Particle-associated DNA Polymerase Research Articles

In vivo antiviral activity and pharmacokinetics of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in woodchuck hepatitis virus-infected woodchucks.

The (-) enantiomer of cis-5-fluoro-1l-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine [(-)-FTC)], a substituted oxathiolane compound with anti-hepatitis B virus activity in vitro, was assessed for its efficacy in woodchucks with naturally acquired woodchuck hepatitis virus (WHV) infection. Pharmacokinetics and in vitro anabolism were also determined. (-)-FTC was anabolized to the 5'-triphosphate in a dose-related fashion, reaching a maximum concentration at about 24 h in cultured woodchuck hepatocytes. Following administration of a dose of 10 mg/kg of body weight intraperitoneally (i.p.), the clearance of (-)-FTC from plasma was monoexponential, the terminal half-life was 3.76 +/- 1.4 h, and the systemic clearance was 0.12 +/- 0.06 liters/h/kg. The antiviral efficacy of (-)-FTC in the woodchuck model was assessed by quantitation of serum WHV DNA levels and by WHV particle-associated DNA polymerase activity at two dosages, 30 and 20 mg/kg given i.p. twice daily (b.i.d.), respectively. The level of WHV DNA in serum was reduced 20- to 150-fold (average, 56-fold) in the 30-mg/kg-b.i.d. treatment group and 6- to 49-fold (average, 27-fold) in the 20-mg/kg-b.i.d. treatment group. Viral DNA polymerase levels diminished accordingly. One week after treatment was discontinued, WHV levels returned to pretreatment levels in both studies. These animals were biopsied before and following treatment with 30 mg of (-)-FTC per kg. Their livers were characterized by a mild increase in cytoplasmic lipid levels, but this change was not associated with altered liver enzyme levels. Serum chemistry and hematology results were within the normal ranges for all treated animals. We conclude that (-)-FTC is a potent antihepadnaviral agent and that it has no detectable toxic effects in woodchucks when given for up to 25 days. Further development of (-)-FTC as an anti-hepatitis B virus therapy for patients is warranted.

DNA polymerase activity of hepatitis B virus particles: differential inhibition by l-enantiomers of nucleotide analogs

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5′-triphosphates of nucleoside analogs. The 1-β- l (1 S,4 R) and 1-β- d (1 R,4 S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA. Previously, both enantiomers of several analogs were found to be substrates for human immunodeficiency virus type 1 reverse transcriptase (HIV RT); the 1-β- d enantiomers of some pairs were preferred as substrates. In contrast, the 1-β- l enantiomers of all pairs tested were the more potent inhibitors of labeled substrate incorporation into hepatitis B virus DNA; the concentration required to inhibit the incorporation of the natural substrate by 50% was 6-fold to several hundred-fold lower than the concentration of the 1-β- d enantiomer required for the same inhibitory effect. This preference for the 1-β- l enantiomers was observed for both RNA-directed synthesis in core particles and DNA-directed synthesis in viral particles. The observed antiviral effect of the nucleoside analogs in cell culture seemed to be limited chiefly by their phosphorylation in cells.

3′ → 5′ and 5′ → 5′ uridine-AZT phosphodiester dimers: Synthesis and in vitro anti-HIV activities

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5′-triphosphates of nucleoside analogs. The 1-β-l (1S,4R) and 1-β-d (1R,4S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA. Previously, both enantiomers of several analogs were found to be substrates for human immunodeficiency virus type 1 reverse transcriptase (HIV RT); the 1-β-d enantiomers of some pairs were preferred as substrates. In contrast, the 1-β-l enantiomers of all pairs tested were the more potent inhibitors of labeled substrate incorporation into hepatitis B virus DNA; the concentration required to inhibit the incorporation of the natural substrate by 50% was 6-fold to several hundred-fold lower than the concentration of the 1-β-d enantiomer required for the same inhibitory effect. This preference for the 1-β-l enantiomers was observed for both RNA-directed synthesis in core particles and DNA-directed synthesis in viral particles. The observed antiviral effect of the nucleoside analogs in cell culture seemed to be limited chiefly by their phosphorylation in cells.

