Detergent Effects on a Reverse Transcriptase Activity and on Inhibition by Rifamycin Derivatives

Thompson, Frances M.; Libertini, Louis J.; Joss, Urs R.; Calvin, Melvin

doi:10.1126/science.178.4060.505

Cited by 34 publications

(10 citation statements)

References 3 publications

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“…We also observed that Trition X-100 stimulated the purified enzyme activity with (dT)12_.18 (rA)n as template-primer (Wu, A. M., Cretta, A. & Gallo, R. C., in preparation), confirming the observation made by Thompson et al with partially purified enzymes (12,13). The magnitude of stimulation is proportional to the concentration of template primer and to the time of incubation.…”

Section: Methodssupporting

confidence: 89%

“…The results in Table 3 also show that the concentration of nonionic detergent (Triton) can drastically affect the magnitude of inhibition by various concentrations of rifamycin SV derivatives, confirming the observation first made by Thompson et al (12,13). Presumably, this is because of micelle formation from the detergent and solubilization of the inhibitor into the micelles.…”

Section: Methodssupporting

confidence: 86%

“…In order to show a tight, but reversible, binding between purified RNA-directed DNA polymerase and Class C in-hibitors (specifically compound no. 120), an experiment was designed based on observations made by Thompson et al that inhibitory activity of rifamycin SV derivatives on the RNA-directed DNA polymerases can be nullified by the presence of Triton X-100 in the reaction mixture (12,13) (Fig. 4).…”

Section: Methodsmentioning

confidence: 99%

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RNA-Directed DNA Polymerase and Virus-Induced Leukemia in Mice

Wu¹,

Ting²,

Gallo³

1973

Proc. Natl. Acad. Sci. U.S.A.

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Rifamycin antibiotics, which inhibit RNAdirected DNA polymerase of Rauscher leuckemia virus, prevent the leukemogenic activity of the virus, and the effect of leukemogenesis correlates with the magnitude of inhibition of the purified enzyme. This inhibition of enzyme activity by rifamycin SV derivatives is due to a relatively tight binding between the enzymes and the inhibitors, yet the binding can be reversed by nonionic detergent. The results of this study suggest that RNAdirected DNA polymerase is essential for induction of leukemia by exogenous virus and correlate with the previous observation that the same derivatives block viral transformation in vitro.Leukemia can be induced in mice by inoculation with RNA leukoviruses, such as Rauscher leukemia virus (RLV) (1), These viruses contain RNA-directed DNA polymerase (2, 3). Some recent experiments have indicated that this enzyme is necessary for successful transformation of normal cells in culture (4-6). However, evidence of involvement of this enzyme in leukemic formation in animals is lacking.Hanafusa and Hanafusa (4) and Hanafusa et al. (5) reported that noninfectious RSV a (0) particles produced from a-type transformed cells did not contain RNA-directed DNA polymerase. To further demonstrate a relationship between RNA-directed DNA polymerase and infectivity, they showed that an infectious pseudotype RLV a (ALV) can be rescued by superinfection of the a-type transformed cells with avian leukosis virus (ALV) and the cells then transformed by RSV a (ALV) produced only RSV a (0) (4). Using different classes of rifamycin SV derivatives (with respect to potency of inhibition of activity of RNA-directed DNA polymerase), we reported that there is a direct correlation of inhibition of viral RNA-directed DNA polymerase and inhibition of focus formation by murine sarcoma virus (MSV) as well as infectivity by RLV (6). This experiment was performed by preincubation of the inhibitors with virus particles at 37°for 30 min in order to prevent cytotoxicity in culture (6, 7). These findings suggested that RNA-directed DNA polymerase was essential to viral transformation in culture. It is of interest, then, to extend these observations to an animal system; leukemia induced by RLV in mice was used. Splenomegaly is a useful index for a quantitative measurement of the development of leukemia in this system (8). Therefore, the degree of splenomegaly was used to measure the inhibitory effect of rifamycin SV derivatives on murine leukemogenesis. The result suggests that RNA-directed DNA polymerase is required for development of virus-induced leukemia. METHODS AND MATERIALSVirus and Animal System. 4-to 6-week-old NIH Swiss Mice of either sex were used. RLV from plasma and tissue culture were obtained through the Special Virus Cancer Program from Hazelton Laboratory (Vienna, Va.) and Electro-Nucleonics, Incorp. (Bethesda, Md.), respectively. Only one stock of plasma viruses (about 5 X 105 PFU/ml) was used for all of the animal experiments. The virus suspension was diluted wi...

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Section: Methodssupporting

confidence: 89%

Section: Methodssupporting

confidence: 86%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

RNA-Directed DNA Polymerase and Virus-Induced Leukemia in Mice

Wu¹,

Ting²,

Gallo³

1973

Proc. Natl. Acad. Sci. U.S.A.

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show abstract

“…Some restriction enzymes, reverse transcriptases, and Taq polymerases are stabilized by Triton X-100 or NP-40 (Weyant et al, 1990), while some other polymerases are activated by detergents (Thompson et al, 1972;Wu and Cetta, 1975;Hirschman et al, 1978). Triton X-100 is used for purification of HCV RdRp (Weng et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Detergent-induced activation of the hepatitis C virus genotype 1b RNA polymerase

Weng

Kohara

Wakita

et al. 2012

Gene

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“…Rifazacyclo-16 is the most effective inhibitor of the RNA-instructed DNA polymerase yet tested (ref. 4, and Thompson, F. M., Libertini, L. J., Joss, U. R. & Calvin, M., Biochemistry, submitted), while the others were less active. However, these drugs were all ineffective against viral transformation of mouse cells, presumably because they were unable to penetrate the cell membrane.…”

mentioning

confidence: 97%