Temperature-independent revertants of adenovirus H5ts125 and H5ts107 mutants in the DNA binding protein: isolation of a new class of host range temperature conditional revertants

H5ts125 and H5ts107 are temperature sensitive mutants of type 5 adenovirus in the structural gene for the DNA binding protein. Spontaneous, temperature-independent revertants of these mutants were isolated by growth in HeLa cells at 39°. Out of 30 independently isolated temperature-independent revertants grown in HeLa cells, two isolates were found to retain their temperature sensitive phenotype when grown or plaqued in 293 cells, which are a human cell line transformed by type 5 adenovirus. These two revertants represent a new class of host range temperature conditional mutants. It is suggested that these revertants arise by either a viral-encoded extragenic suppressor mutation or a second site intragenic mutation that recognizes cellular proteins in HeLa and 293 cells differently.     

Host range temperature-conditional mutants in the adenovirus DNA binding protein are defective in the assembly of infectious virus

R(ts107)202 is a host range temperature-conditional mutant of adenovirus type 5. This mutant is temperature sensitive for replication and plaquing in 293 cells but is temperature independent for growth and plaquing in HeLa cells (J.C. Nicolas, F. Suarez, A. J. Levine, and M. Girard (1981)Virology108, 521–524). The mutant was isolated in HeLa cells as a temperature-independent revertant of the H5ts107 temperature-sensitive mutant that maps in the adenovirus DNA binding protein (DBP). The reasons for the temperature conditional phenotype of this mutant in 293 cells were investigated. The mutant synthesized an unstable DBP in both HeLa and 293 cells at 39°. In 293 cells at 39°, about two- to threefold less viral DNA was synthesized by r(ts107)202 as compared to Ad5wt. R(ts107)202 infected cells at 39° produced normal (wild-type) amounts of all detectable late viral structural proteins. The mutant failed, however, to produce infectious virus or assemble virus particles in 293 cells at 39°. The altered DBP may therefore play a role in the assembly of virus particles, either directly or indirectly via an altered DNA structure. The failure of r(ts107)202 to assemble virion particles in 293 cells at 39° furthermore suggests that virus assembly is dependent upon cellular factors that differ in HeLa and 293 cell.     

Structure and function of adenovirus DNA binding protein: comparison of the amino acid sequences of the Ad5 and Ad12 proteins derived from the nucleotide sequence of the corresponding genes

The adenoviral DNA binding protein (DBP) is a multifunctional protein involved in DNA replication and gene expression. In order to investigate the relation between structure and function of DBP, the amino acid sequences of the serotypes 5 and 12 (Ad5 and Ad12) have been compared. The amino acid sequence of Ad5 DBP was previously established by nucleotide sequence analysis of the Ad5 DBP gene (W. Kruijer, F. M. A. Van Schaik, and J. S. Sussenbach, Nucl. Acids Res. 9, 4439-4457, 1981). In this study the analysis of the Ad5 DBP gene and adjacent regions by determination of the sequence of the first leader in late DBP mRNA's and the splice point between the tripartite leader and the main body of the mRNA encoding the 100-kDa protein has been extended. The nucleotide sequence of the Ad12 DBP gene is also described. From the nucleotide sequence and RNA mapping data of Ad12 DBP mRNA's (I. Saito, J. Sato H. Handa, K. Shiraki, and H. Shimojo, Virology 114, 379-398, 1981) the complete Ad12 DBP amino acid sequence could be deduced. Ad12 DBP contains 484 amino acids and has an actual Mr of 54,992. It is 45 amino acids shorter than Ad5 DBP. Comparison of the Ad12 and Ad5 DBP amino acid sequences shows that several longer deletions are present in the N-terminal 125 amino acid residues of Ad12 DBP. In contrast, only a single amino acid deletion and insertion is found in the C-terminal 359 amino acids of Ad12 DBP. The N- and C-terminal domains of Ad12 and Ad5 DBP are 45 and 80% homologous, respectively. This suggests that both domains of DBP are subjected to different evolutionary pressures. Analysis of various Ad5 mutants with an altered DBP gene, has indicated that the C-terminal domain is involved in DNA replication and early gene expression, while the N-terminal domain has a role in late gene expression in monkey cells. These results are discussed in relation to the structure and function of adenovirus DBP.     

