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T W North

Publications and source records attributed to T W North.

12 recordsLinked to original sources
PubMed1992

Inhibition of reverse transcriptase from feline immunodeficiency virus by analogs of 2'-deoxyadenosine-5'-triphosphate.

The replication of feline immunodeficiency virus (FIV) in cultured cells was inhibited by 2',3'-dideoxyadenosine (ddA) and by 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with IC50 values of 0.98 and 0.95 microM, respectively. The effects of the presumed active forms of these inhibitors, ddATP and PMEA-diphosphate (PMEApp), upon the FIV reverse transcriptase (RT) were examined with two different template-primer systems. Both of these compounds were potent inhibitors of the FIV RT in reactions with primed phi X-174 DNA, yielding Ki values of 8.8 nM for ddATP and 5.0 nM for PMEApp. However, they were both poor inhibitors of the reaction with poly(rU)-oligo(dA); concentrations of ddATP or PMEApp greater than 10 microM were required to inhibit this reaction by 50%. Further analysis of the reaction with poly(rU)-oligo(dA) revealed that even in the absence of inhibitors the primers were extended by less than 20 nucleotides. In contrast, high molecular weight products were obtained in reactions with phi X-174 DNA. These results suggest that the reaction of FIV RT with poly(rU)-oligo(dA) is not highly processive. The high degree of termination encountered during this reaction with poly(rU)-oligo(dA) may be responsible for the low inhibitory potential of ddATP and PMEApp.

Adenine↗
PubMed1992

RNase H activity associated with reverse transcriptase from feline immunodeficiency virus.

Reverse transcription of retroviral genomes requires the action of an RNase H for template switching and primer generation. In this report, we compare enzymatic properties of the RNase H associated with the reverse transcriptase (RT) from feline immunodeficiency virus (FIV) and that from human immunodeficiency virus (HIV). Both enzymes displayed substrate preference for poly[3H](rG) . poly(dC) hybird over poly[3H](rA) . poly(dT) and cation preference for Mg2+ over Mn2+. Activity of the FIV RNase H upon poly(rG) . poly(dC) produced hydrolysis products from 1 to 6 nucleotides in length, similar to that reported for HIV. Dextran sulfates were effective inhibitors of both the FIV and HIV RNase H and RT activities. Nearly identical inhibition constants (0.12 nM) were obtained for all enzyme activities with dextran sulfate 500,000, while different inhibition constants were observed with dextran sulfate 8,000. Our results suggest that FIV and HIV RTs contain a conserved region that is sensitive to the larger dextran sulfate and that dextran sulfate 8,000 may interact at a different site or by a different mechanism.

Animals↗
PubMed1991

Mutants of feline immunodeficiency virus resistant to 3'-azido-3'-deoxythymidine.

We selected 3'-azido-3'-deoxythymidine (AZT)-resistant mutants of feline immunodeficiency virus (FIV) in a cat cell culture system. The characterization of one of these mutants was facilitated by the development of a focal immunoassay which could accurately measure FIV infectivity. This assay was used to quantitate the susceptibility of FIV to various inhibitors. The AZT-resistant mutant was found to be cross-resistant to 3'-azido-2',3'-dideoxyuridine and 3'-azido-2',3'-dideoxyguanosine but remained sensitive to several other inhibitors (2',3'-dideoxyinosine, 2',3'-dideoxy-2',3'-didehydrothymidine, and phosphonoformate). These patterns of cross-resistance and sensitivity were similar to those of the AZT-resistant human immunodeficiency virus (HIV) that has recently been isolated from patients with AIDS (B. A. Larder and S. D. Kemp, Science 246:1155-1158, 1989). Like the AZT-resistant HIV, purified reverse transcriptase from mutant FIV failed to show resistance to the 5'-triphosphate of AZT. This mutant can be used in the FIV model system to study the mechanisms of drug resistance and to determine the pathogenicity of AZT-resistant mutants.

Animals↗
PubMed1990

Characterization of reverse transcriptase from feline immunodeficiency virus.

