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We have developed a method to localize DNA double strand breaks (DSBs) in situ in cultured mammalian cells. Adenoviruses encoding Saccharomyces cerevisiae HO endonuclease and its cleavage site were used to induce site-specific DSBs. Rolling circle amplification (RCA), a sensitive method that allows the detection of single molecular event by rapid isothermal amplification, was used to localize the broken ends in situ. Punctate RCA signals were only seen in the cells that had been infected with both adenoviruses encoding HO endonuclease and HO cleavage site, but not in the cells mock-infected or infected with the site or endonuclease virus only. With use of a chemical crosslinker, in situ RCA and immunofluorescence (IF) can be performed simultaneously on the same sample. This methodology provides a novel approach for investigation of DNA recombination, DNA repair, and checkpoint controls in mammalian cells.

Previous results showed that the structure and function of the adenovirus major late promoter (MLP) can be analyzed genetically in its correct location, despite its essential role in the viral life cycle. This genetic approach was extended to investigate the in vivo role of the initiator (INR), a transcriptional element that surrounds the start site of transcription. The analysis was designed to investigate if the INR is an alternative basal element to the canonical TATA box of the MLP, its relative importance in the functioning of the promoter, and if its function was affected by upstream activating elements. Accordingly, two different mutations in the INR were created and tested in the genome, either by themselves or together with mutations in the TATA box or one of the two upstream activating elements, the upstream promoter element (UPE) and the inverted CAAT box. The mutant viruses were examined first in one-step growth experiments, and then levels of late mRNA accumulation were measured by primer extension, transcription initiation was assayed in isolated nuclei, and viral DNA accumulation was determined by Southern hybridization. Neither mutation in the INR alone had any discernible phenotypic effects but when coupled to a phenotypically silent mutation in the TATA box gave rise to viruses with growth defects that were attributable to a significantly lowered rate of transcription initiation from the MLP. These results suggest that the INR plays a role in vivo and can act as an alternative basal element in the absence of a functioning TATA box. A virus with mutations in both the INR and the UPE, although viable, likewise had a severe deficiency in transcription, suggesting that the function of the INR is affected by that of the UPE. This contrasts with the previous report that a TATA box-UPE double mutation is not recoverable in virus. In addition, the virus with mutations in both the INR and the inverted CAAT box was phenotypically wild type, unlike the previously described TATA box-CAAT box double mutant, which had a severe transcription deficiency. Taken together, the present and previous genetic results can be interpreted as evidence that in the MLP, the TATA box and the UPE are the more important of the two basal and activating elements, respectively, but that the INR and CAAT can function in transcription initiation. We consider the role of the INR in the formation of the preinitiation complex and speculate on possible protein-protein interactions.

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.

An adenovirus major late promoter (MLP) has been constructed with a 4-bp alteration in the sequence which binds the transcription factor known as USF or MLTF. This upstream element has often been considered necessary and sufficient for maximal transcription of the MLP. A duplex oligonucleotide containing the mutant sequence was not capable of binding specific proteins in a band shift assay, nor was it capable of inhibiting such binding by the wild-type sequence. In an in vitro assay, the mutant sequence was incapable of inhibiting transcription from a duplex sequence containing the MLP, whereas the wild-type sequence could. These two pieces of evidence suggest that the sequence is functionally impaired. Surprisingly, a virus containing the mutant MLP had a normal replication phenotype. On more detailed examination however, we show that the mutant viral MLP was deficient in transcription at 9 h postinfection but that the rate of transcription was close to normal by 20 h postinfection. An inverted CAAT box located immediately upstream of the USF-binding element was not previously thought to be of importance to the functioning of the MLP. However, a single point mutation in the CAAT box, placed in the USF mutant background, had a marked effect upon transcription from the MLP. This result suggests that the MLP may exhibit functional redundancy in which either the USF-binding site or the CAAT box can serve as an upstream promoter element. Neither of the mutant viruses displayed any change in the levels of the divergent IVa2 transcription unit, suggesting that the levels of divergent transcription are not determined by competition for limiting transcription factors.