Hexon trimerization occurring in an assembly-defective, 100K temperature-sensitive mutant of adenovirus 2

Analysis of 100K-defective temperature-sensitive adenovirus mutants confirmed the multifunctional character of the nonstructural, virus-coded 100K protein. In addition to its function in hexon trimerization (altered in H5ts1), and its possible direct or indirect role in hexon transport to nucleus (mutated in H2ts118), genetic and biochemical evidence was presented that 100K play some critical role in the scaffolding process of adenovirus capsid. This function appeared to be defective in H2ts107 and to map between coordinates 69.0 and 69.9, leftward from the H5ts1 lesion (70-73 map units; Arrand, 1978). This corresponded to the central domain of the 100K protein, between amino acid 300 and 400 from the N end. DNA sequencing of cloned fragments of H2ts107 DNA overlapping the mutation revealed two point mutations on the same codon at nucleotide 25,082 and 25,083 (GAC----GCA), corresponding to a nonconservative amino acid change (aspartic acid----alanine) at position 324 in the 100K sequence. 100K of adenovirus 2 wild type (WT) was found to bind in significant amounts to novobiocin-affinity column, and to be coeluted with hexon, penton, IIIa, and cellular topoisomerase II activity, by novobiocin- or ATP-Mg2+-containing buffers. H2ts107 100K also bound to novobiocin column, but the elution pattern differed from that of WT, suggesting some alteration in the affinity of the mutated 100K for novobiocin. The same behavior on affinity column as H2ts107 100K was observed for 90K, a cleavage product of the 100K, found in great abundance in H2ts107 at 39.5 degrees and corresponding to the C-terminal moiety of the 100K molecule. This implied that the "novobiocin-binding" domain of the 100K was not confined at its N terminus, and was altered in the H2ts107 mutant.     

Revertants of temperature-sensitive mutants of reovirus: evidence for frequent extragenic suppression.

Twenty-eight independently isolated, spontaneous revertants isolated from temperature-sensitive (ts) mutants of reovirus type 3 representing all the known mutant groups, were examined to determine whether they were intragenic revertants or contained extragenic suppressor mutations. Analysis of the progeny of backcrosses of the revertants to wild type, showed that 25 of the 28 revertants contained is lesions. This result indicated that 25 of the 28 revertants were suppressed pseudorevertants with the suppressor mutation in a gene that could be separated from the parental is lesion by recombination. The nature of the is lesion(s) was examined for a number of the is clones derived from back-crosses. In every case, except one, the parental is lesion was found. In five of the ten suppressed pseudorevertants examined, nonparental is lesions could also be rescued. Two of the nonparental is lesions were in the previously defined recombinant groups. Five of the nonparental is lesions represented a new recombination group or groups since they recombined with the prototype mutants of all of the defined recombination groups. Recombination analysis indicated that the five new mutants fall into two recombination groups for which we propose the designations H and I. The nonparental is lesions rescued from suppressed pseudorevertants may represent suppressor mutations with is phenotype. However, the majority of the suppressor mutations identified had no temperature phenotype and were identified only by their effect on the phenotype of the original is lesion. The fact that a large proportion of revertants were suppressed by extragenic suppressor mutations suggests that mutation events leading to extragenic suppression occur at a much higher frequency than do intragenic events leading to revertant phenotype. These results indicate a general mechanism by which RNA viruses can bypass the effects of deleterious mutations in the absence of intramolecular recombination.     

Multiple effects of the 72-kDa, adenovirus-specified DNA binding protein on the efficiency of cellular transformation

