Letter: Some unusual properties of replicating adenovirus type 2 DNA

Replicating adenovirus type 2 DNA was isolated from KB cells 13 hours after infection. The buoyant density in caesium chloride of the replicating DNA was found to be 5 to 10 mg/cm² greater than that of mature adenovirus type 2 DNA. The single-strand specific nuclease from Neurospora crassa released 25 to 30% of the radioactivity from replicating DNA and the density difference between replicating and mature adenovirus DNA was eliminated after digestion with this enzyme, but not after digestion with RNase or pronase. The results suggest that the complementary strands of adenovirus type 2 DNA are replicated asynchronously.     

Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA

The adenovirus-encoded single-stranded DNA-binding protein (DBP) functions in viral DNA replication and several aspects of RNA metabolism. Previous studies (G. A. M. Neale and G. R. Kitchingman, J. Biol. Chem. 264:3153-3159, 1989) have defined three highly conserved regions in the carboxy-terminal domain of the protein (amino acids 178 to 186, 322 to 330, and 464 to 475) that may be involved in the binding of the protein to single-stranded DNA. We examined the role of conserved region 3 (464 to 475) by constructing nine classes of point mutants with from one to four amino acid changes. The point mutants were tested for their ability to assist adeno-associated virus DNA replication. All nine differed from wild-type DBP; seven were essentially nonfunctional, whereas two had 55 and 145%, respectively, of the wild-type DBP helper activity. Three of the mutants were found to be temperature sensitive, with significantly greater helper activity at 33 degrees C than at 37 degrees C. All nine mutants produced essentially wild-type levels of protein. One monoclonal antibody against the DBP, termed 2/4, did not immunoprecipitate the mutant DBPs as well as wild-type DBP, indicating either that the antibody recognized sequences around CR3 or that the conformation of the protein around the epitope recognized by 2/4 had changed. Two of the three temperature-sensitive DBP mutants bound to single-stranded DNA-cellulose with the same affinity as wild-type DBP at 4 degrees C; the remaining mutants all showed reduced affinity. These results demonstrated that many of the residues within conserved region 3 of the DBP are important for interaction of the protein with nucleic acid.     

Isolation and partial characterization of single-stranded adenoviral DNA produced during synthesis of adenovirus type 2 DNA.

Single-stranded fragments of adenovirus type 2 DNA were isolated from infected KB cells under conditions which retarded reassociation of complementary sequences but did not denature native viral DNA. Of the total intracellular, virus-specific DNA labeled during a 1-h pulse with tritiated thymidine begining 15 h after infection, about 20% was single stranded when fractionated on hydroxylapatite. This DNA shifted predominantly to the double-stranded fraction on hydroxylapatite during an extended chase incubation, suggesting that it may represent single-stranded DNA in replicating intermediates. Furthermore, the single-stranded DNA annealed nearly equally to both strands of the adenovirus genome. These findings indicate that at least portions of both complementary strands of adenovirus type 2 DNA are exposed as single strands during the period of viral DNA synthesis.     

Immunofluorescence study of the adenovirus type 2 single-stranded DNA binding protein in infected and transformed cells.

