Viral Deoxyribonucleic Acid Replication Research Articles

P53: A Cell Cycle Regulator Activated by DNA Damage

Publisher Summary The chapter focuses on p53 —that is, a cell cycle regulator activated by deoxyribonucleic acid (DNA) damage. Mutations in the p53 tumor-suppressor gene occur at high frequency in a large variety of human tumors. These mutations include deletions of the entire gene, leading to complete loss of p53 expression, and missense point mutations that give rise to mutant p53 proteins lacking wild-type (WT) p53 activity. The high frequency of p53 alterations in human cancer suggests that this gene plays a critical role in the normal control of cell growth. A number of biological and biochemical functions have now been ascribed to WT p53 . Importantly, mutant p53 proteins derived from human tumors are defective in some of these functions, suggesting that such functions are relevant for p53 -mediated growth control and tumor suppression. This chapter reviews some of the recent progress in the exciting p53 field and proposes a model for the activation of p53 in response to DNA damage. The expression of exogenous WT p53 in various transformed cells carrying mutant p53 or completely lacking p53 inhibits cell proliferation. Experiments using replication of viral DNA as a model system have indicated that p53 may fulfill a role in the control of DNA replication. The common association between the viral oncoproteins and p53 represents a remarkable case of convergent evolution and provides strong evidence that p53 is a critical cell cycle regulator. P53 is normally an unstable protein with a half-life of 15–30 min. As a result, its levels in normal cells are almost undetectable and probably insufficient to suppress growth.

Involvement of eucaryotic deoxyribonucleic acid polymerases .alpha. and .gamma. in the replication of cellular and viral deoxyribonucleic acid

In an effort to identify the deoxyribonucleic acid (DNA) polymerase activities responsible for mammalian viral and cellular DNA replication, the effect of DNA synthesis inhibitors on isolated DNA polymerases was compared with their effects on viral and cellular DNA replication in vitro. DNA polymerase alpha, simian virus 40 (SV40) DNA replication in nuclear extracts, and CV-1 cell (the host for SV40) DNA replication in isolated nuclei all responded to DNA synthesis inhibitors in a quantitatively similar manner: they were relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), but completely inhibited by aphidicolin, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP), and N-ethylmaleimide. In comparison, DNA polymerases beta and gamma were inhibited by d2TTP but insensitive to aphidicolin and 20--30 times less sensitive to araCTP than DNA polymerase alpha. Herpes simplex virus type 1 (HSV-1) DNA polymerase and DNA polymerase alpha were the only enzymes tested that were relatively insensitive to d2TTP; DNA polymerases beta and gamma, phage T4 and T7 DNA polymerases, and Escherichia coli DNA polymerase I were 100--250 times more sensitive. The results with d2TTP were independent of enzyme concentration, primer-template concentration, primer-template choice, and the labeled dNTP. A specific requirement for DNA polymerase alpha in the replication of SV40 DNA was demonstrated by the fact that DNA polymerase alpha was required, in addition to other cytosol proteins, to reconstitute SV40 DNA replication activity in N-ethylmaleimide-inactivated nuclear extracts containing replicating SV40 chromosomes. DNA polymerases beta and gamma did not substitute for DNA polymerase alpha. In contrast to SV40 and CV-1 DNA replication, adenovirus type 2 (Ad-2) DNA replication in isolated nuclei was inhibited by d2TTP to the same extent as gamma-polymerase. Ad-2 DNA replication was also inhibited by aphidicolin to the same extent as alpha-polymerase. Synthesis of CV-1 DNA, SV40 DNA, and HSV-1 DNA in intact CV-1 cells was inhibited by aphidicolin. Ad-2 DNA replication was also inhibited, but only at a 100-fold higher concentration. We found no effect of 2'-3'-dideoxythymidine (d2Thd) on cellular or viral DNA replication in spite of the fact that Ad-2 DNA replication in isolated nuclei was inhibited 50% by a ratio of d2TTP/dTTP of 0.02. This was due to the inability of CV-1 and Hela cells to phosphorylate d2Thd to d2TTP. These data are consistent with the hypothesis that DNA polymerase alpha is the only DNA polymerase involved in replicating SV40 DNA and CV-1 DNA and that Ad-2 DNA replication involves both DNA polymerases gamma and alpha.

