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Despite the eradication of smallpox four decades ago, poxviruses continue to be a threat to humans and animals. The arsenal of anti-poxvirus agents is very limited and understanding mechanisms of resistance to agents targeting viral DNA polymerases is fundamental for the development of antiviral therapies. We describe here the phenotypic and genotypic characterization of poxvirus DNA polymerase mutants isolated under selective pressure with different acyclic nucleoside phosphonates, including HPMPC (cidofovir), cHPMPC, HPMPA, cHPMPA, HPMPDAP, HPMPO-DAPy, and PMEO-DAPy, and the pyrophosphate analogue phosphonoacetic acid. Vaccinia virus (VACV) and cowpox virus drug-resistant viral clones emerging under drug pressure were characterized phenotypically (drug-susceptibility profile) and genotypically (DNA polymerase sequencing). Different amino acid changes in the polymerase domain and in the 3′-5′ exonuclease domain were linked to drug resistance. Changes in the 3′-5′ domain emerged earlier than in the polymerase domain when viruses acquired a combination of mutations. Our study highlights the importance of poxvirus DNA polymerase residues 314, 613, 684, 688, and 851, previously linked to drug resistance, and identified several novel mutations in the 3′-5′ exonuclease domain (M313I, F354L, D480Y) and in the DNA polymerase domain (A632T, T831I, E856K, L924F) associated with different drug-susceptibility profiles. Furthermore, a combination of mutations resulted in complex patterns of cross-resistance. Modeling of the VACV DNA polymerase bearing the newly described mutations was performed to understand the effects of these mutations on the structure of the viral enzyme. We demonstrated the emergence of drug-resistant DNA polymerase mutations in complex patterns to be considered in case such mutations should eventually arise in the clinic. [ABSTRACT FROM AUTHOR]

The appearance of drug-resistant mutations in UL54 DNA polymerase and UL97 kinase genes is problematic for the treatment of human cytomegalovirus (HCMV) diseases. During treatment of HCMV infection in a pediatric hematopoietic cell transplant recipient, H600L and T700A mutations and E576G mutation were independently found in the UL54 gene. Foscarnet (FOS; phosphonoformic acid) resistance by T700A mutation is reported. Here, we investigated the role of novel mutations in drug resistance by producing recombinant viruses and a model polymerase structure. The H600L mutant virus showed an increase in resistance to ganciclovir (GCV) by 11-fold and to FOS and cidofovir (CDV) by 5-fold, compared to the wild type, while the E756G mutant virus showed an increase in resistance to FOS by 9-fold and modestly to CDV by 2-fold. With the FOS-resistant T700A mutation, only H600L produced increased FOS resistance up to 37-fold, indicating an additive effect of these mutations on FOS resistance. To gain insight into drug resistance mechanisms, a model structure for UL54 polymerase was constructed using the yeast DNA polymerase as a template. In this model, HCMV DNA polymerase contains a long palm loop domain of which H600 and T700 are located on each end and T700 interacts with the FOS binding pocket. Our results demonstrate that H600L and E756G mutations in UL54 polymerase are novel drug-resistant mutations and that the acquisition of both H600L and T700A mutations in the DNA-binding loop confers increased resistance to FOS treatment, providing novel insights for the mechanism acquiring foscarnet resistance. [ABSTRACT FROM AUTHOR]

