Physical mapping of a large-plaque mutation of adenovirus type 2.

We have developed a simple method based on cotransfection of overlapping DNA restriction fragments for construction of recombinants of adenovirus type 2 (Ad2) and Ad5. When Ad2 DNA digested with restriction endonuclease EcoRI was cotransfected with Ad5 DNA digested with SalI, recombination occurred between Ad2 EcoRI-A (map position 0 to 59) and Ad5 SalI-A (map position 45 to 100). Analysis of the recombinant DNAs by digestion with EcoRI or BamHI restriction endonucleases indicated that, as expected, recombination had occurred in overlapping sequences (map position 45 to 59) between the Ad2 EcoRI-A fragment and the Ad5 SalI-A fragment. By using this method, several recombinants were constructed between a large-plaque (lp) mutant of Ad2 and wild-type Ad5. Cleavage of the recombinant genomes with restriction endonucleases BamHI, EcoRI, and HindIII revealed that the lp mutation is located within the left 41% of Ad2 genome.     

Physical mapping of adenovirus type 2 temperature-sensitive mutations by restriction endonuclease analysis of interserotypic recombinants

The genome structures of about 100 interserotypic ts recombinants produced in crosses between human adenovirus type 2 (H2) and 5 (H5) temperature-sensitive mutants were analyzed by cleavage with restriction endonucleases to determine the map coordinates of the following temperature-sensitive mutants: penton base plus fiber-defective H2 ts103, -104, and -136, assembly-defective H2 ts112, fiber-defective H2 ts125, hexon-defective H2 ts118 and -121, and DNA-negative H2 ts111. H5 ts1 (100 K defective), H5 ts36 (DNA negative), H5 ts125 (mutated in the early 72,000-dalton protein), H5 ts22 (fiber defective), H5 ts58 (IIIa defective), and H5 ts18 and -19 were used as one of the parents. The physical locations of the H2 temperature-sensitive mutations thus defined are discussed in relation to the genetic map, the biological function altered, and the positions of the structural genes on the genome.     

Identification of adenovirus 2 early genes required for induction of cellular DNA synthesis in resting hamster cells.

tsAF8 cells are temperature-sensitive (ts) mutants of BHK-21 cells that arrest at the nonpermissive temperature in the G1 phase of the cell cycle. When made quiescent by serum restriction, they can be stimulated to enter the S phase by 10% serum at 34 degrees C, but not at 40.6 degrees C. Infection by adenovirus type 2 or type 5 stimulates cellular DNA synthesis in tsAF8 cells at both 34 and 40.6 degrees C. Infection of these cells with deletion Ad5dl312, Ad5dl313, Ad2 delta p305, and Ad2+D1) and temperature-sensitive (H5ts125, H5ts36) mutants of adenovirus indicates that the expression of both early regions 1A and 2 is needed to induce quiescent tsAF8 cells to enter the S phase at the permissive temperature. This finding has been confirmed by microinjection of selected adenovirus DNA fragments into the nucleus of tsAF8 cells. In addition, we have shown that additional viral functions encoded by early regions 1B and 5 are required for the induction of cellular DNA synthesis at the nonpermissive temperature.     

Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis.

A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late vaccinia virus promoter. The recombinant viruses were used for overexpression of the mutant genes in HeLa cells. In total, 22 different mutant pTP and 10 different Ad pol vaccinia virus recombinants were constructed, including some that expressed carboxyl-terminus-truncated forms of both proteins and one that produced the mutant H5ts149 Ad pol. To investigate the structure-function relationships of both proteins, extracts from cells infected with the recombinant viruses were tested for in vitro complementation of the initiation and elongation steps in adenovirus DNA replication. The results were in accordance with those of earlier in vivo experiments with these insertion mutants and indicate that multiple regions of both proteins are essential for adenovirus DNA replication. The carboxyl termini of both pTP and Ad pol were shown to be essential for proper functioning of these proteins during initiation of adenovirus DNA replication. Three different DNA replication-negative pTP mutants were shown to have residual activity in the initiation assay, suggesting not only that pTP is required for initiation but also that it may play a role in DNA replication after the deoxycytidylation step.     