Reversible Inhibition of Herpes Simplex Virus Replication by Hydroxyurea

Hydroxyurea, at a concentration of 5 x 10(-2) M, inhibits the replication of herpes simplex deoxyribonucleic acid (DNA) in the nuclei of infected cells. As a result, the synthesis of infectious virus progeny was prevented. The presence of parental viral DNA genomes in inhibited cells led to the synthesis of the viral structural peptides. The inhibitory effect of hydroxyurea was reversible; after washing the cells free from hydroxyurea, virus progeny appeared after a lag of 3 h. Upon resumption of viral DNA replication, the content of radioactive viral structural peptides gradually increased in parallel with the increase in mature virions. It is concluded that the information for the synthesis of viral structural peptides is transcribed from the parental DNA genomes.

Gene Expression During the Development of Bacillus subtilis Bacteriophage φ29 I. Analysis of Viral-Specific Transcription by Deoxyribonucleic Acid-Ribonucleic Acid Competition Hybridization

The ribonucleic acid (RNA) specified by bacteriophage phi29 was analyzed to determine its composition at various times in the viral lytic cycle. Although viral-specific RNA was detected immediately after infection, a large increase in the rate was observed at 10 min when DNA synthesis began. phi29 was found to resemble other viruses in that gene expression occurred in two stages which could be defined temporally as "early" and "late." Early RNA appeared before the onset of viral deoxyribonucleic acid (DNA) replication and accounted for approximately 40% of the viral genetic potential. This RNA was also present late in the infectious cycle because of the slow turnover rate of phi29-specific RNA (approximately 10 min half-life) and the continued synthesis of much early viral RNA throughout infection. Late RNA was first detected at approximately the same time as viral DNA replication, although late transcription was not dependent upon DNA synthesis. This RNA was only partially displaced by early RNA in the appropriate competition experiments, suggesting that it contained sequences not present in the early class. Expression of viral genes was sensitive to rifamycin throughout the lytic cycle, the sensitivity resulting from a dependence upon the rifamycin phenotype of the host RNA polymerase.

Transcription During the Development of Bacteriophage φ29: Definition of “Early” and “Late” φ29 Ribonucleic Acid

Bacteriophage phi29 messenger ribonucleic acid (mRNA) production following infection of Bacillus subtilis has been analyzed. Early (e) phi29 RNA, made prior to the onset of phage deoxyribonucleic acid (DNA) replication and exclusively from the light (L) phi29 DNA strand, has been shown by RNA-DNA hybridization-competition experiments to be present throughout the phage latent period. No repression of e RNA production during phi29 development could be demonstrated. Unmodified host RNA polymerase molecules appear to be sufficient for the synthesis of e RNA since phage-specific RNA made in the presence of chloramphenicol hybridizes only to the L strand of phi29 DNA, and this RNA can be effectively competed during hybridization by e RNA. The appearance of late (l) phi29 RNA is coincident with the onset of viral DNA replication. This RNA consists of L DNA strand transcripts which are identical to e RNA and a new class of mRNAs made exclusively from the "heavy" (H) phi29 DNA strand (lH). Protein synthesis in infected cells is required for lH RNA production. Studies with the antibiotic rifamycin demonstrated that synthesis of the major phi29 structural proteins is dependent on production of lH RNA.

Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon.

Three temperature-sensitive (ts) mutants of simian virus 40 (SV40) in complementation group A (tsA7, tsA28, tsA30) have been isolated and characterized in permissive and restrictive host cells. At 41 C in the AH line of African green monkey kidney cells, the mutants are deficient in an early function required to produce infectious viral deoxyribonucleic acid (DNA). Temperature-shift experiments and analysis of SV40 viral DNA replication by gel electrophoresis have provided strong evidence that the ts gene product of the three mutants is directly required to initiate each new round of viral DNA replication but is not required to complete a cycle which has already begun. The synthesis of mutant DNA molecules themselves can be initiated by a nonmutant gene product in viral complementation studies at 41 C. The cell, however, cannot substitute a host function to provide the initiator required for the replication of free viral DNA. The viral initiator is also required to establish the stable transformation of 3T3 cells.