The appearance of drug-resistant mutations in UL54 DNA polymerase and UL97 kinase genes is problematic for the treatment of human cytomegalovirus (HCMV) diseases. During treatment of HCMV infection in a pediatric hematopoietic cell transplant recipient, H600L and T700A mutations and E576G mutation were independently found in the UL54 gene. Foscarnet (FOS; phosphonoformic acid) resistance by T700A mutation is reported. Here, we investigated the role of novel mutations in drug resistance by producing recombinant viruses and a model polymerase structure. The H600L mutant virus showed an increase in resistance to ganciclovir (GCV) by 11-fold and to FOS and cidofovir (CDV) by 5-fold, compared to the wild type, while the E756G mutant virus showed an increase in resistance to FOS by 9-fold and modestly to CDV by 2-fold. With the FOS-resistant T700A mutation, only H600L produced increased FOS resistance up to 37-fold, indicating an additive effect of these mutations on FOS resistance. To gain insight into drug resistance mechanisms, a model structure for UL54 polymerase was constructed using the yeast DNA polymerase as a template. In this model, HCMV DNA polymerase contains a long palm loop domain of which H600 and T700 are located on each end and T700 interacts with the FOS binding pocket. Our results demonstrate that H600L and E756G mutations in UL54 polymerase are novel drug-resistant mutations and that the acquisition of both H600L and T700A mutations in the DNA-binding loop confers increased resistance to FOS treatment, providing novel insights for the mechanism acquiring foscarnet resistance.

Despite the eradication of smallpox four decades ago, poxviruses continue to be a threat to humans and animals. The arsenal of anti-poxvirus agents is very limited and understanding mechanisms of resistance to agents targeting viral DNA polymerases is fundamental for the development of antiviral therapies. We describe here the phenotypic and genotypic characterization of poxvirus DNA polymerase mutants isolated under selective pressure with different acyclic nucleoside phosphonates, including HPMPC (cidofovir), cHPMPC, HPMPA, cHPMPA, HPMPDAP, HPMPO-DAPy, and PMEO-DAPy, and the pyrophosphate analogue phosphonoacetic acid. Vaccinia virus (VACV) and cowpox virus drug-resistant viral clones emerging under drug pressure were characterized phenotypically (drug-susceptibility profile) and genotypically (DNA polymerase sequencing). Different amino acid changes in the polymerase domain and in the 3′-5′ exonuclease domain were linked to drug resistance. Changes in the 3′-5′ domain emerged earlier than in the polymerase domain when viruses acquired a combination of mutations. Our study highlights the importance of poxvirus DNA polymerase residues 314, 613, 684, 688, and 851, previously linked to drug resistance, and identified several novel mutations in the 3′-5′ exonuclease domain (M313I, F354L, D480Y) and in the DNA polymerase domain (A632T, T831I, E856K, L924F) associated with different drug-susceptibility profiles. Furthermore, a combination of mutations resulted in complex patterns of cross-resistance. Modeling of the VACV DNA polymerase bearing the newly described mutations was performed to understand the effects of these mutations on the structure of the viral enzyme. We demonstrated the emergence of drug-resistant DNA polymerase mutations in complex patterns to be considered in case such mutations should eventually arise in the clinic.

CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'- O -methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'- O -methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.

Expression of the human telomerase catalytic component, hTERT, in normal human somatic cells can reconstitute telomerase activity and extend their replicative lifespan. We report here that at twice the normal number of population doublings, telomerase-expressing human skin fibroblasts (BJ-hTERT) and retinal pigment epithelial cells (RPE-hTERT) retain normal growth control in response to serum deprivation, high cell density, G1 or G2 phase blockers and spindle inhibitors. In addition, we observed no cell growth in soft agar and detected no tumour formation in vivo. Thus, we find that telomerase expression in normal cells does not appear to induce changes associated with a malignant phenotype.

It has been obtained and characterized the products of the reaction of phosphorous pentachloride with esters that are the mixture of geometric isomers of dichloroanhydrides of β- chloro-β-alkoxyvinylphosphonic acid, which are intermediates in formation of threechloroanhydries of phosphonchlorocarboxylic acid.It was established, that in excess of phosphorous pentachloride, ethylacetate forms as products showed above so threechloroanhydride of phosphonoacetic acid. Proposed the sequence of obtaining of threechloroanhydrides of phosphonoacetic acid.In opposite of ethylacetate, phosphorylation of ethyl ester of propionic acid with phosphorous pentachloride gives only two products: threechloroanhydride of α-chloro-α-phosphonpropionic acid and dichloroanhydride of α-metyl- β-chloro-β-etoxyvinylphosphonic acid in the form of just one geometric isomer, that were separated and characterized. Formation of every vinylphosphonate can be explained by conformational feature of intermediates phosphorylation.On the base of these reasonings, 1H and 13C NMR spectras of theeethyl ester of α-phosphonpropionic acid and diethylacetal of phosphonoacetic aldehyde were analyzed and using conformational characteristics of called phosphonates an explanation of ambiguity of spectras is given. [ABSTRACT FROM AUTHOR]

