RNA-instructed DNA Polymerase Research Articles

Evidence for type-C retrovirus production by Burkitt's lymphoma-derived cell line.

Burkitt's lymphoma cell line, P3HR-I, was found to secrete virions with properties of known type-C RNA tumor viruses. The viral particles had a buoyant density of 1.16 g/ml in sucrose gradients and contained a high-molecular-weight RNA and an RNA-instructed DNA polymerase. The viral polymerase was active in an endogenous reaction requiring the presence of the four deoxyriboside triphosphates and manganese ions, and was sensitive to RNase. The DNA product of the endogenous reaction specifically hybridized to P3HR-I viral 60 to 70S RNA. Electron microscopic examination of ultrathin sections of P3HR-I cells revealed immature, mature and budding virions typical of type-C retroviridae. Nucleic acid hybridization assays showed no sequence homoblastosis virus, murine oncornaviruses, simian sarcoma virus or RD114 virus.

Biochemical characterization of the type C retrovirus associated with lymphoproliferative disease of turkeys.

Turkeys inoculated with spleen extracts from lymphoproliferative disease (LPD)-affected birds developed viremia, followed by typical LPD lesions. Electron microscopy and biochemical characterization established that the virus present in the blood of infected turkeys is a type C retrovirus. The viral particles possess a buoyant density of 1.17 g/ml in sucrose gradients; they contain high-molecular-weight RNA and an RNA-instructed DNA polymerase with efficient exogenous and endogenous activity. The LPD virus polymerase is preferentially activated by magnesium ions. Cross nucleic acid hybridization assays revealed no sequence homology between the viral genome of LPD and avian myeloblastosis virus or reticuloendotheliosis virus, thus indicating that the LPD virus belongs to a distinct group unrelated to the avian leukosis-sarcoma virus complex or to the reticuloendotheliosis virus group.

Murine and human melanomas containing a high molecular weight RNA associated with an RNA-instructed DNA polymerase.

Murine and human melanomas containing a high molecular weight RNA associated with an RNA-instructed DNA polymerase.

Can a reverse transcriptase be involved in centriole duplication?

Accepting tentatively that the nucleic acid thought to be present in centrioles and basal bodies is RNA rather than DNA, which had earlier been thought to be the case, it is suggested that this RNA represents the primary genome of the organelle which is replicated via a DNA intermediate with the aid of an RNA-instructed DNA polymerase (reverse transcriptase) into more RNA during the course of the duplication cycle. This suggestion is presented as an alternate, and not as a replacement, to one made earlier which states that the organellar RNA is a transcript of the primary DNA genome located in the nucleus. In support of this hypothesis are data, cited from the literature, showing: simultaneous formation of multiple centrioles and basal bodies; cytoplasmic synthesis of thymidine triphosphate; presence in the cytoplasm of particles capable of performing RNA-instructed synthesis of DNA; presence of reverse transcriptase in eukaryotic cells not infected with RNA viruses; formation of centrioles in homogenates of artificially activated surf clam eggs. The relationship of this hypothesis to the postulated generative mechanism for centriole duplication is also discussed.

RNA-dependent DNA polymerase associated with equine infectious anemia virus

Equine infectious anemia (EIAV) is shown to have an associated RNA-instructed DNA polymerase similar in its cofactor requirements and reaction conditions to the RNA tumor virus DNA polymerases. Demonstrating this DNA polymerase activity requires a critical concentration of a nonionic detergent, all four deoxyribonucleoside triphosphates, and a divalent metal ion. The reaction is sensitive to RNase, and a substantial fraction of the FNA synthesized is complementary to viral RNA. The detection of a complex of tritium-labeled polymerase product DNA-template RNA, which sedimented at 60S to 70S, provided evidence that EIAV contains high-molecular-weight RNA. These results, obtained with both virus propagated in cell culture and virus from the serum of an experimentally infected horse, indicate that EIAV may properly be considered a member of the family Retroviridae. They may also be pertinent to the mechanism(s) of viral persistence and periodic recrudescence of disease in chronically infected horses.

