H5ts125-infected Cells Research Articles

Regulation of the biosynthesis of subgroup C adenovirus protein IVa2

The IVa2 gene is located between 16 and 11.3 map units on the left strand of the adenovirus type 5 (Ad5) genome. The coded RNA contains an intron of 277 nucleotides. To determine whether protein IVa2 is synthetized during productive infection and to obtain an immunological reagent to study its function, we prepared antibodies directed to 414 amino acids of protein IVa2 fused to the N-terminal domain of Staphylococcus aureus protein A. Western immunoblot analysis of viral proteins demonstrates that protein IVa2 is a minor component of mature viral particles and that it is also present in assembly intermediates and young virions. Thus, contrary to a previous report (H. Persson, B. Mathisen, L. Philipson, and U. Pettersson, Virology 93:198-208, 1979), protein IVa2 is not related to the 50-kDa polypeptide, a scaffolding protein present in assembly intermediates. The biosynthesis of protein IVa2 during productive infection was examined. Time course studies using immunofluorescence analysis with polyclonal antibodies targeted to protein IVa2 revealed that this protein is first synthesized at 12 h in a few cells exhibiting very striking fluorescence. Synthesis continues until at least 24 h postinfection. When hydroxyurea is added, protein IVa2 is not detected. In cells infected with mutant H5 ts125, blocked at the nonpermissive temperature (40 degrees C) in viral DNA replication, protein IVa2 is overexpressed. These results suggest that protein IVa2 synthesis requires cellular rather than viral DNA replication. RNase protection assay results indicate that hydroxyurea inhibits protein IVa2 synthesis at the transcriptional level. Thus, overexpression of protein IVa2 in H5 ts125-infected cells may be regulated at the translational level.

Temperature-sensitive initiation and elongation of adenovirus DNA replication in vitro with nuclear extracts from H5ts36-, H5ts149-, and H5ts125-infected HeLa cells.

Adenovirus DNA replication was studied in vitro in nuclear extracts prepared from HeLa cells infected at the permissive temperature with H5ts125, H5ts36, or H5ts149, three DNA-negative mutants belonging to two different complementation groups. At the restrictive temperature, H5ts125 extracts, containing a thermolabile 72-kilodalton DNA-binding protein, enable the formation of an initiation complex between the 82-kilodalton terminal protein precursor (pTP) and dCTP, but further elongation of this complex is inhibited. Wild-type DNA-binding protein or a 47-kilodalton chymotryptic DNA-binding fragment can complement the mutant protein in the elongation reaction. No difference in heat inactivation was observed between wild-type extracts and H5ts36 or H5ts149 extracts when the replication of terminal XbaI fragments of adenovirus type 5 DNA-terminal protein complex was studied. In contrast, the formation of a pTP-dCMP initiation complex, as well as the partial elongation reaction up to nucleotide 26, were consistently more temperature sensitive in mutant extracts. The results suggest that the H5ts36/H5ts149 gene product is required for initiation of adenovirus type 5 DNA replication and that the 72-kilodalton DNA-binding protein functions early in elongation.

A mutation in the adenovirus type 5 DNA binding protein that fails to autoregulate the production of the dna binding protein

H5ts125 is a temperature-sensitive mutant of type 5 adenovirus whose defect maps in the structural gene of the viral DNA binding protein. Among a large number of temperature-independent revertants of this mutant, that plaque efficiently at 39° in HeLa cells, one revertant, r(ts125)13, has been studied in more detail. The synthesis and stability of the adenovirus DNA binding protein in HeLa cells infected with Ad5wt, H5ts125, and r(ts125)13 was investigated by pulse-chase experiments with [ 35S]methionine. In H5ts125-infected cells at the nonpermissive temperature about 1 1 2 to 2 1 2 - fold more DNA binding protein was synthesized in the pulse-labeling period than was detected in wild-type infected cells. Unlike the wild-type protein, the H5ts125 DNA binding protein was unstable and it was degraded during the chase period. In r13-infected HeLa cells, the DNA binding protein was overproduced 3- to 5-fold during the pulse-labeling time. The r13 DNA binding protein was stable and accumulated throughout the chase period. The r13 DNA binding protein was not overproduced and remained stable during a chase period, in human 293 cells on monkey CV-1 cells indicating that the faulty autoregulation of the r13 DNA binding protein synthesis was host cell or host range specific. These experiments provide genetic evidence for the adenovirus DNA binding proteins' ability to autoregulate its own synthesis. In addition the results suggest that cellular functions, interacting with the DNA binding protein, play a role in this autoregulatory event.

Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells.

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.

Initiation of adenovirus DNA replication: detection of covalent complexes between nucleotide and the 80-kilodalton terminal protein.

We have previously shown that the 5'-terminal deoxycytidine residue of each nascent adenovirus 5 DNA strand synthesized in vitro is covalently linked to the 80-kilodalton (kd) terminal protein precursor via a phosphodiester bond to a serine residue in the protein. When extracts prepared from adenovirus 5-infected cells are incubated with [alpha-33P]dCTP as the only added deoxynucleoside triphosphate, complexes consisting of nucleotide covalently linked to the 80-kd protein can be detected. The nucleotide moieties present in such complexes include d(pC) and d(pCpA), the 5'-terminal nucleotide and dinucleotide of adenovirus 5 DNA, respectively, as well as some longer oligonucleotides. The formation of these complexes requires the presence of adenovirus DNA containing the attached 55-kd terminal protein and ATP. Extracts from H5ts125-infected cells which are defective in DNA replication catalyze complex formation to the same extent as extracts prepared from wild-type infected cells; thus, the presence of the adenovirus-coded 72-kd DNA-binding protein is apparently not required. Most, if not all, of the 80-kd protein-nucleotide complexes that are formed are noncovalently bound to the input viral DNA. These observations are consistent with the protein-priming model for the initiation of adenovirus DNA replication.

Regulation of early adenovirus transcription: a protein product of early region 2 specifically represses region 4 transcription.

An aspect of the regulation of early adenovirus gene expression has been studied by measuring the rates of transcription of several viral transcription units during infection by wild-type (WT) adenovirus type 5 and a temperature-sensitive mutant, H5ts125. It has previously been shown that two transcription units (regions 2 and 4) are subject to negative control. During the course of early infection, transcription of regions 2 and 4 increased to a maximal rate and then declined. The decline from each of these transcription units appeared to be a specific shutoff of transcription, because the transcription of another early transcription unit (region 1A) remained constant during early infection and, in the absence of protein synthesis, the apparent shutoff of region 2 and region 4 transcription did not occur. At the nonpermissive temperature for the mutant, the kinetics of transcription of early region 2 were identical to the kinetics of transcription of WT. Thus, the repression of transcription of region 2 occurred in H5ts125-infected cells as well as in WT-infected cells. Region 4 transcription, however, was not repressed during H5ts125 infection at the nonpermissive temperature. After a maximal rate of transcription was reached, this rate was maintained throughout the course of the experiment. Furthermore, the repression of region 4 transcription appears to be the result of a block of initiation of transcription rather than the result of an inducement of premature termination of transcription. Therefore, it can be concluded that a product of the adenovirus region 2 gene, which is the site of the mutation for H5ts125, is responsible for the specific shutoff of region 4 transcription.

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.

Adenovirus DNA-binding protein in cells infected with wild-type 5 adenovirus and two DNA-minus, temperature-sensitive mutants, H5ts125 and H5ts149

Studies have been done to characterize further H5ts125, an adenovirus type 5 conditionally lethal, temperature-sensitive (ts) mutant defective in initiation of DNA synthesis and to investigate whether the single-strand-specific DNA-binding (72,000 molecular weight) protein is coded by the mutated viral gene. When H5ts125-infected cells were labeled with [35S]methionine at 32 degrees C and then incubated without isotope at 39.5 degrees C, the mutant's nonpermissive temperature, the 72,000 molecular weight polypeptide was progressively degraded. Immunofluorescence examination of cells infected with wild-type virus, H5ts125, and H5ts149 (a second, unique DNA-minus mutant) showed that immunologically reactive DNA-binding protein was barely detectable in H5ts125-infected cells at 39.5 degrees C, whereas this protein was present in wild-type- and H5TS149-infected cells, that the protein made at 32 degrees C in H5ts125-infected cells lost its ability to bind specific DNA-binding protein antibody when the infected cells were shifted to 39.5 degrees C, and that if H5ts125-infected cells were shifted from the restrictive temperature to 32 degrees C, even in the presence of cycloheximide to stop protein synthesis, immunologically reactive DNA-binding protein reappeared.