Abstract The ocean is an important source of methane, however, the sources of oceanic methane and mechanisms of its release to the atmosphere have only recently begun to be understood. Recent studies have identified methylphosphonate (MPn) as a previously unknown and likely source of methane in the aerobic ocean (Karl et al., 2008), as well as shown the biosynthesis of methylphosphonic acid to be a widespread trait in marine microbes (Metcalf et al., 2012). The mechanisms and reaction pathways from MPn to free methane, however, have not been well studied. Here we present results of laboratory studies on the photo-degradation of MPn, a likely mechanism of methane release to the atmosphere and phosphate release to the surface oceans. Phosphonoacetic acid was also studied as an additional model compound for comparison. We used the multi-labeled water isotope probing (MLWIP) approach, involving 18O-labeled waters to probe the photolytic mechanism of C P bond cleavage in phosphates through analysis of P released from MPn as PO 4. These studies identified distinct reaction pathways involving phosphates compared with other common organophosphorus compounds (e.g., phosphoesters), as well as suggest the involvement of both ambient water and atmospheric oxygen in C P bond cleavage. There is only a small amount of water oxygen incorporated into product PO 4 after cleavage of the C P bond in MPn, suggesting atmospheric O 2 or radicals formed from O 2 under Ultra Violet Radiation (UVR), as the primary source of O that replaces C in the C P bond of MPn. Model calculations suggest that the δ18O P signature of phosphate released via UV-degradation of phosphates is largely (75%) inherited from the original phosphate substrate. This opens up the possibility of tracing and differentiating specific phosphate sources of dissolved phosphate from other organophosphorus (Porg) sources (e.g., phosphoesters) used in primary production, as well as for tracing specific MPn sources of atmospheric methane. Graphical abstract Unlabelled Image Highlights • Photo-degradation of MPn, a likely mechanism of methane release to the atmosphere. • Multi-labeled water isotope probing approach was used to probe the C P bond cleavage. • Both ambient water and atmospheric O 2 involved during photo-degradation of MPn and PA. [ABSTRACT FROM AUTHOR]

Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors. [ABSTRACT FROM AUTHOR]

A coordination-induced film-forming method was developed to construct 2-hydroxyphosphonoacetic acid (HPAA)-Zn 2+ complex conversion layer (HPAA-Zn 2+ layer for short) on cold-rolled steel (CRS) substrate. Moreover, the incorporation of Mo component into the HPAA-Zn 2+ layer can significantly improve the uniformity and anticorrosion properties. The results showed that Mo-incorporated HPAA-Zn 2+ layer displayed the best anticorrosion performance when the molar ratio of HPAA/Zn 2+ was 1:1, the film-forming time was 10 min, and the concentration of molybdate was 0.1 wt%. In addition, energy dispersive spectroscopic (EDS) and X-ray photoelectron spectroscopic (XPS) results demonstrated that the Mo-incorporated HPAA-Zn 2+ layer was mainly composed of ZnO and HPAA-Zn 2+ complex and also confirmed that Mo-O-P bonds occurred between molybdate anion and HPAA molecule. [ABSTRACT FROM AUTHOR]

A facile two-step synthesis of methyl bis(2,2,2-trifluoroethyl) phosphonoacetate (Still-Gennari reagent) has been developed by exploiting Garegg-Samuelsson reaction conditions. Starting from trimethyl phosphonoacetate, Still-Gennari reagent was prepared in 94% yield via methyl 2-{bis[(trimethylsilyl)oxy]phosphoryl}acetate intermediate. This synthetic procedure was also used to prepare some kinds of Horner-Wadsworth-Emmons reagents and related compounds. [ABSTRACT FROM AUTHOR]