Separation of the herpesvirus deoxyribonucleic acid duplex into unique fragments and intact strand on sedimentation in alkaline gradients.

Deoxyribonucleic acid (DNA) extracted from herpes simplex virions forms multiple partially overlapping bands upon denaturation and centrifugation in alkaline sucrose density gradients. The most rapidly sedimenting DNA corresponds to an intact strand 48 x 10(6) daltons in molecular weight. In this study, we analyzed the DNA fragments generated in alkaline sucrose gradients with respect to size and uniqueness of base sequences. The distribution of sedimentation constants of the various fragments obtained in numerous gradients showed that the fragments smaller than the whole strand fall into six distinct classes ranging in molecular weight from 10 x 10(6) to 39 x 10(6) daltons. Four types of DNA strands can be reconstructed from the whole strand and six fragments on the basis of their molecular weights. DNA from each of the bands self-hybridizes to a lower extent than unfractionated viral DNA, indicating that each of the bands preferentially contains sequences from one unique strand. The data permit reconstruction of four possible types of DNA duplexes differing in the positions of the strand interruptions. Analysis of viral DNA extracted from nuclei of cells labeled with (3)H-thymidine for intervals from 3 to 120 min showed that nascent DNA is invariably attached to small fragments and that the fragments become elongated only upon prolonged incubation of cells. The experiments suggest that viral DNA replication begins at numerous initiation sites along each strand and that the elongation beyond the size of the replication unit involves repair or ligation, or both. Since newly made DNA yields more fragments than viral DNA extracted from mature virions, it is suggested that the fragmentation of mature DNA on denaturation with alkali arises from incomplete processing of specific initiation sites. Comparison of viral DNA extracted from nuclei with that extracted from mature cytoplasmic virions in cells labeled for 120 min indicates that packaged DNA is not randomly selected from among the nuclear DNA population but rather represents DNA molecules which in alkaline gradients yield a minimal number of fragments.

Replication of viral deoxyribonucleic acid and breakdown of cellular deoxyribonucleic acid in Epstein-Barr virus infection.

Infection of Raji cells with Epstein-Barr virus (EBV) causes suppression of cellular deoxyribonucleic acid (DNA) synthesis and fragmentation of the cellular DNA. About 1,000 copies of EBV DNA of normal size (about 5 x 10(7) daltons in a single strand, as shown in an alkaline gradient) are synthesized per cell.

Nonproductive infection and induction of cellular deoxyribonucleic acid synthesis by bovine adenovirus type 3 in a contact-inhibited mouse cell line.

Bovine adenovirus type 3 (BAV-3), which has been reported to produce tumors in newborn hamsters, induced cellular deoxyribonucleic acid (DNA) synthesis in a contact-inhibited mouse kidney cell line (C3H2K). In this system, the virus did not multiply, whereas virus-specific tumor antigen (T antigen) was detected in nearly all cells. Replication of viral DNA could not be detected by DNA-DNA hybridization on membrane filters. The cellular DNA synthesis induced by BAV-3 did occur in the absence of added serum. Extent of induction of cellular DNA synthesis was closely correlated with the multiplicity of infection. Cells activated to synthesize DNA in the serum-free medium by the virus infection progressed to cell division without noticeable cell killing.

Ribonucleotide reductase activity of synchronized KB cells infected with herpes simplex virus.