Evidence for a function of the adenovirus DNA-binding protein in initiation of DNA synthesis as well as in elongation of nascent DNA chains

Early in lytic infection of KB cells with adenovirus type 5 (Ad5) a viral-coded single-strand-specific DNA-binding protein with a molecular weight of 72,000 is synthesized. The function of this protein in viral DNA synthesis was studied using two different methods for specific inactivation. First, DNA synthesis was examined in KB cells infected with an early temperature-sensitive mutant, H5ts125, which produces a thermolabile DNA-binding protein. At the nonpermissive temperature (40°) the start of the first and subsequent rounds of DNA replication was inhibited. However, viral DNA synthesis in vitro using a system of isolated nuclei obtained from H5ts125-infected KB cells was not thermosensitive compared to wild type. In this in vitro system, replicating molecules were normally converted to mature DNA at 40° without reinitiation of new replication rounds. These results show that thermal inactivation of the H5ts125 DNA-binding protein destroys its capacity to function in the initiation of new replication rounds but does not impair chain growth. As a second method of inactivation we studied viral DNA synthesis in vitro in the presence of antibody against the purified DNA-binding protein. A 50–60% inhibition of the rate of viral DNA synthesis was observed while cellular DNA synthesis was not affected. Density-labeling experiments showed that the antibody inhibits the duplication of both complementary strands, presumably by a decrease of fork movement in replicating adenovirus DNA. The results suggest that the Ad5 DNA-binding protein functions in initiation of DNA replication as well as in elongation of nascent viral DNA chains.

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

The type 5 adenovirus single-strand-specific DNA-binding protein can be labeled with 32PO 4 added as inorganic phosphate to the medium of infected monkey or human cells. The DNA binding protein in the infected cell can be isolated as a phosphoprotein at early and late times after infection. Most or all of the [ 32P]phosphate can be removed from the DNA-binding protein by alkaline phosphatase or hydrolysis at alkaline pH. The dephosphorylated protein retains the ability to bind specifically to single-stranded DNA. H5 ts125 is a mutation in the structural gene of the 72,000 MW single-strand-specific DNA-binding protein. When cells infected with this viral mutant at the permissive temperature are shifted to the nonpermissive temperature, the phosphorylation of the 72,000 MW protein decreased rapidly (within 30 min) even though approximately normal (wild type) levels of this protein (labeled with 35S]methionine for 30 min) were detected in H5 ts125-infected cells shifted to the nonpermissive temperature. An in vitro protein kinase assay utilizing nuclei from infected or uninfected cells has been developed. This in vitro assay can detect the phosphorylation of at least three adenovirus-induced phosphoproteins (100,000 MW; 72,000 MW; 36,000 MW) that are also observed when adenovirus-infected cells are labeled with inorganic [ 32P] phosphate in vivo. The protein kinase activity detected in this in vitro assay is not dependent upon or stimulated by the addition of exogenous cyclic AMP or cyclic GMP. The 72,000 M W protein phosphorylated in vitro by this phosphokinase activity has been shown to be the adenovirus single-strand-specific DNA-binding protein by a specific immunoprecipitation test.

Characterization of single-stranded viral DNA sequences present during replication of adenovirus types 2 and 5

Replication intermediates of adenovirus DNA apparently contain extensive stretches of single-stranded DNA. Such single-stranded viral DNA sequences homologous to different regions of the viral genome present in adenovirus-infected cells during viral DNA replication have therefore been characterized by hybridization to the separated strands of restriction endonuclease fragments of 32P-labeled adenovirus types 2 and 5 DNA. Saturation hybridization experiments with infected cell DNA extracted at late times suggest that all regions of the adenovirus genome are represented in the single-stranded fraction, but at unequal frequencies. This nonuniform representation has been characterized in more detail with self-annealed, total cell DNA extracted 18 hr after adenovirus type 2 infection: the concentration of single-stranded sequences homologous to different regions of the viral genome was determined by comparing the rates of hybridization of 32P-labeled, single-stranded DNA probes with such self-annealed 18 hr DNA to the rates of hybridization of the same probes with equal concentrations of their complements. This approach allows the concentration of single-stranded viral DNA sequences in excess of their complements to be determined. Such sequences can be represented by two concentration gradients across the viral genome: those homologous to the r strand increase in concentration from 27.8–40.9 units toward the right end, whereas sequences homologous to the I strand increase from an area 27.8–40.9 units toward the left end. The time course of synthesis of single-stranded viral DNA sequences relative to accumulation of total viral DNA during the productive cycle and their behavior following a shift of H5ts125-infected cells in which viral DNA replication has begun from a permissive to a nonpermissive temperature support the contention that these sequences are indeed generated as adenovirus DNA is replicated. These results are therefore discussed in terms of current models of adenovirus DNA replication.