Purification and functional characterization of adenovirus ts111A DNA-binding protein. Fluorescence studies of protein-nucleic acid binding

The adenovirus single-stranded DNA (ssDNA)-binding protein (DBP) is necessary for the elongation step in viral DNA replication. In an attempt to characterize the putative ssDNA-binding domain of the DBP, we purified and characterized the Ad2ts111A DBP, which contains a glycine-to-valine substitution at amino acid 280. This mutation is adjacent to that in the previously studied Ad2+ND1ts23. Ad2+ND1ts23 exhibits a temperature-sensitive defect in DNA replication, and its DBP has previously been shown to bind ssDNA with reduced affinity. Ad2ts111A DBP, like Ad2+ND1ts23, does not support adenovirus DNA replication in vitro at elevated temperatures. However, the Ad2ts111A DBP binds ssDNA more tightly than does Ad2+ND1ts23 and is not temperature sensitive in this function. To determine the nucleic acid-binding properties of DBP, we applied spectrofluorometric techniques, which had not been used previously to study adenovirus DBP. Using the homopolynucleotide poly(1,N6)-ethenoadenylic acid (poly(r epsilon A], we have determined that the binding site size is approximately 16 nucleotides. In 20 mM NaCl, the Ad2wt, Ad2ts111A, and Ad2+ND1ts23 DBP proteins all bound stoichiometrically to poly(r epsilon A) with overall apparent affinities above 108 M-1. Based on titrations carried out at higher salt concentrations, however, the stability of these complexes did appear to increase in the order Ad2+ND1ts23 less than Ad2ts111A less than Ad2wt. By these techniques, we have confirmed also that the DBP of another temperature-sensitive mutant, H5ts107, like the Ad2ts111A DBP, retains its ability to bind ssDNA even at a restrictive temperature utilizing the salt concentration compatible with adenovirus DNA replication in vitro. The H5ts107 DBP, which contains an amino acid substitution at position 413, is defective for in vitro replication at nonpermissive temperature but is not temperature sensitive for binding to ssDNA. In summary, our results indicate that the replication defects of the Ad2ts111A are similar to those of H5ts107 and cannot be attributed to defective, nonspecific ssDNA binding by the DBP. It appears that ssDNA binding by itself is not sufficient to account for the role of DBP in adenovirus DNA replication.     

Palindromic adenovirus type 5-simian virus 40 hybrid.

A family of novel adenovirus type 5-simian virus 40 (Ad5-SV40) recombinants (Ad5++D1) whose genomes consist of symmetrically inverted structures was isolated. Particles of Ad5++D1 could contain one of several recombinant genomes that differed incrementally from one another by a full-length copy of linear SV40 DNA. The members of the Ad5++D1 family appeared to be in genetic equilibrium with one another. In all probability this equilibrium was maintained by homologous recombination, resulting in the loss or gain of one or two unit length copies of the SV40 genome. The genome of the most abundant recombinant from consisted of a giant inverted repeat which was some 35,000 nucleotide pairs in length. Beginning from one end, the recombinant genome consisted of 3,534 nucleotides derived from the left end of the adenovirus type 5 genome; these nucleotides were joined to 2.7 copies of SV40 DNA arranged as head-to-tail tandems. This entire structure was then repeated in the opposite orientation, thereby forming a large inverted repeat whose structure was Ad5-SV40-SV40-04VS-04VS-5dA. The population of hybrid genomes was stable and was maintained through serial rounds of infection.     

Characterization of two temperature-sensitive mutants of type 5 adenovirus with mutations in the 100,000-dalton protein gene.