Penciclovir: A novel nucleoside analogue with potent activity against duck hepatitis B virus in vivo: S Locarnini, E Lin, C Luscombe, P Edwards, D Anderson, T Shaw. Victorian Infectious Diseases Reference Laboratory and Macfarlane Burnet Centre for Medical Research, Fairfield Hospital, Fairfield, Victoria 3078, Australia

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5′-triphosphates of nucleoside analogs. The 1-β-l (1S,4R) and 1-β-d (1R,4S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA. Previously, both enantiomers of several analogs were found to be substrates for human immunodeficiency virus type 1 reverse transcriptase (HIV RT); the 1-β-d enantiomers of some pairs were preferred as substrates. In contrast, the 1-β-l enantiomers of all pairs tested were the more potent inhibitors of labeled substrate incorporation into hepatitis B virus DNA; the concentration required to inhibit the incorporation of the natural substrate by 50% was 6-fold to several hundred-fold lower than the concentration of the 1-β-d enantiomer required for the same inhibitory effect. This preference for the 1-β-l enantiomers was observed for both RNA-directed synthesis in core particles and DNA-directed synthesis in viral particles. The observed antiviral effect of the nucleoside analogs in cell culture seemed to be limited chiefly by their phosphorylation in cells.

Effect of low dosage of interferon on natural killer activity in patients with HBsAg-positive chronic active hepatitis.

The effect of human leukocyte interferon (IFN) on natural killer (NK) activity in 7 patients with HBsAg-positive chronic active hepatitis was investigated. Human leukocyte IFN was intramuscularly given once a week for 4 consecutive weeks (10 X 10(5), 5 X 10(5), 2 X 10(5) and 1 X 10(5) U). 1 week after the initial injection, NK activity showed a significant rise compared with the preinjection levels (p less than 0.001) and remained elevated during the 1-month treatment, while it fell after the cessation of IFN therapy. 4 of 5 patients showed a marked decrease in Dane particle-associated DNA-polymerase activity. Serum HBeAg disappeared in 3 of 6 patients and serum transaminase levels markedly improved in all patients. Only a transient reduction in HBsAg was observed in 2 patients after the treatment period. The findings indicate that a low dosage of human leukocyte IFN such as 18 X 10(5) U, administered in decreasing doses, enhances NK activity in patients with HBsAg-positive chronic active hepatitis, and that the effect of IFN is involved in the immune mechanisms associated with natural cytotoxicity.

Susceptibility of domestic birds to Lymphoproliferative disease virus (LPDV) of Turkeys1

The susceptibility of domestic ducks, geese and chickens to infection with lymphoproliferative disease virus (LPDV) of turkeys was tested. Infection with LPDV was assayed by measuring the particle-associated DNA polymerase in plasma. Ducks and geese were not susceptible to infection with this virus. Chickens were susceptible to the infection with LPDV and some of the birds developed a persistent viraemia. Six serial passages in chickens of viraemic plasma were achieved. Lymphoproliferative gross and microscopic lesions caused by LPDV infection were less frequent and less severe in chickens than in turkeys. LPDV infection in chickens was proven by the reproduction of LPD in turkeys by inoculation of chicken viraemic plasma, and by molecular hybridisation between LPDV (3H)cDNA and RNA extracted from the plasma and organs of inoculated chickens.

Therapeutic effect of a low dosage of human leukocyte interferon on chronic hepatitis B virus infection.

A low dosage of human leukocyte interferon was intramuscularly given to 47 patients with hepatitis B surface antigen (HBsAg) positive chronic active hepatitis once a week for 4 consecutive weeks (10 X 10(5), 5 X 10(5), 2 X 10(5) and 1 X 10(5) U). After treatment, a reduction of serum HBsAg was observed in 26 patients; 3 of them showed no serum HBsAg and 1 of the 3 appeared to produce antibody to HBsAg. 31 of 34 patients investigated showed a significant decrease in Dane particle-associated DNA-polymerase activity (p less than 0.001). Among 17 patients positive for hepatitis B e antigen (HBeAg), 10 of them seroconverted to anti-HBe. Serum transaminase levels also significantly improved in 34 of 38 patients (p less than 0.001). Our findings indicate that a low dosage of human leukocyte interferon such as 18 X 10(5) U, administered in progressively decreasing doses, may be effective in the treatment of chronic hepatitis B virus infection.