High-titer monospecific antiserum against highly purified adenovirus 2 (Ad2) single-stranded DNA binding protein (DBP) was used to study, by indirect immunofluorescence (IF), the synthesis of DBP in Ad2-infected human cells and adenovirus-transformed rat, hamster, and human cell lines. In infected cells the synthesis of DBP was first detected in the cytoplasm at 2 to 4 h postinfection and reached a maximum intensity at 6 h postinfection. At this time DBP began to accumulate in the nucleus, where it reached maximum intensity at about 14 h postinfection. The cytoplasmic IF was diffuse, whereas nuclear IF appeared as dots that coalesced into large globules as infection progressed. In cells treated with 1-beta-d-arabinofuranosylcytosine to inhibit viral DNA synthesis, strong nuclear IF was observed in the form of dots, but the large fluorescent globules were not observed. The Ad2 (oncogenic group C) anti-DBP serum reacted very strongly by IF with Ad5 (group C)-infected, to a lesser extent with Ad7 and Ad11 (group B)-infected, and weakly with Ad12 and Ad18 (group A)-infected KB cells (treated with 1-beta-d-arabinofuranosylcytosine). These results may indicate that Ad2 DBP is closely related immunologically to DBPs induced early after infection by adenovirus serotypes in oncogenic group C, moderately related to DBPs of serotypes in oncogenic group B, and perhaps distantly related to DBPs of serotypes in oncogenic group A. The following adenovirus-transformed cell lines were examined for DBP synthesis by IF with the Ad2 anti-DBP serum: six rat cell lines (T2C4, F17, 8662, 8638, 8617, and F161) transformed by Ad2 virus, three hamster cell lines transformed by Ad2 virus (Ad2HT1) and Ad2-simian virus 40 hybrid virus (ND1HK1 and ND4HK4), and one rat (5RK) and one human (293-31) cell line transformed by transfection with Ad5 DNA. T2C4 and 8662 appeared weakly positive, whereas Ad2HT1 and ND4HK1 were strongly positive. The other transformed cell lines did not produce DBP detectable by IF. Thus, some but not all transformed cell lines produce DBP, which indicates that DBP is not required for maintenance of cell transformation and that transformed cells can express "nontransforming" viral genes as protein.     

The structure of replicating adenovirus 2 DNA molecules

Adenovirus 2 (Ad2)-infected KB cells were exposed to a 2.5 min pulse of ³H-thymidine at 19 hr after infection. The labeled DNA molecules were separated from cell DNA and mature Ad2 DNA by sucrose gradient sedimentation and CsCI equilibrium centrifugation under conditions designed to minimize branch migration and hybridization of single strands. Electron microscopy-of fractions containing radioactivity revealed two basic types of putative replicating molecules: Ad2 length duplex DNA molecules with one or more single-stranded branches (type I) and Ad2 length linear DNA molecules with a single-stranded region extending a variable distance from one end (type II). Length measurements, partial denaturation studies and 3′ terminal labeling experiments were consistent with the following model for Ad2 DNA replication. Initiation of DNA synthesis occurs at or near an end of the Ad2 duplex. Following initiation, a daughter strand is synthesized in the 5′ to 3′ direction, displacing the parental strand with the same polarity. This results in the formation of a branched replicating molecule (type I). Initiations at the right and left molecular ends are approximately equal in frequency, and multiple initiations on the same replicating molecule are common. At any given displacement fork in a type I molecule, only one of the two parental strands is replicated. Two nonexclusive mechanisms are proposed to account for the replication of the other parental strand. In some cases, before completion of a round of displacement synthesis initiated at one end of the Ad2 duplex, a second initiation will occur at the opposite end. In these doubly initiated molecules, both parental strands serve as templates for displacement synthesis. Two type II molecules are generated when the oppositely moving displacement forks meet. Alternatively, displacement synthesis may proceed to the end of the Ad2 duplex, resulting in the formation of a daughter duplex and a parental single strand. Replication of the displaced parental strand is then initiated at or near its 3′ terminus, producing a type II molecule. Daughter strand synthesis proceeds in the 5′ to 3′ direction in type II molecules generated by either mechanism, and completion of synthesis results in the formation of a daughter duplex.     

The nucleotide sequence at the termini of adenovirus type 5 DNA.

The sequences of the first 194 base pairs at both termini of adenovirus type 5 (Ad5) DNA have been determined, using the chemical degradation technique developed by Maxam and Gilbert (Proc. Nat. Acad. Sci. USA 74 (1977), pp. 560-564). The nucleotide sequences 1-75 were confirmed by analysis of labeled RNA transcribed from the terminal HhaI fragments in vitro. The sequence data show that Ad5 DNA has a perfect inverted terminal repetition of 103 base pairs long.     

Presence of protein at the termini of intracellular adenovirus type 5 DNA.

Adenovirus type 5 contains linear double-stranded DNA with protein covalently attached to the ends of the molecules. The presence of protein at the termini of intracellular viral DNA in adenovirus type 5-infected cells was investigated at different stages during the replication process. The intracellular viral DNA was isolated from the nuclei by lysis in 4 M guanidine hydrochloride. Electrophoresis on agarose gels of HsuI restriction enzyme fragments and sucrose gradient centrifugation were used to detect protein on intracellular viral DNA. After uncoating parental DNA still contains protein attached to the termini of the viral genome. Replicating and mature progeny viral DNA can also be isolated in the form of DNA-protein complexes. These complexes exhibit the same properties as the DNA-protein complex isolated from purified virions. These results suggest that the protein at the termini of intracellular viral DNA is identical to the protein attached to the 5'-ends of the DNA extracted from virions and that it is possibly involved in the replication of viral DNA.     

cis and trans requirements for the selective packaging of adenovirus type 5 DNA.