Granzymes are key components of the immune response that play important roles in eliminating host cells infected by intracellular pathogens. Several granzymes are potent inducers of cell death. However, whether granzymes use additional mechanisms to exert their antipathogen activity remains elusive. Here, we show that in adenovirus-infected cells in which granzyme B (gzmB) and downstream apoptosis pathways are inhibited, granzyme H (gzmH), an orphan granzyme without known function, directly cleaves the adenovirus DNA-binding protein (DBP), a viral component absolutely required for viral DNA replication. We directly addressed the functional consequences of the cleavage of the DBP by gzmH through the generation of a virus that encodes a gzmH-resistant DBP. This virus demonstrated that gzmH directly induces an important decay in viral DNA replication. Interestingly, gzmH also cleaves the adenovirus 100K assembly protein, a major inhibitor of gzmB, and relieves gzmB inhibition. These results provide the first evidence that granzymes can mediate antiviral activity through direct cleavage of viral substrates, and further suggest that different granzymes have synergistic functions to outflank viral defenses that block host antiviral activities.

The adenovirus major late promoter (MLP) has played a pre-eminent role in the analysis of transcription initiation in mammalian cells, and is an outstanding example of the ways in which the study of adenovirus has led to fundamental insights into general cellular processes. The aim of this chapter is to give a comprehensive review of the structure and function of this model mammalian promoter. After a brief description of late transcription in the adenovirus replication cycle, the experimental evidence for the current consensus on the genetic structure of the MLP, including a consideration of non-primate adenovirus MLPs, will be reviewed. Next, the functions of the MLP in the viral life cycle will be examined, and some of the problems that remain to be resolved will be addressed. The review ends with some ideas on how the knowledge of the structure and function of the MLP can be used in designing virus vectors for specific experimental purposes.

The adenovirus major late promoter (MLP) has played a preeminent role in the analysis of transcription initiation in mammalian cells, and is an outstanding example of the ways in which the study of adenovirus has led to fundamental insights into general cellular processes. The aim of this chapter is to give a comprehensive review of the structure and function of this model mammalian promoter. After a brief description of late transcription in the adenovirus replication cycle, the experimental evidence for the current consensus on the genetic structure of the MLP, including a consideration of non-primate adenovirus MLPs, will be reviewed. Next, the functions of the MLP in the viral life cycle will be examined, and some of the problems that remain to be resolved will be addressed. The review ends with some ideas on how the knowledge of the structure and function of the MLP can be used in designing virus vectors for specific experimental purposes.

The observation that adenovirus genomes can undergo genetic recombination is almost a quarter of a century old (Williams and Ustacelebi 1971; Takemori 1972; Ensinger and Ginsberg 1972), yet despite its early discovery and its rapid exploitation as a tool for mapping mutations and for creating new genotypes (reviewed in Ginsberg and Young 1977; Young et al. 1984b), the mechanisms underlying it are not well understood at the molecular and biochemical levels. Such an understanding, however, can be expected to shed light not only on the specific features peculiar to adenovirus recombination itself, but also on the more general characteristics of the repair and recombinational capacities of the cell. These cellular aspects are of considerable current theoretical and practical interest, because of recent advances both in uncovering the molecular basis of genetic defects in DNA repair in mammalian cells and in the techniques of gene targeting by homologous recombination in order to investigate mammalian development and differentiation. As with many other fundamental biological phenomena, the study of adenovirus recombination can be expected to yield valuable insights into the normal capacities of the cell.

The adenovirus major late promoter (MLP) has been analyzed by constructing recombinant viral genomes containing mutations in possible promoter elements. Single base pair changes in the TATA box had no effect on viral replication, and MLP expression, as measured by the accumulation of late mRNAs, was at wild type levels. However, a double mutation in the TATA box reduced viral replication and MLP expression, demonstrating that the TATA box is important, although not essential, for maximal activity in virus. Primer extension analysis showed that the mRNAs were initiated at the correct position. A mutation in the CAAT box was viable, and had only minor effects on MLP expression. However, this mutation when coupled to a single mutation in the TATA box, severely reduced viral replication and expression from the MLP. Similarly, a viable mutation in the UPE, shown previously to abolish binding of USF, coupled to a single mutation in the TATA box was lethal. These results suggest that both USF and the CAAT box binding factor CP1 can interact with TFIID to effect activation, and thus that the mechanism of activation is functionally redundant.

To investigate whether early genes other than those involved directly in DNA replication are required for efficient adenovirus recombination, pairs of viruses with deletions in E1a, E1b 496R, E1b 196R, or E4 and containing differing restriction site markers were used to infect both permissive and non- or semipermissive cells. Recombination was assayed among intracellular and extracellular genomes by restriction digestion and blot hybridization. Recombination was delayed in infections of nonpermissive cells with E1a- viruses until a time consistent with the late onset of DNA replication characteristic of the cell type. This shows that E1a expression is not absolutely required for adenovirus recombination. Similar tests with deletion mutations in E1b and E4 also show that these genes are not required for efficient recombination. Taken together with earlier results showing that recombination depends on DNA replication, it is likely that adenovirus recombination is a consequence of cellular repair functions acting on the substrates produced by replication.