RNA-instructed DNA polymerase associated with C-type particles produced in vivo by murine myeloma cells.

C-type particles secreted in vivo by MOPC-315 myeloma cells were characterized. These particles localize at a density of 1-16 g/ml in sucrose and possess a 60 to 70S RNA and an RNA-instructed DNA polymerase. Endogenous enzyme activity requires manganese and is inhibited by ribonuclease or by the omission of any of the deoxynucleoside triphosphates. The enzyme utilizes the virus 60 to 70S RNA as a template to synthesize DNA molecules which specifically hybridize to the homologous RNA.

Purification of RNA-instructed DNA polymerase from human leukemic spleens.

Particles possessing a density of 1.16 g/ml and encapsulating a 70S RNA and a RNA-instructed DNA polymerase (reverse transcriptase) have been prepared from the spleen of a patient with chronic lymphocytic leukemia. These particles have been converted to cores with a density of 1.26 g/ml and containing the enzyme-RNA complex, in complete analogy to the known RNA tumor viruses of avian and murine origin. The reverse transcriptase was purified from the cores by column chromatography to a stage showing a single major protein band of 70,000 daltons in a gel electrophoresis. The enzyme was capable of transcribing heteropolymeric RNA into DNA complements as demonstrated by specific back hybridization to template RNA. The leukemic spleen would appear to represent an important source of this enzyme, as well as other potentially important leukemia-specific reagents.

Oncornavirus-like particles in human skin cancers.

A high-molecular-weight RNA encapsulated with an RNA-instructed DNA polymerase in particles possessing the density characteristic of the RNA tumor viruses has been detected in 13 out of 14 human malignant melanomas. The [3H]DNA synthesized by these particles in an endogenous reaction hybridizes to RNA extracted from the human melanoma particulate structures, but not to RNA from normal skin. Similar particles containing RNA and enzyme have been found in basal cell and squamous cell carcinomas of the skin. The RNA of the melanoma particles is easily distinguishable by hybridization from the RNAs found in the particles of the basal and squamous cell carcinomas.

RNA-instructed DNA polymerase activity in a cytoplasmic particulate fraction in brains from Guamanian patients.

Nervous system tissues from a number of patients with idiopathic neurological disorders were examined for biochemical evidence of RNA tumor virus infection. RNase-sensitive DNA polymerase activity was found in a cytoplasmic particulate fraction from two patients with Guamanian amyotrophic lateral sclerosis (ALS) but not in brains from two normal U.S. individuals. The buoyant density of the enzyme-containing fraction was 1.16-1.18 g/ml and could be converted to a denser region of the gradient (1.24 g/ml) by treatment with the nonionic surfactant, Sterox. The cation and detergent requirements for the endogenous RNase-sensitive DNA polymerase reaction were determined. The early (5 min) endogenous reverse transcriptase product was analyzed by cesium sulfate gradient centrifugation. RNase- and heat-sensitive RNA-DNA hybrids were detected in the product analysis of two ALS, one Parkinsonism-dementia (PD) brain, and two brains from asymptomatic Chamorros but not in brains from normal U.S. individuals and a number of patients with neuro-psychiatric disorders. The DNA product was a 4.5S heteropolymer that hybridized more extensively to RNA extracted from the enzyme-containing pellet from PD brain as compared to a similar fraction from normal U.S. brain. The DNA product appeared to be unrelated to Rausvher or visna virus 70S RNA as determined by RNA-[-3H]DNA hybridization.

Transformation of cultured human embryonic fibroblasts by oncornavirus-like particles released from a human carcinoma cell line.

A fibroblast-like cell culture was established from a stomach biopsy of a patient with metastatic adenocarcinoma. One of the cultures, at the 6th passage level, left unattended for a month at 37 degrees, produced numerous foci of epithelioid cells. Upon subculturing, an epithelioid cell line, designated HCCL (human carcinoma cell line), was established. The HCCL cells released particles possessing the characteristics of oncornaviruses: density 1.175 g/ml, cores with a density of 1.22-1.26 g/ml, high-molecular-weight RNA (60-70S) and RNA-instructed DNA polymerase activity (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7). Inoculation of particles released from HCCL cells into cultures of human embryo muscle fibroblasts resulted in the appearance of foci of transformed cells.