Polar packaging of adenovirus DNA into virions is dependent on the presence of cis-acting sequences at the left end of the viral genome. Our previous analyses demonstrated that the adenovirus type 5 (Ad5) packaging domain (nucleotides 194 to 358) is composed of at least five elements that are functionally redundant. A repeated sequence, termed the A repeat, was associated with packaging function. Here we report a more detailed analysis of the requirements for the selective packaging of Ad5 DNA. By introducing site-directed point mutations into specific A repeat sequences, we demonstrate that the A repeats represent cis-acting functional components of the packaging signal. Additional elements, located outside the originally defined packaging domain boundaries and that resemble the A repeat consensus sequence, also are capable of promoting the packaging of viral DNA. The cis-acting components of the packaging signal appear to be subject to certain spatial constraints for function, possibly reflecting a necessity for the coordinate binding of packaging proteins to these sites. In agreement with this idea, we present evidence that the interaction of a limiting trans-acting factor(s) with the packaging domain in vivo is required for efficient encapsidation of the Ad5 genome.     

In vitro synthesis of adenovirus type 5 T antigens. II. Translation of virus-specific RNA from cells transformed by fragments of adenovirus type 5 DNA.

Virus-specific cytoplasmic RNA was isolated from rat cell lines transformed by fragments of adenovirus type 5 DNA, and the RNAs were translated in cell-free systems derived from wheat germ or rabbit reticulocytes. RNA was isolated from cell lines transformed by the following fragments: XhoI-C (leftmost 15.5%), HindIII-G (leftmost 8%), and HpaI-E (leftmost 4.5%). In addition, the adenovirus type 5-transformed human embryonic kidney line 293.C31 was investigated. The products were immunoprecipitated with serum from tumor-bearing hamsters and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The results show that all transformed cells investigated contain early region 1a-specific RNAs which can be translated into proteins with molecular weights of 34,000 (34K), 36K, 40K, and 42K. Transformed cells that also contain an intact early region 1b synthesized RNA which can be translated into proteins with molecular weights of 19K and 65K. Minor proteins of 15K, 16K, 17.5K, 18K, 25K, and 29K were also observed, but these proteins could not be mapped unambiguously. Cells transformed by the 8% HindIII-G apparently lack RNA encoding the 65K protein, but they do contain RNA coding for the 19K protein.     

Cellular transformation by adenovirus type 5 is influenced by the viral DNA polymerase.

Early region 2B (E2B) of the group C adenoviruses encodes a number of proteins, including the 140-kilodalton DNA polymerase, which plays a role in the initiation of viral DNA replication. Temperature-sensitive (ts) mutants with mutations mapping to E2B are conditionally defective for both DNA replication in human cells and transformation of rat cells. Nucleotide sequence analysis shows that the E2B mutant ts36 possesses a single point mutation specific to the viral DNA polymerase; this transition of a C to a T at position 7623 changes leucine residue 249 in the polymerase to a phenylalanine. A wild-type (ts+) revertant possesses a codon specifying the original leucine at position 249. Phenotypic analysis of revertant and wild-type viruses derived by marker rescue from ts36 shows that these variants are wild type for both viral DNA replication and transformation. Thus, the single point mutation in the polymerase gene of ts36 is responsible for both defects.     

Mechanism of replication of phichi174 single-stranded DNA. IV. The parental viral strand is not conserved in the replicating DNA structure

The role of the infecting viral strand in the replication of bacteriophage φX174 replicative form DNA was studied by [³H]thymidine pulse-labeling Escherichia coli cells infected with ²H¹⁵N density-labeled phage. The products of a round of semi-conservative replicative form replication (in light medium) do not contain the original heavy viral strand by 15 minutes after infection or later in the presence of chloramphenicol. Similar results were obtained at earlier times in the absence of chloramphenicol. We conclude that the parental viral strand need not be conserved in the replicating DNA structure in succeeding rounds of replication.     