We have previously shown that adenovirus recombinants expressing functional ICP0 reactivate latent herpes simplex virus type 2 (HSV-2) in an in vitro latency system. This study demonstrated that ICP0, independent of other HSV gene products, is sufficient to reactivate latent HSV-2 in this in vitro system. To assess the effects of defined mutations in the sequence encoding ICP0 (IE-0) on reactivation, seven in-frame insertion and three in-frame deletion mutants were moved into an adenovirus expression vector. Each recombinant directed the synthesis of stable ICP0 of the correct size. The transactivation activity of the mutated sequences in these recombinants was similar to that when they were tested in plasmids. When these recombinants were examined for their ability to reactivate in the in vitro latency system, mutants with dramatic defects in transactivation (Ad-0/125, Ad-0/89, Ad-0/2/7, and Ad-0/88/93) were unable to reactivate latent HSV-2 independent of the multiplicity of infection. An exception to this correlation was the finding that Ad-0/89, which transactivated poorly, was able to reactivate latent virus after prolonged incubation whereas other transactivation-deficient mutants could not. Moreover, the presence of ICP4 did not compensate for the inability of any of the recombinants tested to reactivate HSV-2. These results show that (i) the transactivation domains of ICP0 are also used in reactivation, (ii) the presence of another essential HSV regulatory protein ICP4 does not alter the pattern of reactivation by ICP0, and (iii) mutations in some regions of IE-0 previously shown to affect viral growth and plaque formation did not alter its ability to reactivate in this in vitro system.

We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up- and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit beta-globin sequences placed in the dispensable E1 region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.

Extrachromosomal elements in a super-suppression system of yeast II. Relations with other extrachromosomal elements

Extrachromosomal elements in a super-suppression system of yeast I. A nuclear gene controlling the inheritance of the extrachromosomal elements

The use of genetics to study the interactions of adenoviruses with permissive and nonpermissive host cells has had two primary goals. One is to enumerate the genes and the functions encoded by them, a task that in recent years has depended more on the ability to map specific messenger RNAs (mRNAs) to particular regions of the genome than on classic genetic analysis. The other is to use mutants of various kinds in functional studies to determine the role of specific genes in such processes as the control of RNA transcription, DNA replication, host-cell transformation, and viral assembly. In these functional roles, adenovirus mutants have been of great practical benefit, as a reading of the following chapters in this volume will show. With the development of in vitro systems to study specific processes, mutants have been and will continue to be of great value in dissecting the steps involved and, importantly, in confirming the faithfulness of the reconstituted systems to the events detected in vivo.

ICP0, one of the five immediate-early (IE) gene products of herpes simplex virus (HSV), is a potent activator of transcription. To assess the biological activities of ICP0 and to explore its mechanisms of action, two helper-independent recombinant adenoviruses were constructed. In each recombinant, the E1 region was substituted with the ICP0-encoding genomic segment under the control of either the adenovirus major late promoter (MLP-0) or the HSV IE-0 promoter (0PRO-0). Infection of HeLa cells or 293 cells (a human embryo kidney cell line expressing adenovirus 5 E1a and -b functions) with the MLP-0 recombinant results in the synthesis of more IE-0 mRNA and ICP0 protein than did infection with the 0PRO-0 recombinant. Although 293 cells infected with MLP-0 accumulate 5- to 10-fold more IE-0 mRNA late in the infection than cells infected with HSV, the level of the protein product, ICP0, increased only slightly. In 293 cells, both recombinants could replicate, albeit at a slower rate and with lower final yields than wild-type adenovirus. Neither virus replicates its DNA in HeLa cells, and thus ICP0 cannot substitute for adenovirus E1a; however, the level of ICP0 that accumulates in MLP-0-infected HeLa cells was comparable to that of HSV-infected HeLa cells. In a functional test, we demonstrated that the adeno-ICP0 recombinant viruses can transactivate a transfected TK-CAT cassette, indicating that the ICP0 is biologically active.

