbers of reiterations could rapidly evolve from the DNA of a single virus particle. These enlarged DNA molecules were capable of replication both in vivo and in vitro. The nucleotide sequence of the mutant Ad34 inverted terminal repetitions contained most of the essential features of the Ad origin of DNA replication. These features include the ATAATATACC sequence which is present between the highly conserved bases 9-18 in all human adenoviruses, as well as the consensus sequences for the binding of nuclear factor I and nuclear factor III. However, the reiterated sequences lacked a dG appropriately placed on the template strand to serve as a potential site for internal initiation. It appears that the rapid amplification of two to eight copies of the reiterated terminal sequences does not arise from internal initiation during replication but probably from homologous recombination.     

In vivo and in vitro phosphorylation of the adenovirus type 5 single strand-specific DNA-binding protein.

Electron microscopy of thin sections of carnation (Dianthus caryophyllus L.) leaves, infected with carnation yellow fleck virus (CYFV), revealed CYFV particles in epidermis, ground parenchyma, and phloem and in mature xylem elements. Sections of CYFV aggregates revealed three distinctive characteristic patterns: (1) cross sections of hexagonal arrays with a center-to-center distance of 109 Å; (2) sections of nearly parallel virus particles; and (3) regions in which no orderly arrangement was evident. Tilting experiments showed that the different appearance of the sections was due only to their orientation and that the characteristic patterns are entirely interchangeable. The results of such tilting experiments lead us to suggest that in vivo the virus aggregates in a hexagonal array.     

Multiple reiteration of a 40-bp nucleotide sequence in the inverted terminal repeat of the genome of a canine adenovirus

The DNA of a vaccine strain of canine adenovirus type 1 [ICHV vaccine; Connaught Laboratories, Ltd.; CAV-1(CLL)] has been cloned in plasmid pAT153 in the form of subgenomic BamHI digestion fragments. Analysis of the nucleotide sequences of cloned terminal fragments has revealed an inverted terminal repeat (ITR) with a minimum length of 198 nucleotides, including a tandem reiteration of the 40-bp nucleotide sequence from positions 14 to 53. The ITRs had the 5'-CATCATCAAT ... sequence typical of adenoviruses and the highly conserved sequence ATAATATAC (nucleotides 9-17) of human strains. Additionally, one BamHI A clone (left terminus) contained three sequential copies of the 40-bp sequence, and two BamHI C clones (right terminus) contained at least seven. These did not appear to be artifacts of cloning, since evidence was obtained that the multiple reiterations also occurred in DNA isolated from intact virus. By analogy with human adenoviruses, the repetitive sequence in the CAV-1(CLL) genome encompasses the entire nuclear factor I (NFI) binding site of the origin of DNA replication. Additionally, the 40-bp nucleotide sequence was found to contain the sequence AGG(N)4GCCTAA (nucleotides 27-39), which closely resembles the concensus sequence of the human adenovirus NFI binding site [TGG(N)6-7GCCAA; nucleotides 25-38]. It appears, therefore, that the Connaught CAV-1 vaccine contains reiterated copies of an essential part of the adenoviral origin of DNA replication. A mechanism is proposed for the generation of multiple reiterations of sequences in the right ITR, given an initial single tandem repeat in the left ITR.     

Host-range mutants of adenovirus type 5 defective for growth in HeLa cells

Host-range mutants of adenovirus type 5 have been selected on human embryo kidney cells transformed by sheared adeno 5 DNA (293 cells). These mutants grow well on 293 cells, but are restricted on HeLa cells. By complementation tests on HeLa cells, these mutants were classified into two groups, neither of which was found to correspond to any of the 17 temperature-sensitive complementation groups. Members of the two complementation groups of host-range mutants differ in their host range on other cell types: Members of one group grow only on 293 cells, while those of the other group on both normal and transformed human embryo kidney cells. In the second group, cell-associated adeno gene functions are not needed for mutant growth, but, with the other group, it is possible that an adeno gene product made in the transformed cell complements the growth of the mutants. Recombination tests carried out with the host-range mutants and Ad5 temperature-sensitive mutants indicate that the host-range mutations map within the left half of the genetic map and that some (hr 1 and hr 2) probably lie very close to the left end of the map. The results presented in this communication are briefly discussed in relation to recent unpublished work concerning the biochemical characteristics of the mutants and the transformed 293 cells and to the role of adeno genes in transformation.     