The early region 2A gene (E2A) of adenovirus types 2 and 5 encodes a 72-kDa DNA binding protein (DBP) which contains two physical domains comprising approximately the amino-terminal one-third and carboxyl-terminal two-thirds of the protein, respectively. Previous work has shown that some Ad5 mutants containing temperature-sensitive (ts) mutations in the carboxyl-terminal domain of DBP, such as Ad5ts125, show a 3- to 8-fold enhanced ability to transform rat cells. We have examined the transformation characteristics of a series of Ad5 E2A deletion mutants, Ad5dl801-5, which encode either no functional DBP or encode truncated, defective DBPs. The E2A deletion mutants transformed rat embryo cells at frequencies similar to wild-type (wt) Ad5. These results suggest that the high transformation phenotype of carboxyl-terminal E2A mutants like Ad5ts125 is not due to the simple inactivation of DBP function, but rather results from an activity possessed by an altered DBP. This hypothesis is supported by the fact that the transformation phenotype of Adsts125 and similar mutants is dominant over the wild-type phenotype. A number of additional Ad2 and Ad5 E2A mutants were examined with respect to their ability to transform primary rat embryo cells. It was found that a carboxyl-terminal E2A mutant, Ad2+ND1ts23, also showed the enhanced transformation phenotype. In contrast, several amino-terminal E2A host-range (hr) mutants, originally isolated on the basis of their ability to replicate in monkey cells, transformed rat embryo cells at a frequency similar to wild-type virus. Ad2ts400, and E2A mutant with alterations in both DBP domains, showed a wild-type frequency of transformation, while two similar mutants, Ad5ts125 X 405 and Ad5ts125 X 404, showed an enhanced frequency. Last, it was found that coinfection of primary rat embryo cells with the hr mutants plus Ad5ts125 or Ad2+ND1ts23 resulted in a wild-type frequency of transformation, demonstrating that the hr mutants are dominant to the ts mutants with regard to transformation phenotype. Thus, DBP can both positively and negatively affect viral transformation in this system.     

Effects of the adenovirus H5ts125 and H5ts107 DNA binding proteins on DNA replication in vitro

Genetic and biochemical studies of adenovirus (Ad) DNA synthesis in vitro demonstrate that the Ad DNA binding protein (Ad DBP) is not necessary for the initiation of Ad DNA synthesis but is required for chain elongation. The DBP, which enhances early elongation to the 26th deoxynucleotide by approximately two- to fourfold, is absolutely required as chain elongation proceeds further. Ad DNA synthesis was assayed in a system requiring Ad DNA covalently linked at each 5' terminus to a protein (Ad DNA-pro), various fractions of Ad-infected cytoplasm, and an extract of uninfected Hela nuclei. Initiation of Ad DNA replication was measured by the formation of a covalent complex between the 80,000 dalton preterminal protein (pTP) and 5' dCMP. DNA binding proteins from two ts mutants, H5ts125 and H5ts107, have been purified and shown to be functional at 30 degrees but inactive at 38 degrees in an in vitro elongation system dependent on purified proteins. Chymotryptic cleavage of the 72K wild-type Ad2 DBP produces a 34K carboxyl terminal fragment which retains full activity in the in vitro elongation of Ad DNA.     

Functional interactions of the domains of the adenovirus DNA binding protein

The 34-kDa fragment of the carboxyl end of the adenovirus (Ad) DNA binding protein (DBP) binds to single-stranded (ss) DNA and is able to replace the intact 72-kDa DBP needed for Ad DNA replication in vitro. A similar fragment prepared from the temperature-sensitive (ts) mutant, H5ts107, which has a single amino acid change in the carboxyl end of the DBP, is temperature sensitive for DNA replication and defective in binding to ssDNA. However, in 20 mM NaCl which is the salt concentration during Ad DNA replication in vitro, the intact 72-kDa H5ts107 DBP is defective only in replication but not binding to DNA at nonpermissive temperatures. These observations indicate that the amino domain of the H5ts107 DBP can stabilize the binding of its carboxyl end to DNA.     

Extragenic and intragenic suppression of a transport mutation in the hemagglutinin gene of an influenza A virus as revealed by backcross and sequence determination

Cooperation of viral proteins, or functional domains within a protein, can be studied by analyzing temperature-sensitive (ts) mutants and revertants carrying suppressor mutations. Accordingly, we have sequenced the hemagglutinin (HA) genes of a ts mutant of fowl plague virus (FPV), with a transport defect in the HA, and of five independent ts+ revertants (R1, R3, R4, R5, and R9). The amino acid replacement in position 480 from Thr to Ile, leading to the loss of a complex carbohydrate side chain, is responsible for the ts phenotype. R3, R4, and R5 are true revertants in that they have Thr in position 480, while R1 and R9 have kept Ile. The sequence of the HA of R1 is exactly the same as that of the ts mutant, while the R9 HA has two additional amino acid replacements in positions 91 (Lys-Thr) and 104 (Gly-Val). By doing a backcross with wild-type virus, it was shown that R1 carries an extragenic suppressor mutation, while R9 is intragenically suppressed. We conclude that the HA is transported from the site of its synthesis in the rough endoplasmic reticulum (RER) to the plasma membrane along with another viral gene product, which by mutation can complement the ts defect. An alternative interpretation is that the ts mutation results from a change in HA which allows an interacting protein to bind HA too soon, holding it back in the RER. The suppressor mutation may remove this premature interaction.     

