Papain-catalyzed oligomerization of diethyl L-glutamate hydrochloride was conducted in phosphate buffer at 40 °C. Because of rapid oligomerization kinetics, high substrate concentrations were not needed to shift the equilibrium for oligomer synthesis. For example, at 0.03 M diethyl L-glutamate hydrochloride, oligo(γ-ethyl L-glutamate) synthesis and precipitation from solution occurred in 55% yield. MALDI-TOF spectra of precipitated products showed two series of ion peaks separated by 157 m/z units, the mass of oligo(γ-ethyl-L-glutamate) repeat units. The most abundant signals were at DP 8 and 9, in excellent agreement with DP avg values determined by 1 H NMR. Lower intensity peaks with m/z less by 28 correspond to hydrolysis of one ester group either at a chain end or a pendant group along chains. Oligo(γ-ethyl-L-glutamate) synthesis at 40 °C in phosphate buffer (0.9 M, pH 7) occurred rapidly so that by 5, 10, and 20 min the yield reached 70 ( 4%, 78 ( 4% and 81 ( 5%, respectively. High product yields were observed over a broad range of pH values. As long as the pH was maintained from 5.5 to 8.5, the product yield was g60%. Ionic strength had no significant effect on oligopeptide yield. The dominant role of phosphate buffer in reactions was its control of pH. Other influences of phosphate ions on papain, such as nonspecific salt interactions or a "salting out" of product, appear to be of little or no importance. Loss in protein concentration and activity in the supernatant was observed after one reaction. A second reaction cycle performed using recovered supernatants resulted in a decrease in oligo(γ-ethyl-L-glutamate) yield from about 75% to 20%.
Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent incorporation of dUMP opposite to A during reverse transcription (“uracilation”). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that monocytes are almost completely devoid of functional UBER, while macrophages are mainly deficient in the initial enzyme uracil DNA glycosylase (hUNG2). Accordingly, dUMP persists in viral DNA during the lifetime of a MC and can only be removed after differentiation of MC into MDM. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (G→T, T→A, T→G, A→C). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. These mutations may arise from the increased mispairing and duplex destabilizing effects of dUMP relative to dTMP during reverse transcription. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection.
Porcine reproductive and respiratory syndrome virus (PRRSV), is a highly mutable RNA virus that affects swine worldwide and its control is very challenging due to its formidable heterogeneity in the field. In the present study, DNA vaccines constructed with PRRSV GP5-Mosaic sequences were complexed to cationic liposomes and administered to experimental pigs by intradermal and intramuscular injection, followed by three boosters 14, 28 and 42 days later. The GP5-Mosaic vaccine thus formulated was immunogenic and induced protection from challenge in vaccinated pigs comparable to that induced by a wild type (VR2332) GP5 DNA vaccine (GP5-WT). Periodic sampling of blood and testing of vaccine-induced responses followed. Interferon-γ (IFN-γ) mRNA expression by virus-stimulated peripheral blood mononuclear cells (PBMCs) of GP5-Mosaic-vaccinated pigs was significantly higher compared to pigs vaccinated with either GP5-WT or empty vector at 21, 35 and 48 days after vaccination. Cross-reactive cellular responses were also demonstrated in GP5-Mosaic vaccinated pigs after stimulation of PBMCs with divergent strains of PRRSV. Thus, significantly higher levels of IFN-γ mRNA were detected when PBMCs from GP5-Mosaic-vaccinated pigs were stimulated by four Genotype 2 strains (VR2332, NADC9, NADC30 and SDSU73), which have at least 10% difference in GP5 amino acid sequences, while such responses were recorded only upon VR2332 stimulation in GP5-WT-vaccinated pigs. In addition, the levels of virus-specific neutralizing antibodies were higher in GP5-Mosaic or GP5-WT vaccinated pigs than those in vector-control pigs. The experimental pigs vaccinated with either the GP5-Mosaic vaccine or the GP5-WT vaccine were partially protected from challenge with VR2332, as measured by significantly lower viral loads in sera and tissues and lower lung lesion scores than the vector control group. These data demonstrate that the GP5-Mosaic vaccine can induce cross-reactive cellular responses to diverse strains, neutralizing antibodies, and protection in pigs.
dUTP is a close structural congener of dTTP and can be readily incorporated into DNA opposite to adenine during DNA replication leading to non-mutagenic dU/A base pairs (‘uracilation’). We find that dU/A pairs located within DNA transcriptional templates optimized for either T7 RNA polymerase (T7 RNAP) or human RNA polymerase II (pol II) have inhibitory and mutagenic effects on transcription. The data for T7 RNAP establishes that even a single dU/A pair can inhibit promoter binding and transcription initiation up to 30-fold, and that inhibitory effects on transcription elongation are also possible. Sequencing of the mRNA transcribed from uniformly uracilated DNA templates by T7 RNAP indicated an increased frequency of transversion and insertion mutations compared to all T/A templates. Strong effects of dU/A pairs on cellular transcription activity and fidelity were also observed with RNA pol II using uracil base excision repair (UBER)-deficient human cells. At the highest levels of template uracilation, transcription by RNA pol II was completely blocked. We propose that these effects arise from the decreased thermodynamic stability and increased dynamics of dU/A pairs in DNA. The potential implications of these findings on gene regulation and disease are discussed.
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