Dane particle DNA polymerase and HBeAg: impact on clinical, laboratory, and histologic findings in hepatitis B-associated chronic liver disease.

Fifty patients with chronic HBs antigenemia and Dane particle-associated DNA polymerase and HBeAg in their serum were contrasted to 46 HBsAg positive patients who had neither serum DNA polymerase or HBeAg. The time from acute onset and the duration of antigenemia were longer in patients who were DNA polymerase and HBeAg negative than in those who had both serum markers. Cirrhosis, hypoalbuminemia, and sequelae of chronic liver disease were more common in DNA polymerase, HBeAg negative patients than in those who were positive. These findings are consistent with the hypothesis that active viral replication is an early, albeit prolonged stage in the development of advanced HBsAg-associated liver disease.

Radioimmunoassay for hepatitis B 'e' antigen and antibody: correlations with viral replication and prognostic value.

The presence of hepatitis B 'e' antigen (HBeAg) and its antibody (anti-HBe was evaluated by radioimmunoassay (RIA) in various groups of HBsAg-positive patients. HBeAg was present in the majority of the sera from patients with acute viral hepatitis at onset of clinical symptoms and disappeared after 1 year. Almost all hemodialysis patients had HBeAg in their sera. 40% of the patients who had chronic active hepatitis and 50% with chronic persistent hepatitis had HBeAg with no relationship to the inflammatory activity of the disease evaluated by the presence of mononuclear infiltration in liver biopsy. The comparison between the presence of HBeAg and Dane particle-associated DNA-polymerase activity showed that HBeAg was consistently found in almost all the sera which presented DNA-polymerase activity. HBeAg, as determined by RIA, may therefore be useful in the screening of highly infective patients with elevated viral replication.

Hepatitis-B-Virus-assoziierte Desoxyribonucleinsäure-Polymerase: eine partielle Charakterisierung mit chemischen Substanzen

The very limited coding capacity of the HBV-DNA led us to study the nature of the HBV (Dane particle) -- associated DNA polymerase. The HBV-associated DNA polymerase met in many respects the characteristics of the repair enzyme of the host: the DNA polymerase beta. It operates under high salt conditions, and exhibits similar salt effects with NaCl, KCL, and PO4(3-). It is insensitive to sulfhydryl group blockers, such as p-hydroxymercuribenzoate and N-ethylmaleimide, is resistant to phosphonoacetic acid, and not inhibited by 5 mol/l urea. It requires a divalent cation (Mg2+) for activity, the Mg2+ concentration revealing optimal activity is somewhat higher than that described for most DNA polymerases beta. The HBV-associated DNA polymerase differs also from most DNA polymerase beta in its sensitivity ot ddTTP and its optimal pH. The fact that DNA polymerase beta of different origin vary considerably in their response to chemical agents and that the DNA polymerase beta from human liver has not been studied allows no definite conclusion as to the nature of the Dane particle-associated DNA polymerase.

Human placentas contain a specific inhibitor of RNA-directed DNA polymerase.

Human placental extracts contain a specific inhibitor of mammalian retroviral RNA-directed DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) activity. This inhibitor copurifies with retrovirus-like particles in human placental extracts. The inhibitor can be removed from these particles by salt extraction, which leads to the recovery of the polymerase activity. Thus, the inhibitor does not irreversibly inactivate the particle-associated RNA-directed DNA polymerase activity. The inhibitory preparation contained no nuclease, protease, or phosphatase activity. Because its inhibitory action can be eliminated by the addition of more virus to the reaction, nonspecific inactivation of enzyme substrate has been ruled out. A partial characterization of the inhibitor indicates that it is (i) insensitive to ether, trypsin, and phospholipase C; (ii) stable to heat and pH 2-12; and (iii) nondialyzable.