The replication of herpes simplex virus (HSV) is unimpeded in KB cells which have been blocked in their capacity to synthesize deoxyribonucleic acid (DNA) by high levels of thymidine (TdR). Studies showed that the presence of excess TdR did not prevent host or viral DNA replication in HSV-infected cells. In fact, more cellular DNA was synthesized in infected TdR-blocked cells than in uninfected TdR-blocked cells. This implies that the event which relieved the TdR block was not specific for viral DNA synthesis but allowed some cellular DNA synthesis to occur. These results suggested that HSV has a means to insure a pool of deoxycytidylate derivatives for DNA replication in the presence of excess TdR. We postulated that a viral-induced ribonucleotide reductase was present in the cell after infection which was not inhibited by thymidine triphosphate (TTP). Accordingly, comparable studies of the ribonucleotide reductase found in infected and uninfected KB cells were made. We established conditions that would permit the study of viral-induced enzymes in logarithmically growing KB cells. A twofold stimulation in reductase activity was observed by 3 hr after HSV-infection. Reductase activity in extracts taken from infected cells was less sensitive to inhibition by exogenous (TTP) than the enzyme activity present in uninfected cells. In fact, the enzyme extracted from infected cells functioned at 60% capacity even in the presence of 2 mm TTP. These results support the idea that a viral-induced ribonucleotide reductase is present after HSV infection of KB cells and that this enzyme is relatively insensitive to inhibition by exogenous TTP.

Mature form of the deoxyribonucleic acid from chick embryo lethal orphan virus.

The deoxyribonucleic acid (DNA) of chick embryo lethal orphan (CELO) virus, an oncogenic avian adenovirus, had a biphasic denaturation profile indicating intramolecular base composition heterogeneity. This was confirmed by shearing the DNA and centrifuging it to equilibrium in Cs(2)SO(4) in the presence of HgCl(2) when two bands were formed. No circular molecules formed when CELO virus DNA was annealed, although lambda DNA formed circles under the same conditions. No circular molecules were found by sedimentation or electron microscopy when the DNA was digested with exonuclease III and then annealed, but 30 to 40% of T7 DNA molecules became circular under similar conditions. The complementary strands of CELO virus DNA both appeared to be continuous, and, when CELO DNA was denatured and then annealed under appropriate conditions, all of the renatured molecules were linear. It is concluded that CELO virus DNA consists of a unique rather than permuted collection of linear molecules that lack exposed single-strand complementary ends or duplex terminal repetitions. These results are discussed in relation to the replication of viral DNA and the transformation of host cells.

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. IV. Characterization of the simian virus 40 ribonucleic acid species induced by wild-type simian virus 40 and by the nondefective hybrid virus, Ad2 + ND 1 .

Ad2(+)ND(1), a nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, has been previously shown to contain a small segment of the SV40 genome covalently linked to Ad2 deoxyribonucleic acid (DNA). The SV40 portion of this hybrid virus has been characterized by relating the SV40-specific ribonucleic acid (RNA) sequences transcribed from the Ad2(+)ND(1) DNA to those transcribed from the DNA of SV40 itself. RNA-DNA hybridization-competition studies indicate that the SV40 component of Ad2(+)ND(1) consists of some, but not all, of that part of the SV40 genome which is transcribed early, i.e., prior to viral DNA replication, in SV40 lytic infection.

A temperature-sensitive mutant of adenovirus 31, defective in viral deoxyribonucleic acid replication

A temperature-sensitive mutant ( ts 13) of adenovirus 31 was isolated. The analysis of the replication of the mutant in human embryonic kidney cells at the nonpermissive temperature (39.5°) revealed that the T antigen, deoxyribonucleic acid (DNA) polymerase, and thymidine kinase were induced, but viral DNA and capsid proteins were not synthesized. When the incubation temperature was shifted-down from 39.5° to 33.5° at 16 or 20 hr post infection, viral DNA synthesis was observed normally but shift-down at later time resulted in decrease of viral DNA synthesis. No DNA synthesis was observed when cycloheximide was added at the time of shift-down. When temperature was shifted-up during viral DNA replication, the rate of DNA synthesis was first accelerated for a short period, then followed by rapid decrease. The mutant produced tumors in hamsters and induced cellular DNA synthesis in nongrowing hamster cells at the nonpermissive temperature.