Vaccinia virus homologues of the shope fibroma virus inverted terminal repeat proteins and a discontinuous orf related to the tumor necrosis factor receptor family

Nucleotide sequencing data from a region extending 35 kb inward from the right inverted terminal repeat (ITR) of the vaccinia virus (VV) genome established the presence of VV homologues of the Shope fibroma virus (SFV) ITR proteins. The nucleotide sequences, comprising a total of 8.6 kb, and the amino acid translations for nine predicted open reading frames (ORFs) (designated SalF4L, SalF 19R, SalF21R, B4R, B8R, B9R, B10R, and B14R) are presented. Eight of the nine VV genes and all the SFV ORFs are transcribed towards their genomic termini. However, the relative positions of the VV genes (genus Orthopoxvirus) are different than those of the corresponding ORFs in SFV (genus Leporipoxvirus), indicating complex rearrangements of DNA in the genome of one or both of these viruses subsequent to their divergence from a common ancestor. Several other features of the VV ORFs were noted. SalF4L, B7R, B8R, and B9R have hydrophobic amino-terminal signal sequences but lack discernible membrane anchor domains suggesting that the proteins may be secreted. VV ORF SalF19R has a single cysteine-rich region homologous to the multiple domains of nerve growth factor receptor (NGFR), CD40, OX40 (a glycoprotein from the surface of activated murine T lymphocytes), and the recently described tumor necrosis factor receptors. Just downstream of the ORF SalF19R and in a different reading frame, there are another two related cysteine-rich domains, indicating that SalF19R was once a larger gene. B4R has homology to the host range gene of cowpox virus and to related genes near the opposite end of the vaccinia virus genome, and contains regions homologous to the repeat domains of erythrocyte ankyrin. In addition, several of the VV ORFs have homology to ORFs from near the opposite end of the VV genome, thus increasing the number of known VV gene families.     

Sequence and repeat structure variants in the long terminal repeat of maedi-visna virus EV1

Diversity in the LTR of maedi-visna virus strain EV1 has been examined by PCR-based gene amplification using DNA from infected cells both in vitro and in experimentally infected animals. In vitro, several variant structures were found in the U3 regions of the LTR which contained repeats of sequences including presumed AP-1 and AP-4 binding sites. Although these repeat variants formed a minor fraction of the LTRs present in the proviral population, they were neither produced nor lost at a significant rate when PCR was performed on cloned viral DNA and so were unlikely to be artefacts of the isolation procedure. When LTRs were isolated from two experimentally EV1 infected sheep, repeat variant structures were found to be present in efferent lymph by 14 days postinfection (p.i.) (although not seen at 9 days p.i.). They were also present at later times and in blood. Overall sequence diversity at 9 days p.i. was reduced compared both with the infecting virus and with later times of infection. When a number of the variant LTR structures were used to drive CAT reporter gene constructs in chondrocytes, all were found to be active, although consistent differences of up to fourfold in activity were seen. However, there is no evidence from these data for strong selective pressure operating on the LTR in vivo.     

Linker mutation scanning of the genes encoding the adenovirus type 5 terminal protein precursor and DNA polymerase

The replication of adenovirus DNA requires, in addition to several host factors, three virus-encoded proteins: a DNA binding protein, the precursor of the terminal protein (pTP), and a DNA polymerase (Ad pol). Ad pol and pTP form a tight complex that is necessary for the initiation step in DNA replication. To perform mutation scanning of the adenovirus type 5 pTP and Ad pol a series of in-frame linker insertions of a 12-mer oligonucleotide d(CCCATCGATGGG) were introduced into cloned viral DNA fragments containing coding sequences of these proteins. The insertions are located at recognition sites for several blunt end-cutting restriction endonucleases. Forty different sites were mutagenized and the mutated genes were transferred to a plasmid that contains the left 42% of the adenovirus genome. They were rebuilt into the viral genome by means of in vivo recombination between plasmid DNA and digested adenovirus DNA-TP complex. The resulting viral genomes were tested for viability and rescued virus was analyzed for the presence of the inserted linker oligonucleotide. This procedure resulted in recovery of a number of viable virus mutants with insertions in the pTP or Ad pol genes, all of which are phenotypically silent. The other mutations did not allow virus production. The positions of these apparent lethal codon insertion mutations were useful to identify regions of functional importance in both proteins. It can be concluded that the precursor-specific region of pTP plays an important role in virus multiplication.     