r(ts107)202 was isolated in HeLa cells as a temperature-independent revertant of H5ts107, an adenovirus mutant that maps in the structural gene of the viral DNA-binding protein. r(ts107)202 is a host-range temperature-conditional mutant: it is temperature independent for growth in HeLa cells but temperature sensitive for growth in 293 cells, a type 5 adenovirus-transformed human cell line. Marker rescue experiments using H5ts107 DNA and restriction enzyme fragments from r(ts107)202 DNA demonstrated that the mutations causing the r(ts107)202 phenotype were localized in Hin dIII fragment A containing the entire DNA-binding protein gene. To obtain a fine structure map of the r(ts107)202 mutations, overlap recombination between Eco RI fragment A (0-75.9 map units) from either Ad5wt or r(ts107)202 and Bam HI fragment B (59.5-100 map units) from either Ad5wt or r(ts107)202 was performed. Segregation of the H5ts107 primary-site mutation away from the accompanying reversion mutation could be demonstrated in 5 of 200 plaques when r(ts107)202 Eco RI fragment A was crossed with the Ad5wt Bam HI fragment. In the reciprocal cross, none of 200 plaques contained the H5ts107 mutant. These results permitted a determination of the order of the primary-site mutation (H5ts107) and secondary-site mutation in r(ts107)202, and the frequency of recombination predicted a distance of 50-340 base pairs between these two mutations. This experimental result agrees with the nucleotide sequence of the r(ts107)202 mutant which shows that 182 base pairs separate the primary-site and secondary-site mutations in r(ts107)202.

Individual colonies of an adenine-requiring strain of the genotype ade 2-1, SUPQ5, [psi minus] exhibit high and uniform frequencies of ADE+ revertants (5-1 times 10-5). The frequencies for strains of the genotypes ade 2-1, SUPQ5+, [psi+] and ade 2-1 SUPQ5+, [psi minus] are by contrast 1-2 times 10-7 and 0-5 times 10-7 respectively. Mutation from [psi minus] to [psi+] can occur. This suggests that the original mutation from [psi+] to [psi minus] is not necessarily a physical loss of, or extensive deletion, the extrachromosomal elements. Although mutation from [psi minus] to [psi+] occurs, the majority of ADE+ revertants from ade 2-1, SUPQ5, [psi minus] strains contain other types of mutation. Crosses between most ADE+ revertants and ade 2-1, SUPO5+, [psi minus] strains yield adenine-requiring diploids and tetrad segregations of 0:4, 1:3 and 2:2 ADE+ :ade minus. It is argued that most mutations give rise to weak recessive suppressors (SUPX) which are themselves incapable of suppressing the ochre allele ade 2-1, unless the allele SUPQ5 is also present. Tests with two suppressors, S-theta and S-zeta, isolated by Dr R. A. Gilmore, which are themselves incapable of suppressing ade 2-1, show that they do not do so even in the presence of SUPQ5. Thus the set of suppressors isolated by us is a special class which can perhaps only be observed in the genetic background ade 2-1, SUPQ5, [psi minus]. There is evidence that some of the new suppressors are capable, in the absence of SUPQ5, of weakly suppressing trp 5-48 and perhaps can 1-100. All ochre alleles are, however, suppressed by the combination SUPQ5, SUPX. SUPQ5 is able to suppress trp 5-48, his 5-2, lys 1-1 and can 1-100 in a [psi minus] background, although it does so variably. It is incapable of suppressing ade 2-1 in such a background. It is argued that the alteration in the [psi] determinant results in a lowered efficiency of suppression by SUPQ5 rather than a changed specificity.

Since the initial discoveries of adenoviruses (Rowe et al., 1953; Hilleman and Werner, 1954), these agents have generated intense interest because of the varied clinical and cellular responses they induce. In vivo, adenoviruses produce infections which range from acute, febrile diseases to latent infections and induction of malignancy in unnatural hosts. Similarly, in cell cultures the reactions vary from acute cytopathic effects to cellular transformation. This array of host responses evolves from the nuclear infection which adenoviruses establish, and thus offers the opportunity to investigate intranuclear replication and transcription of an easily identified and manipulated species of DNA in eukaryotic cells. Since mammalian cells are so complex and contain on the order of 107 genes, it is a most difficult task to study directly the molecular reactions regulating cellular DNA synthesis, transcription of its complex genome, modification of its mRNAs, and translation of its messengers into functional gene products. Study of the smaller adenovirus genome, which contains a maximum of 50 genes and replicates in the nucleus of an eukaryotic cell, offers a simpler model which should yield evidence germane to the molecular biology of mammalian cells as well as to viral biosynthesis. Investigations with bacteriophages, however, pointed to the problem that biochemical techniques alone were insufficient to reveal the controls governing many intracellular reactions. Selected deletion and conditionally lethal mutants were essential to expose the balanced mechanisms regulating the biosynthesis and assembly of bacteriophage components.

Interferon induces protection of enucleated BSC-1 cells against infectious vesicular stomatitis virus production if cells are treated before, but not after, enucleation.