The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus

For hepadnaviruses, the RNA primer for plus-strand DNA synthesis is generated by the final RNase H cleavage of the pregenomic RNA at an 11 nt sequence called DR1 during the synthesis of minus-strand DNA. This RNA primer initiates synthesis at one of two distinct sites on the minus-strand DNA template, resulting in two different end products; duplex linear DNA or relaxed circular DNA. Duplex linear DNA is made when initiation of synthesis occurs at DR1. Relaxed circular DNA, the major product, is made when the RNA primer translocates to the sequence complementary to DR1, called DR2 before initiation of DNA synthesis. We studied the mechanism that determines the site of the final RNase H cleavage in hepatitis B virus (HBV). We showed that the sites of the final RNase H cleavage are always a fixed number of nucleotides from the 5' end of the pregenomic RNA. This finding is similar to what was found previously for duck hepatitis B virus (DHBV), and suggests that all hepadnaviruses use a similar mechanism. Also, we studied the role of complementarity between the RNA primer and the acceptor site at DR2 in HBV. By increasing the complementarity, we were able to increase the level of priming at DR2 over that seen in the wild-type virus. This finding suggests that the level of initiation of plus-strand DNA synthesis at DR2 is sub-maximal for wild-type HBV. Finally, we studied the role of the sequence at the 5' end of the RNA primer that is outside of the DR sequence. We found that substitutions or insertions in this region affected the level of priming at DR1 and DR2.     

Role of RNA synthesis in DNA replication of synchronized populations of cultured mammalian cells

Using the harvesting method of synchronizing L cells, the relationship of RNA synthesis of DNA replication was studied by the use of selective inhibitors of RNA synthesis such as actinomycin D and chromomycin succinate. The synthesis of the early replicating DNA fraction is a process sensitive to the inhibition of RNA synthesis during the G1 period. The synthesis of early replicating DNA was inhibited by chromomycin succinate without affecting the initation of DNA synthesis. However, actinomycin D inhibited the synthesis of early replicating DNA and prevented the initiation of DNA synthesis in 50% of the synchronized cells. However, it was found that the continued synthesis of RNA during the S period is not essential for the synthesis of late replicating DNA. In addition to this specific response of DNA synthesis to the inhibitors of RNA synthesis, another function of early and late replicating DNA was determined relative to the cell viability. Cells synthesizing early replicating DNA were killed more efficiently by chromomycin than at other stages of the cell cycle. This indicates that the early replicating DNA unit plays a more important role in cell reproduction than the late replicating DNA unit.     

RNA-DNA Covalent Bonds Between the RNA Primers and the DNA Products Formed by RNA Tumor Virus DNA Polymerase

Initiation of DNA synthesis by endogenous RNA primer molecules was studied with three different RNA tumor viruses. The influence of the method of virus disruption on the observed RNA-DNA bonds was ascertained. Ether disrupted virions of both murine leukemia virus (MuLV) and the B77 strain of avian sarcoma virus (B77 virus) have rC-dC and rA-dA covalent linkages between RNA primers and newly synthesized DNA. None of the 14 other possible bonds were formed. Ether-disrupted virions of avian myeloblastosis virus (AMV) have rU-dC and rA-dA linkages. In contrast, work reported herein and from other laboratories shows that Nonidet P-40 (NP-40)-disrupted virions of all three viruses have only the rA-dA junction. Studies with virus particles which were first disrupted with ether and then treated with NP-40 indicated that the detergent treatment disallowed the formation of the ribopyrimidine-dC internucleotide bond. The same transfers are found with AMV in the presence or absence of actinomycin D, where only single-stranded DNA is formed. This finding is consistent with the notion that virtually all of the significant primers have been recognized. In contrast to mature virions, transfer experiments with ether-disrupted early harvest (5 min) MuLV showed only the rC-dC bond; the rA-dA bond was absent. The short-time harvest contains a significantly higher proportion of infectious virions than 24-h harvests. Also, since the RNA from early harvest virus is appreciably more homogenous than the RNA of mature MuLV, it is concluded that the ribopyrimidine-dC linkage is the more significant initiation event from a biochemical standpoint.     

A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli

Abstract The 3D gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 3D gene from a serotype Cl virus has been cloned and overexpressed in Escherichia coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells.     