Integration of the deoxyribonucleic acid of adenovirus type 12 into the deoxyribonucleic acid of baby hamster kidney cells.

In a previous report, evidence was presented that the deoxyribonucleic acid (DNA) of adenovirus type 12 (Ad12) is integrated by covalent linkage into the DNA of baby hamster kidney cells (BHK-21 cells). These studies have been extended. The DNA of Ad12 and that of BHK-21 cells grown in medium containing 5-bromodeoxyuridine could be separated by equilibrium centrifugation in alkaline CsCl density gradients. BHK-21 cells were infected with (3)H-labeled Ad12, and the total intracellular DNA was analyzed at various times after infection in alkaline CsCl density gradients. The (3)H label in the position of cellular DNA hybridized predominantly with viral DNA and to a lesser extent also with cellular DNA. Replication of viral DNA could not be detected in BHK-21 cells. The appearance of viral (3)H label in the density stratum of cellular DNA was not significantly affected when DNA synthesis in Ad12-infected BHK-21 cells was inhibited >96% by cytosine arabinoside. These findings provided additional evidence for integration of Ad12 DNA into the DNA of BHK-21 cells. It could be calculated that 5 to 55 Ad12 DNA equivalents per cell are integrated. Replication of viral or cellular DNA was not required for integration. Inhibition of protein or ribonucleic acid synthesis interfered with integration only slightly.

Formation of cellular deoxyribonucleic acid during productive polyoma virus infection.

It was previously shown that the majority of deoxyribonucleic acid (DNA) made in growing mouse embryo cells productively infected at low multiplicity with polyoma virus is cellular in nature and that some of this cell DNA contains discontinuities in the newly synthesized strand. Evidence obtained indicates the following. (i) Induction of cell DNA synthesis precedes the onset of detectable viral DNA replication by approximately 3 hr. (ii) Double-stranded cell DNA molecules, discontinuous in the newly synthesized strands, arise by direct synthesis (rather than by degradation of a high-molecular-weight precursor) only in the cell DNA replicated after initiation of viral DNA synthesis. (iii) This DNA component is continuously formed throughout the "late" stage of infection and is continuously converted into apparently normal cell DNA of high molecular weight without prior degradation to acid-soluble components.

Transcription and replication of viral deoxyribonucleic acid in cells coinfected with adenovirus types 2 and 12.

The yield of infectious virus was determined for KB cells infected with both adenovirus types 2 (ad 2) and 12 (ad 12). It was found that the yield of the former was greatly reduced, whereas that of the latter was not affected significantly. The reduction in virus yield was accompanied by an inhibition of ad 2 virus-specific ribonucleic acid (RNA) and viral deoxyribonucleic acid (DNA) synthesis at various times after infection. On the other hand, the rate of synthesis of ad 12 virus-specific RNA and viral DNA was not inhibited, but advanced in time. The total amount of ad 12 viral DNA synthesized was not affected by coinfection with ad 2. These results suggest that ad 2 infection hastens the maturation of ad 12.

Biochemical studies on adenovirus multiplication. XVI. Transcription of the adenovirus genome during abortive infection of elevated temperatures.

The synthesis of infectious virus is reduced by 99% in adenovirus type 2-infected KB cells maintained at 42 C in suspension. Studies to delineate the steps in virus biosynthesis blocked at 42 C revealed the following. (i) The inhibition of ribosomal ribonucleic acid (RNA) synthesis and the block in the conversion of ribosomal RNA precursors to ribosomal RNA which normally occur in infected cells at 37 C was accentuated at 42 C. (ii) The same species of early and late viral messenger RNA were synthesized at 37 and 42 C. (iii) The shift from host cell deoxyribonucleic acid (DNA) synthesis to viral DNA replication occurred earlier at 42 C than at 37 C. These findings indicate that the thermosensitive block(s) in virion formation does not occur at the level of viral DNA replication or transcription of viral mRNA but probably involves the synthesis of late viral proteins or the maturation of the virion.