Methods of synthesis of 4-functionalyzed (5-trifluoromethylfur-2-yl)methanephosphonates and (5-methyl-2-trifluoromethylfur-3-yl)methanephosphonate are developed. Their formylation with ethyl formate in the presence of sodium foil is studied. Spectral characteristics of tautomers of obtained derivatives of phosphonoacetic aldehyde are evaluated. It is shown that interaction of obtained formyl derivatives with ethoxycarbonylmethylenetriphenylphosphorane regardless of the structure of the furan fragment leads to 4-furylsubstituted alkyl 4-(diethoxyphosphoryl)but-3-enoate, the abnormal product of Wittig reaction. [ABSTRACT FROM AUTHOR]

The bacteriophage T4 DNA polymerase (pol) and the closely related RB69 DNA pol have been developed into model enzymes to study family B DNA pols. While all family B DNA pols have similar structures and share conserved protein motifs, the molecular mechanism underlying natural drug resistance of nonherpes family B DNA pols and drug sensitivity of herpes DNA pols remains unknown. In the present study, we constructed T4 phages containing G466S, Y460F, G466S/Y460F, P469S, and V475W mutations in DNA pol. These amino acid substitutions replace the residues in drug-resistant T4 DNA pol with residues found in drug-sensitive herpes family DNA pols. We investigated whether the T4 phages expressing the engineered mutant DNA pols were sensitive to the antiviral drug phosphonoacetic acid (PAA) and characterized the in vivo replication fidelity of the phage DNA pols. We found that G466S substitution marginally increased PAA sensitivity, whereas Y460F substitution conferred resistance. The phage expressing a double mutant G466S/Y460F DNA pol was more PAA-sensitive. V475W T4 DNA pol was highly sensitive to PAA, as was the case with V478W RB69 DNA pol. However, DNA replication was severely compromised, which resulted in the selection of phages expressing more robust DNA pols that have strong ability to replicate DNA and contain additional amino acid substitutions that suppress PAA sensitivity. Reduced replication fidelity was observed in all mutant phages expressing PAA-sensitive DNA pols. These observations indicate that PAA sensitivity and fidelity are balanced in DNA pols that can replicate DNA in different environments. [ABSTRACT FROM AUTHOR]

Infection of human MRC-5 cells and mouse NIH-3T3 cells with a murine gamma-herpesvirus (MuHV-4 strain 68; MHV-68) photoinactivated by visible light in the presence of methylene blue (MB) resulted in nonproductive infection and the appearance of morphologically transformed cells. Two stably transformed cell lines were derived from both of these cell types and were confirmed to contain both viral DNA and antigen. Next, a quiescent MHV-68 infection in MRC-5 and NIH- 3T3 cells was established after cultivation at 41 ° C in the presence of phosphonoacetic acid. Following the exposure of quiescently infected cells to visible light for 120 s (5 times daily for 6 days) in the presence of MB, both MRC-5 and NIH- 3T3 cells were observed to acquire transformed phenotypes. The cytopathic effect was observed in cells after 4-5 passages, after which the cells degenerated. However, when human interferon (IFN)-α and mouse IFN-β were added to the media of quiescently infected MRC-5 and NIH-3T3 cells during the photoinactivating procedure, 2 stable transformed cell lines containing both viral DNA and the antigen were obtained and resembled those attained following nonproductive infection with photoinactivated virus. [ABSTRACT FROM AUTHOR]