Purification and properties of spleen necrosis virus DNA polymerase.

DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.     

Effect of Ultraviolet Irradiation and Actinomycin D on Polyoma Virus Replication in Mouse Embryo Cell Cultures

Ultraviolet irradiation and actinomycin D impair the capacity of mouse embryo (ME) cells to support the replication of polyoma virus, but not of encephalomyocarditis (EMC) virus. The loss in capacity for polyoma virus synthesis was an “all-or-none” effect and followed closely upon the loss in cellular capacity for clone formation. Cells treated with either agent produced polyoma “T” antigen, but did not synthesize polyoma structural protein. Infection of untreated ME cells with polyoma virus produced marked stimulation of both deoxyribonucleic acid (DNA) synthesis and ribonucleic acid (RNA) synthesis. ME cell cultures irradiated with ultraviolet for 30 sec at 60 μw/cm² or treated with actinomycin D at 0.1 μg/ml for 6 hr prior to infection were incapable of synthesizing DNA or RNA, even after infection with polyoma virus. Irradiation of cells during infection produced cessation of synthesis of both RNA and DNA. Addition of actinomycin D during infection did not inhibit DNA synthesis but abolished RNA synthesis and reduced the yield of polyoma virus to 10% of that in untreated infected cultures. Both agents lost the ability to prevent replication of a full yield of polyoma virus when administered 30 hr after infection or later. The period after which neither agent inhibited polyoma replication corresponded with the period at which maximal RNA synthesis in untreated infected cultures had subsided. It can be concluded on the basis of the data presented that the functional integrity of the mouse embryo cell genome is required for the replication of polyoma virus, but not for EMC virus. Whereas the requirement for cellular DNA-dependent RNA synthesis for polyoma virus replication has been demonstrated, the exact nature of the host-cell function remains to be elucidated.     

Differential in vitro growth properties of cells transformed by DNA and RNA tumor viruses

Mammalian cells transformed by DNA and RNA tumor viruses are shown to display consistently different growth properties. All SV40, adenovirus type 7 and polyoma virus (DNA viruses) transformed cells propagated to high densities. The same cells transformed instead by RNA viruses: MSV strain Kirsten (MSV-Ki) or MSV strain Maloney (MSV-M) grew to densities which were consistently lower than DNA virus-transformed cells but greater than that of untransformed cells. The capacity to synthesize DNA at increasing densities also differentiated the RNA and DNA virus-transformed cells. As growing cultures of untransformed cells neared saturation density, the fraction of cells synthesizing DNA was minimal. The RNA virus-transformed cells were also contact-inhibited but at a significantly higher density. In contrast the DNA virus-transformed cells propagated to still greater densities and continued DNA synthesis at a high rate even at very high densities. Therefore the DNA virus-transformed cells truly are not contact inhibited. It is suggested that the capacity to continue DNA synthesis at high densities explains the attainment of much greater densities by DNA virus-transformed cells. There were no clear-cut differences in the ability to form colonies in agar, although a few of the RNA virus-transformed lines could not be propagated in semi-solid medium. These results may be explained as a persistence of the capacity of DNA tumor viruses to stimulate host cell DNA synthesis.     

Elongation of DNA complementary to the 5'' end of the avian sarcoma virus genome by the virion-associated RNA-dependent DNA polymerase.

RNA-dependent DNA synthesis in a virion-associated reaction has been described as being dependent upon the detergent concentration used for disruption of the virion. In this study, the Triton X-100 concentration was found to affect the elongation of the initially synthesized DNA complementary to the last approximately 100 nucleotides at the 5' end of the RNA (cDNA100). Whereas elongation of cDNA100 increased with time of incubation at the optimal detergent concentration, this process was retarded at higher detergent concentrations. At the optimal detergent concentration, elongated DNA was of low chemical complexity, indicating that extension of cDNA100 occurred at a unique site on the RNA. Higher than optimal detergent concentrations resulted in nonspecific elongation and in DNA of high chemical complexity. This was shown by oligopyrimidine tract analysis. Furthermore, actinomycin D was observed to inhibit the elongation of cDNA100 at the optimal detergent concentration. The nature of the elongation process was elucidated by analysis of DNA synthesized in a virion-associated reaction in the presence of bacteriophage Qbeta RNA. At the optimal detergent concentration DNA complementary only to avian sarcoma virus RNA was synthesized, whereas at higher concentrations DNA was copied from both avian sarcoma virus and Qbeta RNA. We conclude that the elongation mechanism of cDNA100 is affected by the detergent concentration and elongation is unspecific at higher than optimal detergent concentrations. The mechanism by which the nonionic detergent stimulates DNA synthesis has not yet been resolve. We assume that other factors in addition to DNA polymerase are involved in elongation of cDNA100.     

RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus.

The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression.     

Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus

The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DNA (0.35 kb), and it annealed with complementary DNAs specific for the 3' and 5' termini of viral RNA (Varmus et al., J. Mol. Biol. 120:50-82, 1978). Several subgenomic plus-strand fragments (0.94, 1.38, 2.3, and 3.4 kb) annealed with these reagents. At least the 0.94- and 1.38-kb strands were located at the same end of linear DNA as the 0.35-kb strand, indicating that multiple specific sites for initiation were employed to generate strands which overlapped on the structural map. We were unable to detect RNA liked to plus strands isolated as early as 2.5 h postinfection; thus, the primers must be short (fewer than 50 to 100 nucleotides), rapidly removed, or not composed of RNA. To determine whether multiple priming events are a general property of retroviral DNA synthesis in vivo, we also examined plus strands of mouse mammary tumor virus DNA in chronically infected rat cells after induction of RNA and subsequent DNA synthesis with dexamethasone. In this case, multiple, discrete subgenomic DNA plus strands were not found when the same methods applied to avian sarcoma virus DNA were used; instead, the plus strands present in the linear DNA of mouse mammary tumor virus fell mainly into two classes: (i) strands of ca. 1.3 kb which appeared early in synthesis and were similar in size and genetic content to the terminally repeated sequence in linear DNA; and (ii) plus strands of the same length as linear DNA. A heterogeneous population of other strands diminished with time, was not found in completed molecules, and was probably composed of strands undergoing elongation. These two retroviruses thus appear to differ with respect to both the number of priming sites used for the synthesis of plus strands and the abundance of full-length plus strands. On the other hand the major subgenomic plus strand of mouse mammary tumor virus DNA (1.3 kb) is probably the functional homolog of a major subgenomic plus strand of avian sarcoma virus DNA (0.35 kb). The significance of this plus strand species is discussed in the context of current models which hold that it is used as a template for the completion of the minus strand, thereby generating the long terminal redundancy.     

Characterization of endogenous RNA-directed DNA polymerase activity of reticuloendotheliosis viruses.

Reticuloendotheliosis viruses (REV) contain an endogenous RNA-directed DNA polymerase activity. The endogenous DNA polymerase activity can be elicited in purified preparations of REV by treatment with nonionic detergents. The enzyme activity has a strong preference for manganous ions. Therefore, appreciable endogenous DNA polymerase activity can be demonstrated only if the reaction mixture contains appropriate concentrations of manganous ions. Enzyme activity can be inhibited by pretreatment with RNase or deletion of one or more deoxyribonucleoside triphosphates from the reaction mixture. In contrast, actinomycin D has little effect in initial DNA synthesis. The results from both velocity and equilibrium centrifugation indicate that the nascent chains of product DNA are associated with 60S viral RNA. The DNA product of the endogenous DNA polymerase reaction is hybridizable to REV RNA, but not to avian leukosis virus RNA.     

The effect of saffron on intracellular DNA, RNA and protein synthesis in malignant and non-malignant human cells.

Extract of saffron (Crocus sativis) has previously been shown to inhibit colony formation and cellular DNA and RNA synthesis by HeLa cells in vitro. In order to compare the sensitivity of malignant and non-malignant cells to saffron, we examined the effect of the extract on macromolecular synthesis in three human cell lines: A549 cells (derived from a lung tumor), WI-38 cells (normal lung fibroblasts) and VA-13 cells (WI-38 cells transformed in vitro by SV40 tumor virus). We found that the malignant cells were more sensitive than the normal cells to the inhibitory effects of saffron on both DNA and RNA synthesis. There was no effect on protein synthesis in any of the cells.