Induction and regulation of DNA nucleotidyltransferase activity in fish cells infected with frog virus 3

Infection of fathead minnow (FHM) cells with frog virus 3 (FV 3) at 28° induced synthesis of deoxyribonucleic acid (DNA) polymerase and viral DNA in the cell cytoplasm starting between 1 and 2 hours after infection. By 4 hours, increase in DNA polymerase synthesis terminated and enzyme activity in infected cells was 4–15 times greater than in uninfected cells. Viral DNA synthesis was completed by 10–12 hours which corresponded to a single cycle of virus growth. Induction and “swith-off” of enzyme synthesis were not affected by treatment of the infected cells with 1-β- d-arabinofuranosylcytosine, an inhibitor of DNA synthesis and FV 3 growth. Increase of DNA polymerase activity ceased immediately after addition of cycloheximide or actinomycin D during enzyme synthesis, suggesting that enzyme formation required de novo protein synthesis and continuing messenger ribonucleic acid synthesis. It is concluded that synthesis of DNA polymerase is an “early” function of the parental viral genome. Replication of viral DNA is not required to initiate or “switch-off” formation of this early enzyme.

Synthesis and Intracellular Localization of Vaccinia Virus Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase

The time course of vaccinia deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase synthesis and its intracellular localization were studied with virus-infected HeLa cells. Viral RNA polymerase activity could be meassured shortly after viral infection in the cytoplasmic fraction of infected cells in vitro. However, unless the cells were broken in the presence of the nonionic detergent Triton-X-100, no significant synthesis of new RNA polymerase was detected during the viral growth cycle. When cells were broken in the presence of this detergent, extensive increases in viral RNA polymerase activity were observed late in the infection cycle. The onset of new RNA polymerase synthesis was dependent on prior viral DNA replication. Fluorodeoxyuridine (5 x 10(-5)m) prevented the onset of viral polymerase synthesis. Streptovitacin A, a specific and complete inhibitor of protein synthesis in HeLa cells, prevented the synthesis of RNA polymerase. Thus, the synthesis of RNA polymerase is a "late" function of the virus. The newly synthesized RNA polymerase activity was primarily bound to particles which sedimented during high-speed centrifugation. These particles have been characterized by sucrose gradient centrifugation. A major class of active RNA polymerase particles were considerably "lighter" than whole virus in sucrose gradients. These particles were entirely resistant to the action of added pancreatic deoxyribonuclease, and they were not stimulated by added calf thymus primer DNA. It is concluded that these particles are not active in RNA synthesis in vivo, and that activation occurs as a result of detergent treatment in vitro.

Soluble antigens of vaccinia-infected mammalian cells. 3. Relation of "early" and "late" proteins to virus structure.

The structural proteins of vaccinia virus can be divided into two classes on the basis of their times of synthesis in the infected cell. The production of one of these classes of proteins begins prior to the onset of viral deoxyribonucleic acid (DNA) replication. These are referred to as "early" proteins. Synthesis of the second class of structural proteins follows the onset of viral DNA replication; hence, the term "late" proteins for this class. We are able, by immunological procedures, to identify three "early" virus-structural proteins. These materials, when incorporated into virions, appear to be associated with the "core" of the virion and do not elicit production of virus-neutralizing antibody. It would seem, therefore, that those virus-structural proteins synthesized early in the course of infection act as internal components of the virion. The "late" proteins may be subdivided into two groups on the basis of certain physical properties and molecular weight differences. The first of these groups, comprised of at least two proteins, corresponds to the classical LS antigens and elicits production of neutralizing antibodies. These proteins, when incorporated into virions, are found only in the outer ("coat") fraction of the virion. The second group of "late" antigens, also comprised of two proteins, termed the G antigens, do not elicit synthesis of neutralizing antibody. One of these proteins is associated with the virus "core"; the other is found in the "coat" fraction of the virion and appears to occupy an intermediary, subsurface position. Procedures suitable for the isolation of the G antigens are described, in addition to the partial characterization of these antigens.