New tools for the herpes virologist.
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Drug Resistance, Microbial↗
Biomedical subjects
J Harmenberg
Publications and source records attributed to J Harmenberg.
Antiviral activities of guanosine analogs in guinea pig embryonic fibroblasts.
Previous research has shown that certain antiherpes substances which are activated by thymidine kinase are substantially more active in human fibroblasts than in green monkey kidney cells. The difference has been attributed to the presence of large amounts of intracellular thymidine in the latter cell type. Antiviral guanosine analogs but not thymidine analogs show decreased antiviral activity when used in herpes simplex virus type 1-infected guinea pig fibroblasts. We report the intracellular pools of antiviral di- and triphosphate nucleotides, the monophosphate nucleotide phosphorylating enzyme activities, and the antiviral triphosphate nucleotide stability, studied in herpes simplex virus type 1-infected and uninfected guinea pig fibroblasts. The results were compared with results of parallel experiments done with human fibroblasts and green monkey kidney cells.
Animals↗
Antiherpesvirus activity and mechanism of action of indolo-(2,3-b)quinoxaline and analogs.
The antiherpesvirus activity of 14 derivatives of indoloquinoxaline was tested. The most active was 2,3-dimethyl(dimethylaminoethyl)5H-indolo-(2,3-b)quinoxaline, also called B-220. The antiherpesvirus mechanism of B-220 was sought. The compound inhibited replication of herpes simplex virus type 1, cytomegalovirus, and varicella-zoster virus in tissue culture at concentrations of 1 to 5 microM, depending on the cell type used for assay and the amount of virus. Cellular toxicity was seen at a concentration of 10 to 30 microM, and antiviral activity in the human bladder cancer and human embryonic lung fibroblast cell lines tested was found at concentrations 3 to 15 times lower than the concentrations causing cellular toxicity. Viral DNA synthesis, as well as production of early and late viral proteins, was inhibited at 0.5 to 4.5 microM B-220, but viral DNA polymerases tested in vitro were not inhibited at these concentrations. There was no interaction with the pyrophosphate analog foscarnet, and no reversal of the antiviral activity of B-220 occurred with naturally occurring nucleosides. We conclude that the antiviral effect depends on the multiplicity of infection and may occur at the level of viral DNA synthesis and that no interference occurs with pyrophosphate analogs or nucleosides. The more potent activity against viral DNA than against cellular DNA may be caused by a true selectivity for herpesvirus DNA or by the higher metabolism of viral DNA in infected cells.
Acyclovir↗
Effects of ribonucleotide reductase inhibition on pyrimidine deoxynucleotide metabolism in acyclovir-treated cells infected with herpes simplex virus type 1.
The pyrimidine metabolism of fibroblasts infected with herpes simplex virus type 1 was studied. Herpes simplex virus type 1 infection increased the dTTP pool and thymidylate synthetase activity but reduced thymidine excretion. Addition of acyclovir to infected cells increased thymidine excretion, the dTTP pool, and thymidylate synthetase activity. Addition of a virus-specific ribonucleotide reductase inhibitor (A723U) decreased all three. The synergy between the two compounds is discussed.
Acyclovir↗
Inhibition of varicella-zoster virus-induced DNA polymerase by a new guanosine analog, 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine triphosphate.
The triphosphates of the antiherpesvirus acyclic guanosine analogs 9-[4-hydroxy-2(hydroxymethyl)butyl] guanine (2HM-HBG), 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV]), and 9-(3,4-dihydroxybutyl)guanine (buciclovir) were examined for their effects on partially purified varicella-zoster virus (VZV) DNA polymerase as well as cellular DNA polymerase alpha. The triphosphate of 2HM-HBG competitively inhibited the incorporation of dGMP into DNA catalyzed by the VZV DNA polymerase. 2HM-HBG-triphosphate (2HM-HBG-TP) had a higher affinity for the dGTP-binding site on the VZV DNA polymerase than did dGTP; apparent Km and Ki values of dGTP and 2HM-HBG-TP were 0.64 and 0.034 microM, respectively. ACV-triphosphate (ACV-TP) was found to be the most potent inhibitor of VZV DNA polymerase. ACV-TP had a 14 and 464 times better direct inhibitory effect than 2HM-HBG-TP and buciclovir-triphosphate, respectively. The cellular (human embryonic lung fibroblast) DNA polymerase alpha inhibition was related to viral polymerase inhibition as efficacy ratios: 2HM-HBG-TP had a ratio of more than 1,000, which appeared to be similar to that of ACV-TP.