Spontaneous reversion of a C/T transition mutation in the adenovirus endoproteinase gene

A temperature-sensitive mutation (H2ts1) that abolishes the viral endoproteinase activity at the nonpermissive temperature has been mapped by marker rescue between map coordinates 59.8 and 61.9 on the adenovirus type 2 genome. The mutation has been identified by sequencing to be a C/T transition at coordinate 61.1 changing a proline residue to a leucine residue and eliminating a HaeIII restriction enzyme cleavage site (L. Yeh-Kai, G. Akusjarvi, P. Alestrom, U. Pettersson, M. Tremblay, and J. Weber, 1983, J. Mol. Biol. 167, 217-222). This feature of the mutation offered a convenient assay to distinguish between true revertants and suppressor mutations among phenotypic revertants of H2ts1. Seventeen spontaneous revertants were isolated in three independent experiments by picking plaques at 39 degrees after three passages of H2ts1 at 39 degrees in HEp2 cells. All revertants grew like wild-type virus and regained normal endoproteinase activity. The Ncol fragment encompassing the H2ts1 region was terminally labeled and subcleaved with HaeIII to determine the presence or absence of the HaeIII site at 61.1 for each revertant. All revertants had regained the HaeIII site by true reversion. We conclude that the H2ts1 mutation probably lies in a critical domain of the enzyme and is therefore not suppressible, and that the C/T transition at coordinate 61.1 is the sole cause of the H2ts1 phenotype.     

Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.     

Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein

Two hybridoma cell lines were obtained, A1 and B6, which produced monoclonal antibodies reacting with the 44,000-MW C-terminal domain of the adenovirus type 5 DNA-binding protein (DBP). Clone A1 antibodies reacted with the native form of the DBP, but failed to recognize this protein after denaturation (by exposure to sodium dodecylsulfate, or production of the DBP at 39.5 degrees by H5ts107, a temperature-sensitive DBP mutant). Clone B6 antibodies bound to both the native and denatured forms of the DBP. Immunofluorescent staining of wild-type-virus infected cells revealed the DBP located in discrete nuclear patches. A1 and B6 antibodies detected this patched localization of the DBP in nuclei of H5ts107-infected cells grown at 32 degrees. However, at the nonpermissive temperature of 39.5 degrees, A1 antibodies failed to detect the DBP, and B6 antibodies gave a uniform nuclear fluorescent distribution of the DBP. Thus the nuclear pattern of localization for the DBP synthesized by H5ts107 was temperature dependent in this mutant.     

UL5, a protein required for HSV DNA synthesis: genetic analysis, overexpression in Escherichia coli, and generation of polyclonal antibodies

The mutations in two DNA-negative ts mutants of herpes simplex virus type 1 (HSV-1), tsK13 and tsM19, have been previously mapped to a 2.0-kb fragment (coordinates 0.095-0.108) at the left end of the genome (S. Weller, D. Aschman, W. Sacks, D. Coen, and P. Schaffer, 1983, Virology 130, 290-305). Sequence analysis of the HSV-1 genome has revealed the existence of two open reading frames, UL5 and UL6, within this fragment (D. McGeoch, M. Dalrymple, A. Dolan, D. McNab, L. Perry, P. Taylor, and M. Challberg, 1988, J. Virol. 62, 444-453). In this paper we report fine mapping and sequence analysis of the mutations in tsK13 and tsM19 which unambiguously localize the mutations to UL5, predicted to encode a 99-kDa polypeptide. The mutation in tsK13 was shown to result in a single amino acid substitution, Pro236 to Leu, whereas tsM19 contains two substitutions, Pro236 to Ser and Ala249 to Val. Thus, both mutants are altered in Pro236. Temperature-shift experiments indicated that the UL5 gene product is required continuously during viral DNA synthesis, suggesting a direct role for the 99K protein in viral DNA synthesis. The UL5 gene product was overexpressed in Escherichia coli and used to generate polyclonal antibodies which detected proteins in HSV-1-infected cell extracts from 4 hr postinfection. Although a faint band of the predicted size (99 kDa) was observed, the majority of the immunoreactive material migrated as smaller bands which represent either proteolytic degradation during extraction or post-translational proteolytic modification of the UL5 gene product. Indirect immunofluorescence staining revealed that the UL5 gene product localizes to the nucleus in two patterns: diffuse staining throughout the nucleus and in discrete globules which appear at the periphery of the nucleus. Sequence analysis of the UL5 gene predicts that the 99-kDa protein contains a consensus sequence for an ATP binding site. Possible roles of this protein in viral DNA synthesis are discussed.     

