De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn(2+) than in the presence of Mg(2+). When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3' end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>/=50 microM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.     

Greenhouse gas emissions and global warming potential of traditional and diversified tropical rice rotation systems

Global rice agriculture will be increasingly challenged by water scarcity, while at the same time changes in demand (e.g. changes in diets or increasing demand for biofuels) will feed back on agricultural practices. These factors are changing traditional cropping patterns from double‐rice cropping to the introduction of upland crops in the dry season. For a comprehensive assessment of greenhouse gas (GHG) balances, we measured methane (CH₄)/nitrous oxide (N₂O) emissions and agronomic parameters over 2.5 years in double‐rice cropping (R‐R) and paddy rice rotations diversified with either maize (R‐M) or aerobic rice (R‐A) in upland cultivation. Introduction of upland crops in the dry season reduced irrigation water use and CH₄ emissions by 66–81% and 95–99%, respectively. Moreover, for practices including upland crops, CH₄ emissions in the subsequent wet season with paddy rice were reduced by 54–60%. Although annual N₂O emissions increased two‐ to threefold in the diversified systems, the strong reduction in CH₄ led to a significantly lower (P < 0.05) annual GWP (CH₄ + N₂O) as compared to the traditional double‐rice cropping system. Measurements of soil organic carbon (SOC) contents before and 3 years after the introduction of upland crop rotations indicated a SOC loss for the R‐M system, while for the other systems SOC stocks were unaffected. This trend for R‐M systems needs to be followed as it has significant consequences not only for the GWP balance but also with regard to soil fertility. Economic assessment showed a similar gross profit span for R‐M and R‐R, while gross profits for R‐A were reduced as a consequence of lower productivity. Nevertheless, regarding a future increase in water scarcity, it can be expected that mixed lowland–upland systems will expand in SE Asia as water requirements were cut by more than half in both rotation systems with upland crops.     

Immunobiology of the human placenta. I. IgGFc receptors in trophoblastic villi.

Qualitative and quantitative studies were performed on IgGFc receptors in the trophoblastic villi of human placentae ranging in gestational age from less than 4 weeks to full term. IgGFc receptors were detected on cells of the syncytiotrophoblast (ST) using two different assay systems; EA rosette formation and direct immunofluorescence with deaggregated human IgG. The ST IgGFc receptors had high affinity for native IgG molecules (deaggregated IgG) as well as affinity for antigen-antibody complexes (EA). The receptors for deaggregated IgG were present on a majority of ST cells in first and second trimester trophoblast, but were significantly less frequent on ST cells in older placentae. Similar receptors also were detected on cells lining some fetal vessels in the trophoblastic villi. Not only were the IgGFc receptors expressed on ST cells, but in vivo bound IgG was detected in association with the ST and the two patterns of IgG binding were essentially identical. In contrast to the qualitative nature of the receptors on ST cells, cells in the stromal (central) region of the trophoblastic villi expressed IgGFc receptors that had high affinity for EA but failed to bind the deaggregated IgG except at a high concentration. The results are discussed with respect to the possible role of the IgGFc receptors in the specific transfer of IgG from maternal to fetal circulations.     

Immunobiology of the human placenta: II. Localization of macrophages,in vivo bound IgG and C3

Human placentae ranging in gestational age from less than 4 weeks to full term were studied for the localization of IgGFc receptor bearing cells by the EA adsorption method. Most cells populating the area beneath the syncytiotrophoblast in trophoblastic villi, e.g., the stromal region, adsorbed EA. When cell suspensions of trophoblastic tissue were prepared and the individual cells were rosetted and characterized, most IgGFc receptor bearing cells were determined to be macrophages by morphology, phagocytosis and nonspecific esterase staining. A few of the syncytiotrophoblast cells also were IgGFc receptor positive. When esterase staining was applied to sections of trophoblastic tissue, the pattern of esterase positivity paralleled the pattern of EA adsorption in that most cells in trophoblastic villi were positive while decidual cells were negative. Furthermore, the pattern of IgG bound in vivo as determined by direct immunofluorescence paralleled the EA adsorption and esterase staining patterns. The bound IgG was removed by prolonged washing of the trophoblastic tissue at 37 °C which suggested that most IgG was bound to IgGFc receptors in trophoblastic villi. The results are discussed with respect to the possible functions of large numbers of macrophages located at the interphase between maternal and fetal circulations.     

Specific inhibition of bovine viral diarrhea virus replicase

Compound-1453 was identified and characterized as a specific inhibitor of bovine viral diarrhea virus (BVDV). The concentration of compound-1453 which results in 50% protection from virus-induced cytopathic effect is approximately 2.2 microM, with a therapeutic index of 60, and it is not active against a panel of RNA and DNA viruses. A time-of-addition experiment suggested that compound-1453 targets a stage of the viral life cycle after viral entry. To determine the target of compound-1453, resistant virus was generated. Resistant variants grew efficiently in the presence or absence of 33 micro M compound-1453 and exhibited replication efficiency in the presence of compound-1453 approximately 1,000-fold higher than that of the wild-type (wt) virus. Functional mapping and sequence analysis of resistant cDNAs revealed a single amino acid substitution (Glu to Gly) at residue 291 in the NS5B polymerase in all eight independently generated cDNA clones. Recombinant virus containing this single mutation retained the resistance phenotype and a replication efficiency similar to that of the original isolated resistant virus. Since compound-1453 did not inhibit BVDV polymerase activity in vitro (50% inhibitory concentration > 300 microM), we developed a membrane-based assay that consisted of a BVDV RNA replicase complex isolated from virus-infected cells. Compound-1453 inhibited the activity of the wt, but not the drug-resistant, replicase in the membrane assay at concentrations similar to those observed in the viral infection assay. This work presents a novel inhibitor of a viral RNA-dependent RNA replicase.     

Quantification of plant water uptake by water stable isotopes in rice paddy systems

Aims

Maintaining variation in germination response provides a selective advantage, by spreading risk during recruitment. In fire-prone regions, physically dormant (PY) species vary their response to dormancy-breaking fire-related heat cues at the intra-population level. However little is known about physiologically dormant (PD) species, which respond to smoke cues. These contrasting dormancy types reflect different evolutionary developmental pathways and we considered whether intra-population variation in germination of Boronia floribunda (PD) occurs in response to smoke.

Methods

Seeds were collected from individual plants. We assessed germination magnitude and rate of seeds from each individual in response to a single aerosol smoke treatment, and three concentrations of smoke water, using replicate seed lots in temperature-controlled incubators.

Results

The magnitude and onset of germination differed significantly among individuals in response to the same smoke treatment. Seeds from different individuals varied in their sensitivity to smoke water concentration, with some responding to very low doses, and others obligated to high doses.

Conclusions

Variation in germination response to smoke highlights a mechanism by which PD species spread risk, by allowing some seeds to emerge quickly, while others remain dormant in the soil seed bank. The similarity to heat-cued variation displayed by PY species suggests that this could represent a convergent functional response.