Heparan sulfate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell interactions

Gene therapy vectors derived from subgroup C adenoviruses of the serotype 5 (Ad5) and 2 (Ad2) resulted in inefficient infection of well differentiated respiratory cells, both in vitro and in vivo. The level of expression and localization of the primary receptor for Ad5 and Ad2, termed CAR, do not completely explain why the infection efficiency varies greatly in different experimental conditions. The possibility that additional receptors like proteoglycans are involved in the infection of Ad5 and Ad2 was investigated, because several pathogenic microorganisms use heparan sulfate-glycosaminoglycans (HS-GAGs) as coreceptors for multistep attachment to target cells. The HS-GAG analog heparin decreased Ad5- and Ad2-mediated infection and binding starting from the concentration of 0.1 microgram/ml, up to a maximum of 50%. A similar reduction in Ad5 binding and infection was obtained by treatment of cells with heparin lyases I, II, and III but not with chondroitin ABC lyase. The effect of heparin on Ad5 binding has not been observed in surface GAG-defective Raji cells and after treating A549 cells with heparin lyases I, II,and III. The binding of Ad5 was completely abolished when both CAR was blocked with RmcB antibody and HS-GAGs were competitively inhibited by heparin. Parallel experiments demonstrate that HS-GAGs are irrelevant to binding and infection of the subgroup B adenovirus type 3. Collectively, these results demonstrate for the first time that HS-GAGs expressed on the cell surface are involved in the binding of Ad5 and Ad2 to host cells.     

Individual Mycobacterium tuberculosis resuscitation-promoting factor homologues are dispensable for growth in vitro and in vivo

Mycobacterium tuberculosis possesses five genes with significant homology to the resuscitation-promoting factor (Rpf) of Micrococcus luteus. The M. luteus Rpf is a secreted approximately 16-kDa protein which restores active growth to cultures of M. luteus rendered dormant by prolonged incubation in stationary phase. More recently, the Rpf-like proteins of M. tuberculosis have been shown to stimulate the growth of extended-stationary-phase cultures of Mycobacterium bovis BCG. These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function of M. tuberculosis Rpf homologues in vivo. To address these questions, we have disrupted each of the five rpf-like genes in M. tuberculosis Erdman, and analyzed the mutants for their growth in vitro and in vivo. In contrast to M. luteus, for which rpf is an essential gene, we find that all of the M. tuberculosis rpf deletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection. Analysis of rpf expression in M. tuberculosis cultures from early log phase through late stationary phase indicates that expression of the rpf-like genes is growth phase-dependent, and that the expression patterns of the five M. tuberculosis rpf genes, while overlapping to various degrees, are not uniform. We also provide evidence that mycobacterial rpf genes are expressed in vivo in the lungs of mice acutely infected with virulent M. tuberculosis.     

Kinetics of recombinant adenovirus type 5, vaccinia virus, modified vaccinia ankara virus, and DNA antigen expression in vivo and the induction of memory T-lymphocyte responses

While a new generation of vaccine vectors has been developed for eliciting cellular immune responses, little is known about the optimal routes for their administration or about the ramifications of the kinetics of in vivo vaccine antigen expression for immunogenicity. We evaluated the kinetics of vaccine antigen expression by real-time in vivo photon imaging and showed dramatic differences in these kinetics using different vectors and different routes of administration. Further, using a gamma interferon enzyme-linked immunospot assay to measure T-lymphocyte immune responses, we observed an association between the kinetics of vaccine antigen expression in vivo and the magnitude of vaccine-elicited memory T-lymphocyte responses. These results highlight the utility of the real-time in vivo photon-imaging technology in evaluating novel immunization strategies and suggest an association between the kinetics of vaccine antigen clearance and the magnitude of vaccine-elicited T-lymphocyte memory immune responses.     

A host range mutant of human adenovirus type 5 defective for growth in hamster cells

A host-range mutant of adenovirus type 5, which grows in human cells but not in hamster cells, has been isolated. This mutant is complemented in mixed infection in hamster cells at 38.5 degrees C by temperature-sensitive mutants of type 5 belonging to seventeen complementation groups, and may constitute a new group. In mixed infection of human cells at 32 degrees C with the host-range and temperature-sensitive mutants, recombination takes place and by a series of two factor crosses the host-range mutation has been approximately located on the adenovirus genetic map.