The activity, metabolism, and mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) were studied. Compared to acyclovir (ACV), H2G has superior activity against VZV (50% inhibitory concentration of 2.3 M) and Epstein-Barr virus (50% inhibitory concentration of 0.9 M), comparable activity against HSV-1, and weaker activity against HSV-2. The antiviral effect on HSV-1 showed persistence after removal of compound. H2G was metabolized to its mono-, di-and triphosphate derivatives in virus-infected cells, with H2G-triphosphate being the predominant product. Only small amounts of H2G-triphosphate were detected in uninfected cells (1 to 10 pmol/10 6 cells), whereas the level in HSV-1-infected cells reached 1,900 pmol/10 6 cells. H2G was a substrate for all three viral thymidine kinases and could also be phosphorylated by mitochondrial deoxyguanosine kinase. The intracellular half-life of H2G-triphosphate varied in uninfected (2.5 h) and infected (HSV-1, 14 h; VZV, 3.7 h) cells but was always longer than the half-life of ACV-triphosphate (1 to 2 h). H2G-triphosphate inhibited HSV-1, HSV-2, and VZV DNA polymerases competitively with dGTP (K i of 2.8, 2.2, and 0.3 M, respectively) but could not replace dGTP as a substrate in a polymerase assay. H2G was not an obligate chain terminator but would only support limited DNA chain extension. Only very small amounts of radioactivity, which were too low to be identified by high-performance liquid chromatography analysis of the digested DNA, could be detected in purified DNA from uninfected cells incubated with [ (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) is an acyclic guanosine analog with structural similarities to acyclovir (ACV){9-[(2-hydroxyethoxy)methyl]guanine}, the established treatment for herpesvirus infections (6), and penciclovir (PCV) [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine], a compound recently licensed in some countries for the treatment of herpes zoster (29). The racemic mixture of (RS)H2G [previously abbreviated (Ϯ)2HM-HBG (1, 3, 17)] was originally shown to have good activity against herpes simplex virus (HSV) (18) and varicella-zoster virus (VZV) in vitro (3) and in vivo (17), and subsequent studies with the separated isomers showed that this activity resided predominantly with the R isomer (1, 25), now called H2G (25,27). H2G has activity against HSV type 1 (HSV-1) and type 2 (HSV-2) (1) and has been reported to have activity against human herpesvirus type 6 (4).Such a broad-spectrum anti-herpesvirus activity makes H2G an interesting candidate for clinical development, particularly for the treatment of VZV infection, where its impressive activity could lead to improved efficacy over current therapies.Strains of VZV that are deficient in thymidine kinase (TK) are resistant to H2G (1, 3), suggesting a mechanism of activation involving selective phosphorylation by herpesvirus-induced TKs, as is the case for ACV and PCV. This is support...