Acyclovir↗
Comparison of sample preparation methods for the high-performance liquid chromatographic analysis of cell culture extracts for triphosphate ribonucleosides and deoxyribonucleosides.
We compared four different procedures for the purification and concentration of nucleoside triphosphates in cell extracts prior to HPLC analysis. Two methods involved precipitation, with either acetonitrile or calcium fluoride. The acetonitrile procedure yielded reasonable recovery and sufficient purity for the subsequent HPLC analysis. The calcium fluoride coprecipitation procedure gave both good recovery and purity; but the recovery was shown to be dependent on the concentration of the nucleoside triphosphates. The other two methods involved small Sep-Pak cartridges. The silica cartridge procedure yielded unfavorable recoveries in periodate-treated cell extracts, apparently due to poor solubility of nucleoside triphosphates in the requisite solvents. The strong anion exchange cartridge procedure yielded both good recovery and purity. This procedure was found to be fast, efficient, and reliable for purifying and concentrating nucleotides in cell extracts.
Acetonitriles↗
Inhibiting effect of (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine on varicella-zoster virus replication in cell culture.
The activity and mode of action of the new nucleoside analog (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (2HM-HBG) against varicella-zoster virus (VZV) were determined. In cell culture, replication of different strains of VZV was inhibited to 50% by 0.4 to 0.7 microM 2HM-HBG, while 685 microM was required to inhibit 50% of the DNA synthesis in uninfected human lung fibroblasts. A thymidine kinase-negative VZV strain was not inhibited by 100 microM 2HM-HBG. Inhibition of VZV replication was not reversible after 7 to 14 days of incubation, depending on the multiplicity of VZV. 2HM-HBG was shown to be selectively phosphorylated by purified VZV thymidine kinase, with an inhibition constant of 32.5 microM. The antiviral activity of 2HM-HBG in cell culture was decreased by the addition of deoxythymidine and deoxycytidine but not by other ribo- or deoxyribonucleosides.
Antiviral Agents↗
Comparative methods for detection of thymidine kinase-deficient herpes simplex virus type 1 strains.
Four methods for analyzing viral susceptibility to antiviral substances were compared. In two methods viral products were measured: late viral proteins were measured by an enzyme-linked immunosorbent assay and viral DNA was measured by DNA hybridization. Infectious virus was quantified in the other two assays as the number of plaques and the yield of virus. The enzyme-linked immunosorbent assay procedure in our hands detected the smallest amounts (lowest proportions) of thymidine kinase-deficient herpes simplex virus type 1 mixed with wild-type virus. The thymidine kinase-deficient proportion of the herpes simplex virus type 1 isolate increased rapidly in the presence of acyclovir in cell culture.
Acyclovir↗
[New aspects in the diagnosis and treatment of herpes infections].
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Antiviral Agents↗
Deoxythymidine pools of human skin and guinea pig organs.
The activity of antiviral nucleoside analogues like acyclovir is influenced by a number of cellular factors, one being the deoxythymidine (dThd) concentration. We therefore analysed the dThd concentration in human plasma and skin and in organs of guinea pig, the common experimental animal. High-performance liquid chromatography showed low amounts of dThd in human skin, 0.20-1.15 nmol/g, whereas guinea pig skin and spleen had 20-30 nmol/g and the concentration in guinea pig plasma was 10-times higher than in human plasma. These animals are therefore in this respect less suitable as accurate models for antiviral nucleoside activity in humans.
Animals↗
Urinary modified nucleosides as tumor markers in cancer of the urinary organs or female genital tract.
Using a sensitive and specific method involving high-performance liquid chromatography, urinary levels of four modified nucleosides--pseudouridine (psi), 1-methylinosine (m1I), 1-methyladenosine (m1A), and 1-methylguanosine (m1G)--were investigated before and after treatment in 31 patients with cancer of the urinary organs or the female genital tract. Before treatment m1I was the most frequently elevated nucleoside (77%). Pretreatment urinary levels of psi, m1I, and m1A in patients with stage 2-4 cancer of the female genital tract were significantly elevated compared to human healthy volunteers (p less than 0.005). Compared with the other nucleosides, psi appeared to correlate more closely with the clinical outcome (progression or regression) of patients with cancer of the female genital tract. In the case of patients with cancer of the urinary organs, m1I followed the clinical outcome better than the other nucleosides measured. Therefore psi and m1I seem to be useful for monitoring genito-urinary cancers.
Adenosine↗
Pseudouridine and uridine in normal kidney and kidney cancer tissues.