No evidence for particles encapsulating RNA-instructed DNA polymerase and high molecular weight virus-related RNA in herpesvirus induced tumours of non-human primates.

The simultaneous detection test gave no evidence for the presence of RNA tumour viruses in herpesvirus induced malignant lymphomas of non-human primates. The 12 tumours tested were obtained from three different monkey species inoculated with Herpesvirus saimiri or herpesvirus ateles. Particles encapsulating RNA-instructed DNA polymerase and high mol. wt. virus-related RNA were easily demonstrated in tumours of the mouse induced by type-C or type-B oncornaviruses and in human lymphoid cells infected with simian sarcoma virus type I which were examined in parallel. Attempts to demonstrate partial expression of an oncornavirus genome in the herpesvirus induced tumours and attempts to detect an interspecies antigen related to monkey oncornaviruses were negative and strengthened the observations made with the simultaneous detection test.

Oncorna-viral information in human glioblastoma.

Two of the main biochemical features characteristic for oncogenic RNA (oncorna) viruses have been detected in human glioblastoma. In all tumors tested so far a RNA instructed DNA polymerase (“reverse transcriptase”) activity was present which exhibited the criteria specific for oncorna viruses. It was stimulable by the synthetic polynucleotide poly-rA: oligo-dT, it was almost insensitive to actinomycin D but very sensitive to ethidiumbromide. Using the simultaneous detection test, a high molecular weight RNA species was found in the subcellular fraction with the highest reverse transcriptase activity.

Rifamycin derivatives as inhibitors of a ribonucleic acid instructed deoxyribonucleic acid polymerase function. Effect of lipophilicity.

Several new rifamycin derivatives have been synthesized and tested as inhibitors of an RNA-instructed DNA polymerase (RDP) function in an effort to determine the effect of rifamycin structure on RDP inhibition. It was found, in general, that RDP inhibition is favored by lipophilic tails bound to the 3 positions of rifamycin SV. Relative lipophilicities were measured by a reversed phase thin-layer chromatographic technique.

Herpes Simplex Virus Superinfection of Mouse Cells Chronically Infected with Rausher Leukemia Virus

Although host-controlled fractors are known to alter biological properties of bacteriophage and animal viruses, little is known of the interaction between viruses themselves in a permissive host cell. Some biochemical and morphological evidence has accumulated associating both herpes virus and RNA tumor virus types as the etiological agents of Marek's disease in chickens. Further, Epstein-Barr herpesvirus, the agent associated with Burkitt's lymphoma in humans has been shown to contain RNA tumor virus-like particulate material with RNA-instructed DNA polymerase activity. The following study was initiated to determine the possible interaction of herpes simplex virus type-1 (HSV-1) and Rauscher leukemia virus (RLV) in a dually infected host cell.

Oncorna-viral information in human melanoma

Human malignant melanomas were investigated for the presence of oncornaviral information by chemical and electron microscopical methods. In all primary and metastatic tumors a RNA-instructed DNA polymerase was detected. This enzymic activity exhibits features characteristic for that of oncogenic RNA viruses. A high molecular weight ( 70S) RNA species, a further diagnostic feature for oncorna virions, was found in the subcellular fraction with the highest RNA-instructed DNA polymerase activity. In addition, by electron microscopy particles of a morphology similar to C-type virions were found in this subcellular fraction.

Inhibition of three nucleotide polymerases by rifamycin derivatives.

It is possible to design rifamycin derivatives which can distinguish between viral RNA-instructed DNA polymerase and other nucleotide polymerases.

Mason-Pfizer virus characterization: a similar virus in a human amniotic cell line.

