Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro

Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s) may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis

Protein methylation is required to maintain optimal HIV-1 infectivity

BACKGROUND: Protein methylation is recognized as a major protein modification pathway regulating diverse cellular events such as protein trafficking, transcription, and signal transduction. More recently, protein arginine methyltransferase activity has been shown to regulate HIV-1 transcription via Tat. In this study, adenosine periodate (AdOx) was used to globally inhibit protein methyltransferase activity so that the effect of protein methylation on HIV-1 infectivity could be assessed. RESULTS: Two cell culture models were used: HIV-1-infected CEM T-cells and HEK293T cells transfected with a proviral DNA plasmid. In both models, AdOx treatment of cells increased the levels of virion in culture supernatant. However, these viruses had increased levels of unprocessed or partially processed Gag-Pol, significantly increased diameter, and displayed reduced infectivity in a MAGI X4 assay. AdOx reduced infectivity equally in both dividing and non-dividing cells. However, infectivity was further reduced if Vpr was deleted suggesting virion proteins, other than Vpr, were affected by protein methylation. Endogenous reverse transcription was not inhibited in AdOx-treated HIV-1, and infectivity could be restored by pseudotyping HIV with VSV-G envelope protein. These experiments suggest that AdOx affects an early event between receptor binding and uncoating, but not reverse transcription. CONCLUSION: Overall, we have shown for the first time that protein methylation contributes towards maximal virus infectivity. Furthermore, our results also indicate that protein methylation regulates HIV-1 infectivity in a complex manner most likely involving the methylation of multiple viral or cellular proteins and/or multiple steps of replication

Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases

Background Bioethanol production from sustainable sources of biomass that limit effect on food production are needed and in a biorefinery approach co-products are desirable, obtained from both the plant material and from the microbial biomass. Fungal biotransformation of steroids was among the first industrial biotransformations allowing corticosteroid production. In this work, the potential of yeast to produce intermediates needed in corticosteroid production is demonstrated at laboratory scale following bioethanol production from perennial ryegrass juice. Results Genes encoding the 11?-steroid hydroxylase enzymes from Aspergillus ochraceus (11?-SHAoch) and Rhizopus oryzae (CYP509C12) transformed into Saccharomyces cerevisiae for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11?-SHAoch and AH22:p509C12) exhibited efficient progesterone bioconversion (on glucose minimal medial containing 300 ?M progesterone) producing either 11?-hydroxyprogesterone as the sole metabolite (AH22:p11?-SHAoch) or a 7:1 mixture of 11?-hydroxyprogesterone and 6?-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11?-SHAoch and AH22:p509C12 were comparable resulting in ?75% conversion of glucose to alcohol. Co-production of bioethanol together with efficient production of the 11-OH intermediate for corticosteroid manufacture was then demonstrated using perennial ryegrass juice. Integration of the 11?-SHAoch gene into the yeast genome (AH22:11?-SHAoch+K) resulted in a 36% reduction in yield of 11?-hydroxyprogesterone to 174 ?mol/L using 300 ?M progesterone. However, increasing progesterone concentration to 955 ?M and optimizing growth conditions increased 11?-hydroxyprogesterone production to 592 ?mol/L product formed. Conclusions The progesterone 11?-steroid hydroxylases from A. ochraceus and R. oryzae, both monooxygenase enzymes of the cytochrome P450 superfamily, have been functionally expressed in S. cerevisiae. It appears that these activities in fungi are not associated with a conserved family of cytochromes P450. The activity of the A. ochraceous enzyme was important as the specificity of the biotransformation yielded just the 11-OH product needed for corticosteroid production. The data presented demonstrate how recombinant yeast could find application in rural biorefinery processes where co-production of value-added products (11?-hydroxyprogesterone and ethanol) from novel feedstocks is an emergent and attractive possibility.publishersversionPeer reviewe

Australian bat lyssavirus infection in two horses

In May 2013, the first cases of Australian bat lyssavirus infections in domestic animals were identified in Australia. Two horses (filly-H1 and gelding-H2) were infected with the Yellow-bellied sheathtail bat (YBST) variant of Australian bat lyssavirus (ABLV). The horses presented with neurological signs, pyrexia and progressing ataxia. Intra-cytoplasmic inclusion bodies (Negri bodies) were detected in some Purkinje neurons in haematoxylin and eosin (H&E) stained sections from the brain of one of the two infected horses (H2) by histological examination. A morphological diagnosis of sub-acute moderate non-suppurative, predominantly angiocentric, meningo-encephalomyelitis of viral aetiology was made. The presumptive diagnosis of ABLV infection was confirmed by the positive testing of the affected brain tissue from (H2) in a range of laboratory tests including fluorescent antibody test (FAT) and real-time PCR targeting the nucleocapsid (N) gene. Retrospective testing of the oral swab from (H1) in the real-time PCR also returned a positive result. The FAT and immunohistochemistry (IHC) revealed an abundance of ABLV antigen throughout the examined brain sections. ABLV was isolated from the brain (H2) and oral swab/saliva (H1) in the neuroblastoma cell line (MNA). Alignment of the genome sequence revealed a 97.7% identity with the YBST ABLV strain