Inhibition of hepatitis-B-virus DNA polymerase by phosphonoformate: Studies on its mode of action

Phosphonoformate (PFA) and phosphonoacetate (PAA) were tested for their ability to inhibit the hepatitis-B-virus associated DNA polymerase. The HBV DNA polymerase was inhibited by 100 microM/liter PFA 50% while it was highly resistant to PAA. The inhibition of the Dane particle-associated DNA polymerase by PFA was not competitive to substrates and not affected by changes in the magnesium concentration. PFA was active also after initiation of the DNA polymerase reaction. Competition studies revealed that PFA had a higher affinity to a proposed pyrophosphate binding site than PAA or--alternatively--that both compounds bind to different sites.

Regulation of expression of type C virion DNA polymerase (reverse transcriptase) in human x mouse and human x rat hybrid cells.

Human cells from 13 different individuals were fused to mouse and rat cells producing abundant type C viral particles. The results demonstrate that incorporation of active DNA polymerase (reverse transcriptase) into mature type C particles is suppressed in many of the hybrid clones but not in the parental mouse cell clones. This low particle-associated DNA polymerase phenotype was a heritable trait for over 100 cell generations but reversion to a high particle-associated DNA polymerase phenotype was possible. In contrast, no evidence for suppression of viral p30 antigen was found. These results suggest that human cells contain a factor(s) capable of interfering with the normal maturation of the mouse retrovirus DNA polymerase protein; however, it was not possible to assign this function to any of 20 different human chromosomes tested. It is suggested that these somatic cell hybrids may be useful in examining individual events in retrovirus packaging and release.

Quantitative correlation between the Dane particle-associated DNA polymerase and the hepatitis B e antigen.

In order to evaluate the potential infectivity of blood of hepatitis B patients, the Dane particle associated DNA polymerase was determined, which is a reliable marker for the presence of complete viral particles. Enzyme activities were compared with hepatitis B e antigen (HBeAg) titers determined by radioimmunoassay. Detectable DNA polymerase activity was only present in HBeAg positive blood, preferentially in samples with high antigen titers (1 : 1000 and above). These samples therefore have to be considered as highly infectious. However, blood with low HBeAg levels and free of detectable polymerase activity can still be infectious, since the polymerase reaction is rather insensitive compared to the radioimmunological HBeAg determination.

RNA-directed DNA polymerase from particles released by normal goose cells.

Cells from a goose embryo were shown to release particle-associated RNA-directed DNA polymerase and RNase H activities that required the presence of Nonidet P-40 for detection. The particles were not infectious and did not have endogenous DNA synthesis. The goose particle DNA polymerase was related to the DNA polymerase of spleen necrosis virus with respect to size and was inhibited by immunoglobulin G to spleen necrosis virus DNA polymerase. However, goose cells producing DNA polymerase-containing particles did not contain reticuloendotheliosis virus-related nucleotide sequences in their DNA.

Prevalence of HBeAg, anti-HBe and Dane particle-associated DNA polymerase activity in asymptomatic carriers of HBsAg.

HBeAg/anti-HBe and Dane particle-associated DNA polymerase activity were detected in serum samples from 358 HBsAg asymptomatic carriers found during normal routine screening of 11,200 blood donors (HBsAg prevalence 3.1%). Since virus specific DNA polymerase activity and HBeAg seem to be associated in some way with hepatitis B virus infectivity and liver damage, 5% of the HBsAg carriers examined, as detected by the presence of HBeAg, and 9.5%, as shown by DNA polymerase activity, can be expected to have liver damage and a potential risk of transmitting hepatitis B to contacts. On the other hand, 48% of subjects were theoretically healthy and non-infective because of the presence of anti-HBe in their blood. The differentiation of groups of asymptomatic HBsAg carriers, on the basis of these serological markers, may have important clinical and epidemiological implications.