Mason-Pfizer monkey virus (MP-MV) is a RNA virus with an RNA-instructed DNA polymerase first isolated from a rhesus monkey mammary adenocarcinoma in 1970. Until recently, there have been no other isolates. A continuous human amnion cell line, AO, was found to be producing a virus indistinguishable or closely related to the Mason-Pfizer virus as measured by morphological, immunological, and biochemical methods. By thin-section electron microscopy, the extracellular virus particle in AO line is 115 to 130 nm in diameter and has a preformed nucleoid (80 to 90 nm) before budding, properties which are also characteristic of MP-MV. Two proteins of the virus from the AO line were studied. By immunodiffusion, sera which react specifically with MP-MV give a line of identity with virus from the AO line. The AO viral RNA-instructed DNA polymerase purified by phosphocellulose chromatography was specifically inhibited by anti-MP-MV polymerase sera, and the AO cells contained both DNA and RNA sequences related to MP-MV (3)H-DNA. Viruses thus far indistinguishable from MP-MV have also recently been found by others in different human lines, raising again the question of the species of origin of MP-MV. Because the virus in the AO cells cannot be differentiated from MP-MV, we attempted to determine the origin of MP-MV virus by measuring DNA sequences related to MP-MV (3)H-DNA in uninfected human and rhesus monkey cells. The quantity of MP-MV-like DNA sequences in uninfected primate tissues was found to be much lower than the amount of DNA sequences of murine type-B or type-C viruses in uninfected murine tissues. Thus, it was not possible to determine whether the virus produced by AO cells or MP-MV was of human or monkey origin, or both.

Synthesis of some rifamycin derivatives as inhibitors of an RNA-instructed DNA polymerase function.

Synthesis of some rifamycin derivatives as inhibitors of an RNA-instructed DNA polymerase function.

Further evidence for oncornaviruses in human milk: the production of cores.

Cores, or nucleoids, have been isolated from particles in human milk. The cores have a density of 1.26-1.27 g/ml and contain a 60-70S RNA in association with an RNA-instructed DNA polymerase. The data offer further evidence of similarity between human milk particles and animal RNA tumor viruses. In addition, core isolation provides a new method for detection of these particles by minimizing the difficulties generated by the presence of cell-associated debris often found in the density region (1.16-1.19 g/ml) characteristic of the intact virion. The procedures described now make available preparations of purified subviral components of a putative human RNA tumor virus.

Molecular evidence for a viral etiology of human leukemias, lymphomas, and sarcomas.

The experiments described were designed to probe further the etiologic significance of the earlier findings in human leukemia and lymphoma by Hehlmann and associates13,14 and in sarcoma by Kufe and colleagues16 of RNA homologous to that of the Rauscher leukemia virus (RLV). The data obtained indicate that at least a portion of the viral-related RNA we were detecting is in the form of a 70S-RNA template physically associated with a reverse transcriptase, two of the diagnostic features of the RNA tumor viruses. Further, the DNA synthesized by the leukemic reverse transcriptase on its own endogenous template is related in sequence to RLV-RNA. This last finding completes the logic of our earlier experiments in which the DNA synthesized on RLV-RNA was used as a probe to find the viral-related information in leukemic cells. Finally, the data show that a reverse transcriptase and 70S RNA are associated with a particle possessing the exact density of oncogenic RNA viruses. The data we have detailed show that human leukemic cells contain a 70S RNA encapsulated with RNA-instructed DNA polymerase in a particle possessing the density characteristic of RNA viruses. Further, the DNA synthesized by the human RNA-enzyme complex hybridizes specifically with the RNA of a murine leukemogenic agent. Similar data are now available with human mammary cancers. Thus, for both leukemias and adenocarcinomas of the breast, four diagnostic features of the corresponding animal tumor viruses are exhibited by particles found in these analogous human neoplasias. The accumulating evidence for the involvement of RNA tumor viruses in at least some forms of human cancer is becoming more convincing. Final proof still requires the demonstration that the particles identified in human neoplastic tissues are infectious and transforming agents.