The Investigational Drug VT-1129 Is a Highly Potent Inhibitor of Cryptococcus Species CYP51 but Only Weakly Inhibits the Human Enzyme

Cryptococcosis is a life-threatening disease often associated with HIV infection. Three Cryptococcus species CYP51 enzymes were purified and catalyzed the 14α-demethylation of lanosterol, eburicol, and obtusifoliol. The investigational agent VT-1129 bound tightly to all three CYP51 proteins (dissociation constant [K(d)] range, 14 to 25 nM) with affinities similar to those of fluconazole, voriconazole, itraconazole, clotrimazole, and ketoconazole (K(d) range, 4 to 52 nM), whereas VT-1129 bound weakly to human CYP51 (K(d), 4.53 μM). VT-1129 was as effective as conventional triazole antifungal drugs at inhibiting cryptococcal CYP51 activity (50% inhibitory concentration [IC(50)] range, 0.14 to 0.20 μM), while it only weakly inhibited human CYP51 activity (IC(50), ∼600 μM). Furthermore, VT-1129 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. Finally, the cellular mode of action for VT-1129 was confirmed to be CYP51 inhibition, resulting in the depletion of ergosterol and ergosta-7-enol and the accumulation of eburicol, obtusifolione, and lanosterol/obtusifoliol in the cell membranes

Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51)

Herein, we have investigated the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kd of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 μM), whereas inhibition by fluconazole was weak (IC50, 30 μM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections

Small‐Molecule Inhibitors Targeting Sterol 14α‐Demethylase (CYP51): Synthesis, Molecular Modelling and Evaluation Against Candida albicans

Fungal infections are a global issue affecting over 150 million people worldwide annually, with 750 000 of these caused by invasive Candida infections. Azole drugs are the frontline treatment against fungal infections; however, resistance to current azole antifungals in C. albicans poses a threat to public health. Two series of novel azole derivatives, short and extended derivatives, have been designed, synthesised and investigated for CYP51 inhibitory activity, binding affinity and minimum inhibitory concentration (MIC) against C. albicans strains. The short derivatives were more potent against the C. albicans strains (e. g., MIC 2‐(4‐chlorophenyl)‐N‐(2,4‐dichlorobenzyl)‐3‐(1H‐imidazol‐1‐yl)propanamide (5 f ) <0.03 μg/mL, N‐(4‐((4‐chlorophenyl)sulfonamido)benzyl)‐2‐phenyl‐3‐(1H‐1,2,4‐triazol‐1‐yl)propanamide (12 c ), 1 μg/mL, fluconazole 0.125 μg/mL) but both displayed comparable enzyme binding and inhibition (5 f K d 62±17 nM, IC50 0.46 μM; 12 c K d 43±18 nM, IC50 0.33 μM, fluconazole K d 41±13 nM, IC50 0.31 μM, posaconazole K d 43±11 nM, IC50 0.2 μM). The short series had poor selectivity for CaCYP51 over the human homologue, whereas the selectivity of the extended series, for example, compound 12 c , was higher (21.5‐fold) than posaconazole (4.7‐fold) based on K d values, although posaconazole was more selective (615‐fold) than 12 c (461‐fold) based on IC50 values. Based on inhibitory activity and selectivity profile, the extended series are the better of the two series for further development

Impact of Boron doping to the tunneling magnetoresistance of Heusler alloy Co2FeAl

Heusler alloys based magnetic tunnel junctions can potentially provide high magnetoresistance, small damping and fast switching. Here junctions with Co2FeAl as a ferromagnetic electrode are fabricated by room temperature sputtering on Si/SiO2 substrates. The doping of Boron in Co2FeAl is found to have a large positive impact on the structural, magnetic and transport properties of the junctions, with a reduced interfacial roughness and substantial improved tunneling magnetoresistance. A two-level magnetoresistance is also observed in samples annealed at low temperature, which is believed to be related to the memristive effect of the tunnel barrier with impurities.Comment: 9 pages, 4 figure

Discovery and structure-activity relationships of a novel isothiazolone class of bacterial type II topoisomerase inhibitors

There is an urgent and unmet medical need for new antibacterial drugs that tackle infections caused by multidrug-resistant (MDR) pathogens. During the course of our wider efforts to discover and exploit novel mechanism of action antibacterials, we have identified a novel series of isothiazolone based inhibitors of bacterial type II topoisomerase. Compounds from the class displayed excellent activity against both Gram-positive and Gram-negative bacteria with encouraging activity against a panel of MDR clinical Escherichia coli isolates when compared to ciprofloxacin. Representative compounds also displayed a promising in vitro safety profile