Characterization of an adenovirus early protein required for viral DNA replication: A single strand specific DNA binding protein

1. The human adenoviruses types 2, 5 and 12 code for the production of a single strand specific DNA binding protein. The molecular weights of these proteins were 72,000 for types 2 and 5 and 60,000 for type 12. In all three cases proteolytic breakdown fragments of these binding proteins (48,000 MW) were also observed. 2. Analysis of the methionine containing tryptic peptides of these proteins indicate that the types 2 and 5 proteins are similar and clearly distinguishable from the type 12 protein. The peptide maps of these three viral proteins are clearly different from a similar protein found in mock infected cells. 3. Temperature sensitive mutants of type 5 (H5ts125) and type 12(H12tsA275) adenoviruses fail to produce these proteins at the nonpermissive temperature. H5ts125 infected cells grown at the permissive temperature produce a 72,000 MW protein that is thermolabile, for continued binding to DNA, when compared to type 5 wild type adenovirus 72,000 MW protein. An analysis of the phenotype of this adenovirus mutant indicates that it codes for a viral function at early times after infection that is required for viral DNA replication. 4. The in vitro translation of adenovirus specific m-RNA results in the synthesis of a small amount of a 72,000 MW protein that binds to single stranded DNA just like the authentic adenovirus DNA binding proteins produced in infected cells. 5. Adenovirus anti-Tumor antigen (T) anti-serum from hamsters carrying independently derived adenovirus tumors, have been tested for the presence of antibody to purified DNA binding proteins. One antiserum is positive for these antibodies while the other is negative. These results indicate that some, but not all, adenovirus tumors contain large enough levels of the DNA binding proteins to elicit an antibody response. 6. The type 5 adenovirus temperature sensitive mutant, H5ts125, that codes for a thermolabile DNA binding protein, was complemented or suppressed at the nonpermissive temperature, for the replication of adenovirus DNA, by SV40. SV40tsA temperature sensitive mutants, defective in SV40 DNA replication, do not suppress or complement H5ts125 at the nonpermissive temperature.

Viral transcription in KB cells infected by temperature-sensitive "early" mutants of adenovirus type 5.

RNA:DNA hybridization was used to study the synthesis of viral RNA in two DNA-minus, temperature-sensitive mutants of type 5 adenovirus (H5ts125 and H5ts149) belonging to two different, non-overlapping complementation groups. Hybridization competition analysis showed that both mutants transcribed all early gene sequences at the restrictive temperature (41 C). In mutant-infected cells at 41 C, the rate of viral transcription was similar to the rate of early RNA synthesis in wild-type virus infection and was dependent on the multiplicity of infection; little or no late transcription was detected. The shutoff of class I early RNA transcription was shown to be a late function during wild-type virus infection and did not occur at 41 C in mutant-infected cells. When mutant-infected cells were incubated at the permissive temperature (32 C) for 25 h and then shifted to 41 C, the rate of viral DNA synthesis decreased rapidly for H5ts125 and slowly for H5ts149. However, the rate of viral transcription remained unchanged in H5ts125-infected cells for at least 3 h after the temperature shift; although the synthesis of viral DNA had stopped by this time, the synthesis of late viral RNA sequences continued.

An adenovirus type 5 gene function required for initiation of viral DNA replication

Adenovirus type 5 (Ad5) DNA replication was studied after infection of human or monkey cells with two DNA-negative temperature-sensitive mutants belonging to different complementation groups (H5ts125 and H5ts36). When infection was carried out at the permissive temperature (32°) followed by a shift to the nonpermissive temperature (39.5°) viral DNA synthesis in H5ts125-infected cells was reduced 90% within 1 hr after shift-up, while a decline in DNA synthesis in H5ts36-infected cells is only observed after 6 hr. Analysis of the various forms of DNA synthesized under conditions of inhibition showed a constant ratio of replicating to mature viral DNA for both mutants, while no accumulation of replicating molecules was observed. When H5ts125-infected cells were pulse-labeled with [ 3H]thymidine at 32 or 39.5° followed by a chase of the label at 39.5°, replicating DNA was converted into mature DNA at the same rate as in wild-type-infected cells. This indicates that chain propagation and termination could occur normally under nonpermissive conditions. The results of density labeling experiments performed at 39.5° are in agreement with an initiation block in H5ts125-infected cells at the nonpermissive temperature. It is concluded that the H5ts125 gene product and possibly also the H5ts36 gene product are required for the initiation of new rounds of replication. The potential role in initiation of the adenovirus-specific DNA binding protein, which is coded for by the H5ts125 gene, is discussed.