Altered Drug Sensitivity Research Articles

Genetic characterization of the vaccinia virus DNA polymerase: identification of point mutations conferring altered drug sensitivities and reduced fidelity

We determined that 85 microM aphidicolin was sufficient to block macroscopic plaque formation by vaccinia virus and to cause a 10(4)-fold reduction in viral yield from a wild-type infection. A chemically mutagenized viral stock was passaged sequentially in the presence of drug, and plaque-purified viral stocks resistant to aphidicolin were isolated and characterized. By use of a marker rescue protocol, the lesion in each mutant was found to map within the same 500-bp fragment within the DNA polymerase gene. All of the mutants were found to contain a single nucleotide change in the same codon. In nine of these mutants, the alanine residue at position 498 was changed to a threonine, whereas a 10th mutant sustained a valine substitution at this position. Congenic viral strains which carried the Aphr lesion in an unmutagenized wild-type background were isolated. The Thr and Val mutations were found to confer equivalent levels of drug resistance. In the presence of drug, viral yields were 25% of control levels, and the levels of viral DNA synthesized were 30 to 50% of those seen in control infections. The two mutations also conferred an equivalent hypersensitivity to the cytosine analog 1-beta-D-arabinofuranosylcytosine (araC); strains carrying the Thr mutation were moderately hypersensitive to the pyrophosphate analog phosphonoacetic acid and the adenosine analog araA, whereas the Val mutation conferred acute hypersensitivity to these inhibitors. The Val mutation also conferred a mutator phenotype, leading to a 20- to 40-fold increase in the frequency of spontaneous mutations within the viral stock.

Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition

Eucaryotic, viral, and bacteriophage DNA polymerases of the alpha-like family share blocks of sequence similarity, the most conserved of which has been designated region I. Region I includes a YGDTDS motif that is almost invariant within the alpha-like family and that is similar to a motif conserved among RNA-directed polymerases and also includes adjacent amino acids that are more moderately conserved. To study the function of these conserved amino acids in vivo, site-specific mutagenesis was used to generate herpes simplex virus region I mutants. A recombinant virus constructed to contain a mutation within the nearly invariant YGDTDS motif was severely impaired for growth on Vero cells which do not contain a viral polymerase gene. However, three recombinants constructed to contain mutations altering more moderately conserved residues grew on Vero cells and exhibited altered sensitivities to nucleoside and PPi analogs and to aphidicolin. Marker rescue and DNA sequencing of one such recombinant demonstrated that the region I alteration confers the altered drug sensitivity phenotype. These results indicate that this region has an essential role in polymerase function in vivo and is involved directly or indirectly in drug and substrate recognition.

In vitro mutagenesis of the herpes simplex virus type 1 DNA polymerase gene results in altered drug sensitivity of the enzyme

A mutation (asparagine 815 to serine 815) was introduced into the herpes simplex virus type 1 (HSV-1) DNA polymerase (pol). The HSV-1 pol enzyme in lysates of Saccharomyces cerevisiae cells expressing the mutant protein showed increased resistance to acyclovir triphosphate and increased sensitivity to phosphonoacetate but was not substantially altered with respect to sensitivity to phosphonoformate or aphidicolin. These results directly demonstrate that both resistance to acyclovir triphosphate and sensitivity to phosphonoacetate can be conferred by this mutation in the absence of other viral factors and that the yeast expression system can be used for structure-function studies on HSV-1 pol.

Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition.

Herpes simplex virus (HSV) encodes a DNA polymerase that is similar in several respects to the replicative mammalian DNA polymerase alpha. Recently, these and other DNA polymerases have been shown to share several regions of protein sequence similarity. Despite these similarities, antiviral drugs that mimic natural polymerase substrates specifically inhibit herpesvirus DNA polymerases. To study amino acids involved in substrate and drug recognition, we have characterized and mapped altered drug sensitivity markers of nine HSV pol mutants and sequenced the relevant portions of these mutants. The mutations were found to occur within four relatively small regions. One such region, which we designate region A, has sequence similarity only to DNA polymerases that are sensitive to certain antiviral drugs. The other three regions contain sequences that are similar among various DNA polymerases. The multiple mutations occurring within two of these regions make it likely that the regions interact directly with drugs and substrates. Our results lead us to favor a model in which protein folding allows interactions among the four regions to form the substrate and drug binding sites.