Identification of defects in the neuraminidase gene of four temperature-sensitive mutants of A/WSN/33 influenza virus

Four influenza (A/WSN/33) mutants, temperature sensitive (ts) for neuraminidase (NA) (Sugiura et al., 1972, 1975) were analyzed. All four ts mutants were found to be defective at the nonpermissive temperature (39.5 degrees) both in enzymatic activity and in transport to the cell surface. Upon shift down to the permissive temperature (33 degrees), enzymatic activity and transport to the cell surface were both restored suggesting that the mutational defect is reversible. Comparative sequence analysis of the NA gene from ts mutants, their revertants and wild type WSN viruses revealed that in each case single point mutations causing amino acid substitutions were associated with the ts defect. The positions of each point mutation when mapped in the three-dimensional structure of NA varied. However, all four amino acid substitutions were located in beta-sheet strands of the head region. Several other amino acid changes not essential for the ts phenotype were found in each mutant NA. The nonessential changes were localized either in the stalk region or in the loop structures of the head, but none in the beta-sheet strands. Because both enzymatic activity and transport of NA were affected in all four mutants, we propose that the mutational phenotype is caused by a change in overall conformation rather than a localized change in the sialic acid binding site.     

Mutations affecting conformation or sequence of neutralizing epitopes identified by reactivity of viable plaques segregate from syn and ts domains of HSV-1(F) gB gene

Three classes of HSV-1(F) mutants expressing a resistance phenotype to two highly potent-type common monoclonal antibodies, H126-5 and H233, to glycoprotein B (gB) were selected. Class 1 mutants, selected for resistance to neutralization from nonmutagenized virus stocks, expressed a gB which reacted in biotin-avidin-enhanced surface immunoassays and in immune precipitation tests with the selecting antibodies. Class 2 and 3 mutants were selected for nonreactivity in the biotin-avidin-enhanced surface immunoassay from BUdR-mutagenized, preneutralized virus stocks, but differ in that the selecting antibodies immune precipitated the gB of Class 2 but not that of Class 3. Mutants expressing a resistance phenotype to one monoclonal antibody (H126-5 or H233) invariably retained reactivity in all tests with the heterologous antibody, and recombinants resistant to both antibodies were produced by cotransfection of intact DNA of one mutant with a cloned DNA fragment from another mutant. Class 1 mutations were mapped by marker transfer to a 1734-bp DNA fragment. Class 2 and 3 mutations were mapped to a region defined by a maximum of 377 bp and a minimum of 46 bp, in a biotin-avidin-enhanced surface immunoassay with a panel of DNA fragments of HSV-1(F) BamHI G carrying staggered deletions across the region encoding gB. This region does not overlap the neutralizing antibody determinant site mapped by T.C. Holland, R.M. Sandri-Goldin, L.E. Holland, S.D. Marlin, M. Levine, and J. Glorioso (1983, J. Virol. 46, 649-652) and is located 3' to the ts lesion of HSV-1(HFEM)tsB5 and 5' to the syn3 locus of that virus. It was concluded that (i) inasmuch as the biotin-avidin-enhanced surface immunoassay does not destroy the virus contained in the plaque, it is a rapid and convenient method for both identification and selection of mutants reactive and nonreactive to specific monoclonal antibodies. (ii) gB may contain multiple domains carrying epitopic sites of neutralizing monoclonal antibodies. (iii) The resistance phenotype may arise from mutations which alter the conformation or the amino acid sequence of the epitope. These mutations might be differentiable on the basis of reactivity of mutated gB with selecting monoclonal antibody in nondenaturing and denaturing environments, respectively.     