Bidirectional replication of adenovirus type 2 DNA.

After short periods of labeling with [3H]thymidine, recently completed adenovirus DNA molecules were isolated and cleaved with restriction endonucleases. The strands (heavy and light) of most of the restriction endonuclease fragments were separated. The pattern of labeling clearly shows an asymmetry of radioactivity on the isolated strands of each restriction endonuclease piece. The data is consistent with replication proceeding in the 5' to 3' direction on each strand. Thus, there is an initiation point placed at or near each end of the molecule.     

Complementation of human adenovirus type 5 ts mutants by human adenovirus type 12.

Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.     

Characterization of type 5 adenovirus fiber protein.

Type 5 adenovirus fiber protein was purified and subjected to chemical characterization. Equilibrium sedimentation ultracentrifugation analysis indicated that the intact fiber has a molecular weight of approximately 183,000. Denaturation and chemical analyses implied that the fiber consists of three polypeptide chains, each of about 61,000 mol wt. Mapping of tryptic peptides and electrophoretic separation of the constituent chains suggested that the intact fiber consists of two identical and one unique polypeptide chains.     

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.     

Herpesvirus infection modifies adenovirus RNA metabolism in adenovirus type 5-transformed cells.

The effect of herpes simplex virus (HSV) infection of mRNA metabolism was examined in a system where the fate of specific RNA sequence can be assayed. Adenovirus type 5-transformed rat embryo cell line 107 synthesizes adenovirus-specific RNA (ad-RNA), which functions in the cytoplasm as mRNA. We have utilized ad-RNA as a model for mRNA metabolism, and in a preliminiary study we characterized ad-RNA in the nucleus and cytoplasm by hybridization to filter-bound adenovirus DNA. The results indicated the as-RNA accumulates in the nucleus and that cytoplasmic polyadenylic acid [poly(A)]-containing ad-RNA turns over with a half-life of a few hours. Pulse-chase experiments confirmed these observations and a half-life of about h was determined for the poly(A)-containing cytoplasmic ad-RNA. A second class of ad-RNA remains in the nucleus, where it turns over with a longer hlaf-life (about 24 h). The infection of 107 cells by HSV was restricted at 37 degree C, giving a burst size of 5 PFU per cell and allowing continued host DNA synthesis. Protein synthesis was inhibited greater than 50% by 7 h after infection, and total RNA synthesis was 50% inhibited by 4 h after infection. During the first 8 h after infection, HSV has little effect on the rate of synthesis of ad-RNA as determined by hybridization of nuclear RNA samples, but,during the same period, HSV inhibits the accumulation of poly(A)-containing ad-RNA in the cytoplasm. The degree of this inhibition increases steadily throughout this period and reaches 60% by 6.5 to 8 h after infection. Nosignificant effect was seen on the accumulation of total cellular poly(A)-containing RNA. It was concluded from these experiments that HSV infection alters the metabolism of ad-RNA so as to prevent the normal appearance of the poly(A)-containing mRNA in the cytoplasm. The result for ad-RNA may not represent the behavior of total cellular poly(A)-containing RNA under conditions where infection is restricted.     

Multiple origins and circular structures in replicating T5 bacteriophage DNA.

Replicating T5 phage DNA was gently isolated using NaI density gradient centrifugation and examined by electron microscopy. At the beginning of phage DNA synthesis, linear unit-length T5 DNA molecules containing from one to four replicating "eye-loops" were consistently observed. Replication in these molecules was found to proceed bidirectionally from multiple, internal origins. A primary origin of replication is located near the center of the T5 genome, which does not coincide with the location of any of the nicks (single-strand breaks) found in mature T5 DNA. The initiation of replication at the various origins within an individual molecule does not appear to follow any definite temporal sequence. At later times in the infection, we have observed a significant number of circular T5 DNA molecules-both replicating and nonreplicating-whose average circumference is approximately the length of mature T5 DNA minus the terminal redundancy. The replicating circular molecules appear to be either in a theta configuration, a sigma configuration with the tails all being less than the length of the circle, or a combination of theta and sigma forms.