Controlled release of particle-associated RNA-dependent DNA polymerase by primary chick embryo cell cultures

Chick embryo cell cultures release a particle-associated RNA-dependent DNA polymerase into the culture medium. The release shows a characteristic time course with a maximum on the 3rd or 4th day in culture. The release of enzyme decreases when the cells are further cultivated and passaged. The enzyme was characterized as an RNA-dependent DNA polymerase by its ability to transcribe heteropolymeric RNA into DNA. It is different from the polymerase of the avian leukosis/sarcoma virus group and indistinguishable from an RNA-dependent DNA polymerase from normal embryonated chicken eggs described previously [1, 2]. The release of enzyme is independent of the genetic systems regulating the complete or partial expression of the endogenous avian leukosis virus genome. The amount of enzyme released is dependent on the age of the embryo from which the cell cultures are prepared. Cells prepared from 6-day-old embryos release maximal enzyme activity.

Purification and characterization of particles containing RNA-dependent DNA polymerase, in the allantoic fluid of uninfected leukosis virus-free chicken eggs

The allantoic fluid of embryonated chicken eggs regularly contains particle-associated RNA-dependent DNA polymerase, as shown by its reaction with homopolymeric and heteropolymeric RNA and by the characterization of the products. The purification of the particles is described. The purified particles are different from the known avian RNA tumour viruses in their protein composition and their sedimentation constant. They do not exhibit biological properties typical for RNA tumour viruses, such as helper activity, interfering properties or infectivity and do not show endogenous DNA synthesis. The particles are discussed as non-viral elements.

Bvr-1, a restriction locus of a type C RNA virus in the feline cellular genome: identification, location, and phenotypic characterization in cat X mouse somatic cell hybrids.

Somatic cell hybrids were constructed between BALB/c-RAG mouse cells and feline lymphoma cells by the hypoxanthine-aminopterin-thymidine selection scheme. RAG cells spontaneously produce an endogenous B-tropic type C virus. Cat-mouse hybrids preferentially segregate feline chromosomes and retain murine chromosomes-demonstrable by karyotypic and isozyme analyses. Despite the presence of the complete mouse genome, including the viral genome, virus production was diminished to 1-5% of the levels observed in RAG parents based upon particle-associated RNA-dependent DNA polymerase (reverse transcriptase) activity in the culture fluid. Thirty-seven hybrids made on four different occasions had suppressed virus levels, and no hybrids expressed parental virus levels. Reverse selection experiments on 6-thioguanine demonstrated that a restriction gene, tentatively named Bvr-1, was linked to the feline structural genes for hypoxanthine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase; EC 2.4.4.8) and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase; EC 1.1.1.49) in cats, probably on the X-chromosome. The genetic mode of action of Bvr-1 is trans dominant in restriction of murine leukemia virus. The restriction locus results in a block late in virus maturation but prior to release, since expression of antigens for viral structural proteins and matrue budding particles is apparent on surfaces of restriced hybrid cells but not in high-speed pellets from culture fluid of restricted cells.

Dane particle-associated DNA polymerase and e antigen: relation to chronic hepatitis among carriers of hepatitis B surface antigen.

Thirty-nine carriers of hepatitis B surface antigen (HBs Ag) were studied with respect to e antigen and Dane particle-associated DNA polymerase activity and their relation to chronic hepatitis. Most of these individuals were followed for four or five years. A strong correlation between e antigen and DNA polymerase activity was found. Of the 22 e antigen-positive patients, 21 showed polymerase activity; none of the 13 e antigen-negative patients (one of whom had antibody to e antigen) had such activity. Three of four patients who became e antigen-negative after being e antigen-positive showed loss of polymerase activity. An independent clinical evaluation showed a strong correlation between chronic hepatitis and positive reactions in the tests for e antigen and DNA polymerase. The results emphasize the possibility of differentiating between groups of chronic carriers of HBs Ag by testing for e antigen and Dane particle-associated DNA polymerase activity. The differentiation may have important clinical implications.