Complementation analysis assigned the mutations of strains H5ts115 and H5ts116, two hexon-minus mutants, to the 100,000-dalton (100K) protein gene. Heterotypic marker rescue (i.e., type 5 adenovirus [Ad5] temperature-sensitive mutants DNA X EcoRI restriction fragments of Ad2 DNA) confirmed the results of previous marker rescue mapping studies, and the heterotypic recombinants yielded unique hybrid (Ad5-Ad2) 100K proteins which were intermediate in size between Ad5 and Ad2 proteins and appeared to be as functionally active as the wild-type 100K protein. Phenotypic characterization of these mutants showed that both the hexon polypeptides and the 100K polypeptides were unstable at the nonpermissive temperature, whereas fiber and penton were not degraded, and that the 100K protein made at 39.5 degrees C could not be utilized after a shift to the permissive temperature (32 degrees C). The role of the 100K protein in the assembly of the hexon trimer was also examined by in vitro protein synthesis. Normally, hexon polypeptides synthesized during an in vitro reaction are assembled into immunoreactive hexons. However, this assembly was inhibited by preincubation of the cell extract with anti-100K immunoglobulin G; neither anti-fiber immunoglobulin G nor normal rabbit immunoglobulin G inhibited hexon assembly. It is postulated that an interaction between the 100K protein and hexon polypeptides is required for effective assembly of hexon trimers.     

Recombination in hamster cell nuclear extracts between adenovirus type 12 DNA and two hamster preinsertion sequences.

A cell-free system of nuclear extracts from BHK21 cells has been developed to catalyse recombination in vitro between the DNA of adenovirus type 12 (Ad12) and two different hamster preinsertion sequences. The pBR322 cloned 1768 bp fragment p7 and the 3.1 kbp fragment p16 from BHK21 hamster DNA had previously been identified as the preinsertion sites corresponding to the junctions between Ad12 DNA and hamster DNA in cell line CLAC1 and in the Ad12-induced tumour T1111(2), respectively. Preinsertion sequences, which had recombined previously with foreign (Ad12) DNA, might again be recognized by the recombination system even in a cell-free system. PstI cleaved Ad12 DNA and the circular or the EcoRI linearized p7 or p16 preinsertion sequences were incubated with nuclear extracts. Recombinants were isolated by transfecting the DNA into recA- Escherichia coli strains and by screening for Ad12 DNA-positive colonies. Without a selectable eukaryotic marker, all Ad12 DNA positive recombinants were registered. Out of a total of greater than 90 p7-Ad12 DNA recombinants, 21 were studied by restriction-hybridization, and four by partial nucleotide sequence analyses. Among the p16-Ad12 DNA recombinants, four were analysed. The sites of linkage between Ad12 DNA and p7 or p16 hamster DNA were all different and distinct from the original CLAC1 or T1111(2) junction site between Ad12 and hamster DNA. The in vitro recombinants were not generated by simple end-to-end joining of the DNA fragments used in the reaction but by genetic exchange. Thirteen of the 25 recombinants were derived from the 61-71 map unit fragment of Ad12 DNA. Recombination experiments between Ad12 DNA and four randomly selected unique or repetitive hamster DNA sequences of 1.5-6.2 kbp in length did not yield recombinants. Apparently, the p7 and p16 hamster preinsertion sequences recombined with Ad12 DNA with a certain preference.     

Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein.

We have determined the nucleotide sequence of the gene encoding adenovirus type 2 (Ad2) DNA binding protein (DBP). From the nucleotide sequence the complete amino acid sequence of Ad2 DBP has been deduced. A comparison of the amino acid sequences of Ad2 and Ad5 DBP, both 529 residues long, reveals that the C-terminal 354 residues of both sequences are identical. Within the N-terminal 175 amino acid residues Ad2 and Ad5 show nine differences. The site of mutation in Ad2 ND1ts23, a mutant with a temperature-sensitive DNA replication, was mapped at the nucleotide level. A single nucleotide alteration in the DBP gene, resulting in a leucine leads to phenylalanine substitution at position 282 in the amino acid sequence is responsible for the temperature-sensitive character of this mutant. Previously, we localized the mutation of another DBP mutant with a temperature-sensitive DNA replication (H5ts125) at position 413 in the amino acid sequence of the DBP molecule (Nucleic Acids Res. 9 (1981) 4439-4457). These mapping data are discussed in relation to the structure and function of the DBP molecule.     