The tissue concentrations of a modified nucleoside, pseudouridine, and a normal nucleoside, uridine, were measured with high-performance liquid chromatography. Human kidneys were obtained from five patients with renal cell carcinoma and divided into a noncancerous part and a cancerous part. The pseudouridine concentration in the cancerous part of the kidneys ranged between less than 2-2.8 nmoles/g and in the noncancerous part 4.3-19.4 nmoles/g (mean 10,9 nmoles/g). The uridine concentration in the cancerous and noncancerous parts of the kidney ranged between 19.6-179.1 nmoles/g (mean 110.7 nmoles/g) and 117.5-235.6 nmoles/g (mean 191.5 nmoles/g), respectively. The pseudouridine concentration appeared to be approximately seven times higher in the noncancerous part as compared to the cancerous part of the kidney. In the case of uridine, the difference was less pronounced.
Carcinoma, Renal Cell↗
Deoxythymidine pools in animal and human skin with reference to antiviral drugs.
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Acyclovir↗
Nucleoside pools of acyclovir-treated herpes simplex type 1 infected cells.
Nucleoside pools of herpes simplex type 1 (HSV-1)-infected and uninfected African green monkey kidney (GMK) cells and human fetal lung fibroblasts (HL) have been analysed with high-performance liquid chromatography (HPLC). The only nucleosides found in measurable amounts were deoxythymidine (dThd) and adenosine (Ado). The dThd pool seemed to be greater in GMK cells than in HL cells. dThd was also the only nucleoside excreted into the medium. HSV-1 infection reduced the dThd concentration of GMK cells. Addition of acyclovir (ACV) to HSV-1-infected GMK cells inhibited virus replication. This resulted in a dThd concentration similar to that of uninfected GMK cells. dThd added to HSV-1-infected GMK and HL cells reduced the antiviral action of ACV but not that of phosphonoformic acid (PFA). ACV is known to be activated mainly by HSV-induced deoxythymidine kinase (dTK), an enzyme which utilizes dThd as a substrate, while the action of PFA is independent of dTK. The low antiviral activity of ACV in GMK cells as compared to HL cells may be explained by the presence of high amounts of dThd in GMK cells.
Acyclovir↗
Multiplicity dependence and sensitivity of herpes simplex virus isolates to antiviral compounds.
An immunoassay that enables one to assess both viral multiplicity and sensitivity to antiviral drugs, was used to determine the sensitivity of untreated patients' virus isolates. The upper border levels for judging clinical isolates sensitive to Ara-A, ACV, PFA or IDU were calculated from a total of 48 primary herpes simplex isolates. Although five isolates were considered less sensitive to one drug and one isolate to two drugs, all were sensitive at a lower virus multiplicity. Analysis of these isolates showed that no isolate was genetically resistant, but that multiplicity dependence of drugs was high with ACV and Ara-A, lower with PFA. In view of the multiplicity dependence of HSV sensitivity to different drugs, it is recommended that isolates from treatment failures or isolates considered resistant should be assayed in detail.
Acyclovir↗
Interactions between virus and antiviral compounds in the host cell.
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Acyclovir↗
A novel method for determining the sensitivity of herpes simplex virus to antiviral compounds.
A rapid and sensitive assay was developed to analyse the sensitivity of wild type HSV-1 and HSV-2 isolates with respect to a battery of antiviral substances. In the viral sensitivity assay, human embryonic lung fibroblasts are incubated with the virus isolate and different concentrations of the antivirals. After 1-3 days, the cells are disrupted and analysed for HSV type 1 or 2 antigens by an enzyme-linked immunosorbent assay. Antigens corresponding to 17 plaque-forming units were detectable after 1 day of incubation. After 3 days, HSV antigens derived from less than one plaque-forming unit were measurable. The sensitivities of 22 HSV-1 and 19 HSV-2 primary isolates from untreated patients were tested against adenosine arabinoside, acyclovir, phosphonoformic acid and iododeoxyuridine. Each isolate was found to have an individual pattern of sensitivities to the different antivirals. Five isolates were judged to be relatively resistant to one or more of the drugs tested.
Antigens, Viral↗
Computer-based virus sensitivity assay and neutralization method. Applications for herpesviruses.
A method for computerized analysis of the antiviral efficiency of drugs and immunoglobulins was developed. The sensitivities of herpes simplex virus (HSV), cytomegalovirus (CMV) and varicella-zoster virus (VZV) to antiviral drugs were determined. A new type of neutralization assay could be performed rapidly and accurately with HSV and CMV. Combining immunological methods for herpesvirus antigen detection with a computerized analysis permitted rapid, sensitive measurement of drug sensitivities, comparison of antiviral activities at various virus concentrations and selection of potent neutralizing antisera.
Antibodies, Viral↗