The SV40 A gene product suppresses the adenovirus H5ts125 defect in DNA replication

The adenovirus H5ts125 mutation is a temperature sensitive defect in the structural gene for a DNA-binding protein required for viral DNA replication. Infection of primary African green monkey kidney cells with SV40, followed by superinfection with H5ts125 at the nonpermissive temperature, permits the replication of adenovirus DNA even in the absence of detectable adenovirus DNA binding protein. The SV40 A gene product is required continuously for the replication of H5ts125 adenovirus DNA. The rate of adenovirus DNA replication in SV40 and H5ts125 doubly infected cells at nonpermissive temperature is about one-half that observed for either H5ts125 singly or SV40-H5ts125 doubly infected cells at the permissive temperature. H5ts125 progeny virus are also produced in the SV40-H5ts125-infected cells at the nonpermissive temperature but the yields are only 3% of those observed in doubly infected cells at the permissive temperature. The suppression of the H5ts125 phenotype by SV40 requires a prior infection of primary or secondary African green monkey kidney cells with a high multiplicity of SV40 (50–100 PFU/cell) and H5ts125 DNA replication is optimally observed between 10 and 15 hr after adenovirus superinfection. These observations suggest that the SV40 A gene product could be involved in the initiation of adenovirus DNA replication thereby bypassing the need for the adenovirus DNA-binding protein.     

Selection for temperature-sensitive mutations in specific vaccinia virus genes: isolation and characterization of a virus mutant which encodes a phosphonoacetic acid-resistant, temperature-sensitive DNA polymerase

Seven temperature-sensitive mutants of vaccinia virus have been isolated after preselection for virus resistant to phosphonoacetic acid (PAA). In all seven mutants, the PAA-resistant (PAAr) and ts lesions represent separate mutations. In one mutant, NG26, the PAAr (NG26-PAAr) and ts (NG26-ts) mutations are very closely linked. Both NG26-ts and NG26-PAAr map in the HindIII E DNA fragment. NG26 has a DNA-negative phenotype at 40 degrees. NG26-ts is in the same complementation group as ts42, another DNA-negative mutant which maps in the HindIII E DNA fragment (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128, 000-000, 1983). The order of the mutations is (NG26-ts)-(NG26-PAAr)-ts42. The virus-coded DNA polymerase has been partially purified from wt- and NG26-infected cells. The DNA polymerase encoded by NG26 is temperature sensitive and PAA resistant in vitro as compared to the wt enzyme.     

Characterization of virus DNA synthesized in KB cells infected with two temperature-sensitive mutants of adenovirus type 5.

KB cells were infected with H5ts36 or H5ts125, two adenovirus type 5 (Ad5) mutants with a temperature-sensitive synthesis of virus DNA. Infection was started at the nonpermissive temperature and at 16 h p.i. the temperature was shifted down to the permissive temperature. Shortly after the shift-down H5ts125-infected cells showed an accumulation of purely single-stranded DNA of virus origin, which was not observed in H5ts36-infected cells. This single-stranded DNA has been characterized by hybridization and sedimentation analysis. It was found that the single-stranded DNA was derived from both complementary strands and consisted of short fragments. The observation that the single-stranded DNA accumulates in H5ts125-infected cells under conditions in which the amount of DNA binding protein is reduced, suggests that the DNA-binding protein is not only involved in initiation, but also in elongation of nascent strands.     

Transformation by adenovirus early region 2A temperature-sensitive mutants and their revertants

H5ts107 is a temperature-sensitive mutant of adenovirus type 5 whose alteration maps in the structural gene for the viral DNA binding protein. Temperature-independent revertants of this mutant that form plaques at 39° in HeLa cells have been isolated. These revertants fall into two classes: (1) ts⁺ revertants for growth and plaque formation at 39° in both HeLa cells and 293 cells, a human cell line transformed by type 5 adenovirus; (2) ts⁺ for growth and plaque formation at 39° in HeLa cells, but temperature-sensitive (ts) for growth and plaque formation in 293 cells. The frequency of rat cell transformation by H5tsl07 was fivefold higher than that of wild-type adenovirus. A class 1 revertant, r(tsl07)127, transformed cells at the wild-type virus frequency while a class 2 revertant, r(tsl07)202, retained the ability to transform cells at the higher frequency. When the XhoI-C DNA fragment from these viruses, which contains the left end 15.5% of the viral genome, was employed to transform rat cells, then Ad5 wt, H5tsl07, r(tsl07)127, and r(ts107)202 DNAs all transformed with approximately equal frequencies.