Murine L5178Y Cells Resulting in Altered Drug Sensitivities From Fractionated Radiation Exposure In Vitro

Exposure of murine L5178Y cells in vitro to fractionated X-irradiation (DXR) produced a subline that exhibited significantly altered responses to the cytotoxic effects of a range of antitumor drugs. A DXR schedule of 2 Gy per fraction for 10 fractions (total dose, 20 Gy) was used. Characterization of this subline revealed no marked differences when compared with the parent line in terms of growth rate, cloning efficiency, cell volume, chromosome number, macromolecular content, cell cycle distribution, or response to acute DXR exposure. Drug sensitivities were assessed following 24-hour exposures by colony formation in soft agar. Three distinct patterns of response were found: increased sensitivity of DXR-pretreated cells to bleomycin, cis-diamminedichloroplatinum (II), or dibromodulcitol; comparable responses in both lines to doxorubicin, 5-fluorouracil, or hydroxyurea; and decreased sensitivity of DXR-pretreated cells to vincristine or VP 16213.

Overview of physiology in senescence.

As one ages, blood pressure and hemodynamic changes become apparent. This is reflected in a fall in cardiac output and a gradual increase in total peripheral-vascular resistance. Systolic hypertension is one of the fundamental abnormalities of the cardiovascular system in the elderly. Baroreceptor function and cerebral blood flow likewise may be compromised and result in altered drug sensitivity. Renal function decreases with age and is manifested by a decrease in renal blood plasma flow and in glomerular filtration rate. There is a gradual blunting of sympathetic nervous system responsiveness demonstrated by a decrease in the activity of the renin-angiotensin-aldosterone system. Finally, pathways of drug biotransformation may be altered, resulting in adverse drug reactions.

Overview of Physiology in Senescence

As one ages, blood pressure and hemodynamic changes become apparent. This is reflected in a fall in cardiac output and a gradual increase in total peripheral-vascular resistance. Systolic hypertension is one of the fundamental abnormalities of the cardiovascular system in the elderly. Baroreceptor function and cerebral blood flow likewise may be compromised and result in altered drug sensitivity. Renal function decreases with age and is manifested by a decrease in renal blood plasma flow and in glomerular filtration rate. There is a gradual blunting of sympathetic nervous system responsiveness demonstrated by a decrease in the activity of the renin-angiotensin-aldosterone system. Finally, pathways of drug biotransformation may be altered, resulting in adverse drug reactions.

Exposure of Rats to Alcohol in Utero Alters Drug Sensitivity in Adulthood

Pregnant rats were intubated with alcohol (ethanol, 3 grams per kilogram) twice daily throughout gestation. Control animals received solutions of isocaloric sucrose. At birth, offspring were placed with untreated surrogate dams. Beginning at 6 months of age, the offspring were tested for their thermogenic responsiveness to various drugs and to cold. Prenatal exposure to alcohol resulted in tolerance to alcohol and cross-tolerance to pentobarbital and diazepam but did not affect responsiveness to cold. This pattern of effects suggest that prenatal exposure to alcohol produces specific long-term effects on the neural mechanisms underlying drug tolerance.

Mechanism of DNA synthesis inhibition by arabinosyl cytosine and arabinosyl adenine.

1-β-D-ARABINOFURANOSYL CYTOSINE (ara-C) and 9-β-D-arabinofuranosyl adenine (ara-A) are potent antileukaemic and antiviral agents which inhibit DNA replication in various organisms1–3. Two plausible hypotheses for their cytotoxic action centre on the replicative DNA polymerase: in model 1 the triphosphate derivative of ara-C or ara-A inhibits competitively the utilisation of the corresponding deoxyribonucleoside triphosphate in the DNA polymerase reaction, and in model 2 the analogue is incorporated into DNA and this lesion impedes synthesis by, for example, providing a poor primer for further chain elongation. Although both of these effects have been demonstrated in vitro, the target in vivo for the analogues has not been firmly established, in part because metabolites of these drugs inhibit a number of purified nucleic acid enzymes1–4. Bacterial mutants with altered drug sensitivity could be decisive in determining the mechanism of inhibition and are the subject of this report.