Mutations in the YMDD motif of the hepatitis B virus (HBV) DNA polymerase result in reduced susceptibility of HBV to inhibition by lamivudine, at a cost in replication fitness. The mechanisms underlying the effects of YMDD mutations on replication fitness were investigated using both a cell-based viral replication system and an in vitro enzyme assay to examine wild-type (wt) and YMDD-mutant polymerases. We calculated the affinities of wt and YMDD-mutant polymerases for each natural deoxyribonucleoside triphosphate (dNTP) and determined the intracellular concentrations of each dNTP in HepG2 cells under conditions that support HBV replication. In addition, inhibition constants for lamivudine triphosphate were determined for wt and YMDDmutant polymerases. Relative to wt HBV polymerase, each of the YMDD-mutant polymerases showed increased apparent K m values for the natural dNTP substrates, indicating decreased affinities for these substrates, as well as increased K i values for lamivudine triphosphate, indicating decreased affinity for the drug. The effect of the differences in apparent K m values between YMDD-mutant polymerase and wt HBV polymerase could be masked by high levels of dNTP substrates (>20 M). However, assays using dNTP concentrations equivalent to those measured in HepG2 cells under physiological conditions showed decreased enzymatic activity of YMDD-mutant polymerases relative to wt polymerase. Therefore, the decrease in replication fitness of YMDDmutant HBV strains results from the lower affinities (increased K m values) of the YMDD-mutant polymerases for the natural dNTP substrates and physiological intracellular concentrations of dNTPs that are limiting for the replication of YMDD-mutant HBV strains.The YMDD motif (tyrosine, methionine, aspartate, aspartate) is a highly conserved amino acid sequence involved in deoxynucleoside triphosphate (dNTP) binding in the catalytic site of a number of RNA-dependent DNA polymerases, including hepatitis B virus (HBV) DNA polymerase (38). During lamivudine therapy for treatment of chronic HBV infection, diminished therapeutic responses may occur in some patients due to the emergence of mutant HBV species containing amino acid substitutions in the YMDD motif and in the proximal FLLAQ motif (phenylalanine, leucine, leucine, alanine, glutamine) of the viral polymerase (1,9,10,19,30,46). The amino acid changes in the HBV polymerase that affect lamivudine sensitivity involve the methionine at position 552 (M552) within the YMDD motif and the proximal leucine at position 528 (L528) in the upstream FLLAQ motif. The substitutions observed in clinical isolates at M552 include the hydrophobic amino acid substitutions isoleucine (designated M552I or MI) and valine (designated M552V or MV). Although the M552I mutant has been observed in clinical isolates both as a single substitution (MI) and in combination with L528M (LMMI), the M552V substitution is observed in clinical isolates almost exclusively in combination with L528M (LMMV) (1,10,18,25,31,46,53,54).Changes in the ...

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Process-based models are increasingly used to study agroecosystem interactions and N O emissions from agricultural fields. The widespread use of these models to conduct research and inform policy benefits from periodic model comparisons that assess the state of agroecosystem modeling and indicate areas for model improvement. This work provides an evaluation of simulated N O flux from three process-based models: DayCent, DNDC, and EPIC. The models were calibrated and validated using data collected from two research sites over five years that represent cropping systems and nitrogen fertilizer management strategies common to dairy cropping systems. We also evaluated the use of a multi-model ensemble strategy, which inconsistently outperformed individual model estimations. Regression analysis indicated a cross-model bias to underestimate high magnitude daily and cumulative N O flux. Model estimations of observed soil temperature and water content did not sufficiently explain model underestimations, and we found significant variation in model estimates of heterotrophic respiration, denitrification, soil NH , and soil NO , which may indicate that additional types of observed data are required to evaluate model performance and possible biases. Our results suggest a bias in the model estimation of N O flux from agroecosystems that limits the extension of models beyond calibration and as instruments of policy development. This highlights a growing need for the modeling and measurement communities to collaborate in the collection and analysis of the data necessary to improve models and coordinate future development.

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Soil texture is known to have an influence on the physical and biological processes that produce NO emissions in agricultural fields, yet comparisons across soil textural types are limited by considerations of time and practicality. We used the DayCent biogeochemical model to assess the effects of soil texture on NO emissions from agriculturally productive soils from four counties in Wisconsin. We validated the DayCent model using field data from 2 yr of a long-term (approximately 20-yr) cropping systems trial and then simulated yield and NO emissions from continuous corn ( L.) and corn-soybean ( L.) cropping systems across 35 Wisconsin soil series classified as either silt loam, sandy loam, or loamy sand. Silt loam soils had the highest NO emissions of all soil types, exhibiting 80 to 158% greater mean emissions and 100 to 282% greater emission factors compared with loamy sand and sandy loam soils, respectively. The model predicts that for these soils under these cropping systems, denitrification constituted the majority of the NO flux only in the silt loam soils. However, across all soil textures, locations, and years, denitrification explained the most variation (74-98%) in total NO emissions. Our results suggest that soil texture is an important factor in determining a range of NO emission characteristics and is critical for estimating future NO emissions from agricultural fields.

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