The location of the genes coding for hexon and fiber proteins in adenovirus DNA.

A serological analysis has been made of the capsid antigens hexon and fiber from 17 Ad5-Ad2+ND1 recombinants that enables us to determine the phenotype of the recombinants. By correlation of this data with the genetic and physical maps of the adenovirus genome, obtained by recombination and restriction endonuclease analysis, the genes coding for the hexon and fiber have been assigned to specific locations on the adenovirus DNA.     

Species dependence of the major late promoter in adenovirus type 12 DNA.

In hamster cells human adenovirus type 12 (Ad12) is deficient in DNA replication and late gene expression whereas adenovirus type 2 (Ad2) can replicate. Functions located in the E1 region of the Ad2 or adenovirus type 5 (Ad5) genome can complement the deficiencies of the Ad12 genome in hamster cells, but, infectious viral particles are not produced. We have now investigated the activity of the major late promoter of Ad2 and of Ad12 DNA in human and hamster cells. This promoter governs the expression of most of the late viral functions. We have inserted the major late promoter (MLP) of Ad2 or of Ad12 DNA in front of the chloramphenicol acetyl transferase gene in the pSVO-CAT construct. Upon transfection into uninfected human and hamster cells, the pAd12MLP-CAT construct shows no significant activity; the pAd2MLP-CAT construct exhibits low activity. In Ad12-infected human cells, both constructs are active. These findings support the notion that other viral factors are required for MLP activity of Ad2 or Ad12 DNA in permissive human cells. In Ad2-infected hamster cells, both the pAd2MLP-CAT and the pAd12MLP-CAT constructs are active. Apparently, the Ad12 MLP can be activated by Ad2 functions, as already demonstrated for the entire Ad12 genome in double-infected cells or in Ad2- or Ad5-transformed cells superinfected with Ad12. In Ad12-infected hamster cells, however, the MLP of Ad12 DNA is inactive but that of Ad2 DNA shows activity. Thus the MLP of Ad12 DNA somehow differentiates between cellular auxiliary functions of different species.(ABSTRACT TRUNCATED AT 250 WORDS)     

cyt gene of adenoviruses 2 and 5 is an oncogene for transforming function in early region E1B and encodes the E1B 19,000-molecular-weight polypeptide

A total of 59 cytocidal (cyt) mutants were isolated from adenovirus 2 (Ad2) and Ad5. In contrast to the small plaques and adenovirus type of cytopathic effects produced by wild-type cyt+ viruses, the cyt mutants produced large plaques, and the cytopathic effect was characterized by marked cellular destruction. cyt mutants were transformation defective in established rat 3Y1 cells. cyt+ revertants and cyt+ intragenic recombinants recovered fully the transforming ability of wild-type viruses. Thus, the cyt gene is an oncogene responsible for the transforming function of Ad2 and Ad5. Genetic mapping in which we used three Ad5 deletion mutants (dl312, dl313, and dl314) as reference deletions located the cyt gene between the 3' ends of the dl314 deletion (nucleotide 1,679) and the dl313 deletion (nucleotide 3,625) in region E1B. Restriction endonuclease mapping of these recombinants suggested that the cyt gene encodes the region E1B 19,000-molecular-weight (175R) polypeptide (nucleotides 1,711 to 2,236). This was confirmed by DNA sequencing of eight different cyt mutants. One of these mutants has a single missense mutant, two mutants have double missense mutations, and five mutants have nonsense mutations. Except for one mutant, these point mutations are not located in any other known region E1B gene. We conclude that the cyt gene codes for the E1B 19,000-molecular-weight (175R) polypeptide, that this polypeptide is required for morphological transformation of rat 3Y1 cells, and that simple amino acid substitutions in the protein can be sufficient to produce the cyt phenotype.     