Many microorganisms produce phosphonates, molecules characterized by stable carbon-phosphorus bonds that store phosphorus or act as antimicrobials. The role of phosphonates in the marine biosphere is well characterized but the role of these molecules in the intestine is poorly understood. Salmonella enterica uses its virulence factors to influence the host immune response to compete with the host and normal microflora for nutrients. Salmonella cannot produce phosphonates but encodes the enzymes to use them suggesting that it is exposed to phosphonates during its life cycle. The role of phosphonates during enteric salmonellosis is unexplored. We have previously shown that STM3602, encoding a putative regulator of phosphonate metabolism, is needed for colonization in calves. Here, we report that the necessity of STM3602 in colonization of the murine intestine results from multiple factors. STM3602 is needed for full activation of the type-3 secretion system-1 and for optimal invasion of epithelial cells. The ∆STM3602 mutant grows poorly in phosphonoacetic acid (PA) as the sole phosphorus source, but can use 2-aminoethylphosphonate. PhnA, an enzyme required for PA breakdown, is not controlled by STM3602 suggesting an additional mechanism for utilization of PA in S. Typhimurium. Finally, the requirement of STM3602 for intestinal colonization differs depending on the composition of the microflora. Our data suggest that STM3602 has multiple regulatory targets that are necessary for survival within the microbial community in the intestine. Determination of the members of the STM3602 regulon may illuminate new pathways needed for colonization of the host. [ABSTRACT FROM AUTHOR]

Blue single crystals of Cu[μ2-OOC(CH2)PO3H] ·2H2O ( 1) and Cu1.5[μ3-OOC(CH2)PO3] ·5H2O ( 2) were prepared in aqueous solution. In compound 1 [space group C2/ c (no. 15) with a = 1623.3(2), b = 624.0(1), c = 1495.5(2) pm, β = 122.45(1)°], Cu is coordinated by three oxygen atoms stemming from the hydrogenphosphonoacetate dianion and three water molecules to form a distorted octahedron. The Cu-O bonds range from 190.4(3) to 278.5(3) pm. The connection between the Cu2+ cations and the hydrogenphosphonoacetate dianions leads to a two-dimensional structure with layers parallel to ( 101). The layers are linked by hydrogen bonds. In compound 2 [space group P 1 (no. 2) with a = 608.2(1), b = 800.1(1), c = 1083.6(1) pm, α = 94.98(1)°, β = 105.71(1)°, γ = 109.84(1)°], two crystallographically independent Cu2+ cations are coordinated in a square pyramidal and an octahedral fashion, respectively. The Cu-O bonds range from 192.9(2) to 237.2(2) pm. The coordination of the phosphonoacetate trianion to Cu(1) results in infinite polyanionic chains parallel to [100] with a composition of {Cu(H2O)[OOC(CH2)PO3]} nn-. Hydrated Cu(2) cations are accommodated between the chains as counterions. 1 and 2 show structural features of cation exchangers. Magnetic measurements reveal a paramagnetic Curie-Weiss behavior. Compound 2 shows antiferromagnetic coupling between Cu2+ ions due to a super-superexchange coupling. The UV/Vis spectra of 1 suggest three d-d transition bands at 763 nm (2B1 → 2E), 878 nm (2B1 → 2B2), and 1061 nm (2B1 → 2A1). Thermoanalytical investigations in air show that compound 1 is stable up to 165 °C, whereas decomposition of 2 begins at 63 °C. [ABSTRACT FROM AUTHOR]

Pavlovian stimuli predictive of food can markedly amplify instrumental responding for food. This effect is termed Pavlovian-instrumental transfer (PIT). The ventral tegmental area (VTA) plays a key role in mediating PIT, however, it is yet unknown whether N-methyl- d-aspartate (NMDA)-type glutamate receptors in the VTA are involved in PIT. Here, we examined the effects of an NMDA-receptor blockade in the VTA on PIT. Immediately prior to PIT testing, rats were subjected to intra-VTA infusions of vehicle or of the NMDA-receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5) (1, 5 µg/side). In rats that received AP-5 at the lower dose, the PIT effect was intact, i.e. presentation of the Pavlovian stimulus enhanced instrumental responding. By contrast, in rats that received AP-5 at the higher dose, the PIT effect was blocked. The data suggest that NMDA receptors in the VTA mediate the activating effects of Pavlovian stimuli on instrumental responding. [ABSTRACT FROM AUTHOR]