Reverse transcriptase has been purified from feline immunodeficiency virus (FIV) by DEAE-cellulose and phosphocellulose chromatography. The purified enzyme consists of a single protein with a Mr of 67,000. When proteolysis is not minimized during purification, a fragment of Mr 54,000 is also observed. This is similar to the reverse transcriptase from human immunodeficiency virus type 1 (HIV), which consists of a polypeptide of Mr 66,000; when proteolysis is not minimized during purification, a fragment of Mr 51,000 is also observed. In direct comparisons, the FIV reverse transcriptase is very similar to the HIV reverse transcriptase in template specificity and requirements for Mg2+. In contrast to these similarities, the FIV and HIV reverse transcriptases are substantially different in primary sequence, as determined by peptide mapping.

Animals↗
PubMed1990

Direct comparisons of inhibitor sensitivities of reverse transcriptases from feline and human immunodeficiency viruses.

The sensitivities of reverse transcriptases (RTs) from feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV) were directly compared. The two enzymes had similar sensitivities to three analogs of dTTP, namely, 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate. Each of these analogs demonstrated competitive inhibition of both enzymes. Ki values for inhibition of FIV RT by these three inhibitors were 3.3, 6.7, and 1.8 nM, respectively; Ki values for inhibition of the HIV enzyme were 6.5, 5.9, and 8.3 nM, respectively. Ratios of the Ki for the inhibitor to the Km for the substrate were also determined for each inhibitor, and no differences between the two enzymes greater than threefold were observed. Inhibition constants for 3'-amino-3'-deoxythymidine 5'-triphosphate and 3'-fluoro-3'-deoxythymidine 5'-triphosphate were determined for FIV RT, and these were similar to published values for HIV RT. The activities of three dideoxynucleoside 5'-triphosphates against FIV RT were determined; ddGTP was slightly more potent than ddTTP, whereas both were much more effective than ddCTP. The activity of a noncompetitive inhibitor, phosphonoformate, was also examined with the FIV enzyme; it was much more active with poly(rA)-oligo(dT) as the template-primer than with poly(rC)-oligo(dG) or poly(rI)-oligo(dC).

Animals↗
PubMed1989

Feline immunodeficiency virus, a model for reverse transcriptase-targeted chemotherapy for acquired immune deficiency syndrome.

Feline immunodeficiency virus (FIV), formerly called feline T-lymphotropic lentivirus, causes an immune deficiency in cats that is very similar to the acquired immune deficiency syndrome in humans (N. C. Pedersen, E. M. Ho, M. L. Brown, and J. K. Yamamoto, Science 235:790-793, 1987). We have examined the reverse transcriptase of this virus to determine whether it is similar enough to the reverse transcriptase of the human immunodeficiency virus type 1 (HIV-1) to enable its use as a model for chemotherapy for acquired immune deficiency syndrome. The FIV reverse transcriptase is similar to that of HIV-1 in sensitivity to the noncompetitive inhibitor phosphonoformate (Ki, 0.3 microM) and relative insensitivity to phosphonoacetate. This enzyme was also sensitive to two competitive inhibitors, the 5'-triphosphates of 2', 3'-dideoxythymidine (Ki, 3.4 nM) and 3'-azido-3'-deoxythymidine (AZT; Ki, 6.2 nM). The ratios of Ki/Km for these two competitive inhibitors are similar to the ratios calculated from previously reported data for the HIV-1 enzyme assayed under identical conditions. In contrast, the FIV enzyme is different from the reverse transcriptase of avian myeloblastosis virus in sensitivity to those inhibitors. The replication of FIV in Crandell feline kidney cells was inhibited by AZT; virus production was inhibited more than 95% by 1.0 microM AZT.

Acquired Immunodeficiency Syndrome↗
PubMed1987

DNA synthesis in promastigotes of Leishmania major and L. donovani.