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.     

Use of Nondefective Adenovirus-Simian Virus 40 Hybrids for Mapping the Simian Virus 40 Genome

A series of viable recombinants between adenovirus 2 (Ad2) and simian virus 40 (SV40) (nondefective Ad2-SV40 hybrids) have been isolated. The members of this series (designated Ad2(+)ND(1) through Ad2(+)ND(5)) differ from one another in the early SV40-specific antigens and the SV40-specific RNA species which they induce in infected cells. They also contain different amounts of SV40 DNA as shown by RNA-DNA hybridization techniques. We have examined the structure of the DNA molecules from these hybrids, using electron microscope heteroduplex mapping techniques. Each hybrid was found to contain a single segment of SV40 DNA of characteristic size covalently inserted at a unique location in the adenovirus 2 DNA molecule. The SV40 segments of the various hybrids formed an overlapping series with a common end point. When the results of the electron microscopic study were combined with data on antigen induction, it was found that a self-consistent map could be constructed which related specific regions of the SV40 genome to the induction of specific antigens. The order of these early SV40 antigen inducing regions in the SV40 DNA segments contained in the nondefective hybrids is: U antigen, tumor specific transplantation antigen, and T antigen with the U antigen region being nearest the common end point.     

Effect of deletions in adenovirus early region 1 genes upon replication of adeno-associated virus.

The growth of adeno-associated virus (AAV) is dependent upon helper functions provided by adenovirus. We investigated the role of adenovirus early gene region 1 in the AAV helper function by using six adenovirus type 5 (Ad5) host range mutants having deletions in early region 1. These mutants do not grow in human KB cells but are complemented by and grow in a line of adenovirus-transformed human embryonic kidney cells (293 cells); 293 cells contain and express the Ad5 early region 1 genes. Mutants having extensive deletions of adenovirus early region 1a (dl312) or regions 1a and 1b (dl313) helped AAV as efficiently as wild-type adenovirus in 293 cells, but neither mutant helped in KB cells. No AAV DNA, RNA, or protein synthesis was detected in KB cells in the presence of the mutant adenoviruses. Quantitative blotting experiments showed that at 20 h after infection with AAV and either dl312 or dl313 there was less than one AAV genome per cell. In KB cells infected with AAV alone, the unreplicated AAV genomes were detected readily. Apparently, infection with adenovirus mutant dl312 or dl313 results in degradation of most of the infecting AAV genomes. We suggest that at least an adenovirus region 1b product (and perhaps a region 1a product also) is required for AAV DNA replication. This putative region 1b function appears to protect AAV DNA from degradation by an adenovirus-induced DNase. We also tested additional Ad5 mutants (dl311, dl314, sub315, and sub316). All of these mutants were inefficient helpers, and they showed varying degrees of multiplicity leakiness. dl312 and dl313 complemented each other for the AAV helper function, and each was complemented by Ad5ts125 at the nonpermissive temperature. The defect in region 1 mutants for AAV helper function acts at a different stage of the AAV growth cycle than the defect in the region 2 mutant ts125.     

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.     

Restriction maps of human adenovirus types 2, 5 and 3 for BclI, ClaI, pvul and SphI endonucleases

The sites of cleavage by BclI, ClaI, PvuI and SphI in the DNA from adenovirus (Ad) serotypes 2, 5 and 3 have been located. Certain site coordinates were in accord with nucleotide sequences already published. A small difference in size between the PvuI-E fragments from Ad2 and Ad5 confirmed the existence of a deletion in the N-terminal moiety of Ad5 hexon gene, as previously implied by interserotypic recombinants (Boursnell and Mautner, Virology 112 (1981) 198-209) and more recently by amino acid sequencing (Von Bahr-Lindstr?m et al., Virology 118 (1982) 352-362).