The requirement of protozoan parasites for pre-formed purines affords the opportunity for quantitation of nucleic acid synthesis from incorporation of radioactively labeled purines into DNA and RNA. We have developed rapid and simple assays to quantitate DNA and RNA synthesis in promastigotes of Leishmania major and L. donovani from the incorporation of [3H]hypoxanthine. DNA but not RNA synthesis in L. major or L. donovani promastigotes was inhibited by aphidicolin (50% inhibition by 0.2-0.3 microM) and by hydroxyurea (50% inhibition by 0.3-0.5 mM). The inhibition of DNA synthesis by aphidicolin or hydroxyurea was reversible when the inhibitor was removed 2, 4 or 24 h after its addition. Several well-characterized agents that inhibit DNA synthesis in mammalian cells, 1-beta-D-arabinofuranosylcytosine (araC), 9-beta-D-arabinofuranosyladenine (araA), phosphonoacetic acid, novobiocin and N2-(p-n-butylphenyl)guanine (BuPG), failed to inhibit DNA synthesis in promastigotes of L. major even when used at very high concentrations, demonstrating differences between DNA replication components of parasite and host.

Animals↗
PubMed1978

Erythro-9-(2-hydroxy-3-nonyl)adenine as a specific inhibitor of herpes simplex virus replication in the presence and absence of adenosine analogues.

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; erythro-9-[3-(hydroxynonyl)]adenine), a reversible inhibitor of adenosine deaminase, significantly inhibits replication of herpes simplex virus (HSV), whereas the more active inhibitor of the deaminase, 2'-deoxycoformycin, does not. At 10 micron EHNA, which does not affect viability, growth, or DNA synthesis of uninfected HeLa cells, production of HSV and HSV-specific DNA is inhibited 75-90% and 60%, respectively. HSV multiplies normally in cells pretreated with EHNA and washed to remove this inhibitor. EHNA (10 micron) also markedly potentiates the toxicity of adenine arabinonucleoside and of cordycepin (3'-deoxyadenosine) against HeLa cells and against the production of HSV in those cells. Cordycepin alone (10 micron) does not inhibit HSV replication whereas in combination with 10 micron EHNA there is a greater than 99% inhibition of virus production. Under these conditions, RNA synthesis is inhibited by more than 80% whereas protein and DNA synthesis are inhibited to a lesser extent; in this system, virtually all of the DNA synthesis in infected cells is that of host DNA. Thus, EHNA appears to affect the synthesis of HSV DNA specifically in two different ways, depending on whether it is used alone or in the presence of cordycepin.

Adenine↗
PubMed1976

Biochemistry of DNA-defective mutants of bacteriophage T4. VI. Biological functions of gene 42.

Bacteriophage T4 gene 1 and 42 amber mutants (defective in deoxynucleoside monophosphate kinase and deoxycytidylate hydroxymethylase, respectively) are able to synthesize DNA in cell-free lysates prepared as described by Barry and Alberts (1972), in contrast to their inabliity to do so in plasmolyzed and toluenized cell systems. Addition of extracts containing an active gene 1 or 42 product has no effect on synthesis in lysates defective in the respective gene. Thus, if these enzymes do play additional direct roles in replication, these roles are not manifest in the lysed-cell system. The gene 42 mutant am N122/m, a double mutant bearing an additional defect in DNA polymerase, is unable to synthesize DNA in these lysates. This inability is overcome by addition of extracts containing an active T4 DNA polymerase. m is a leaky amber mutation which reduces DNA polymerase activity to a very low level. However, this level is high enough to allow positive genetic complementation tests with gene 43 mutants. Two other gene 42 amber mutants contain additional defects: am 269 induces only half the normal level of DNA polymerase, and am C87 fails to induce a detectable level of thymidylate synthetase. These defects do not result from pleiotropic effects of the gene 42 mutations. In plasmolyzed cells, temperature-sensitive gene 42 mutants fail to synthesize DNA under conditions where replication forks and 5-hydroxymethyl-dCTP are present. This supports the idea that the gene 42 protein is directly involved in DNA synthesis.

Cell-Free System↗