Novel fully automated prototype assays for specific detection of phosphorylated and non-phosphorylated Hepatitis B core antigens.

BACKGROUND: Hepatitis B core antigen (HBcAg) has been proposed as a surrogate marker to reflect transcriptional activity of HBV covalently closed circular DNA (cccDNA) during active infections and may be a valuable tool to monitor the efficacy of antiviral therapies. However, HBcAg-specific immunoassays are unavailable, and current assays that measure hepatitis B core-related antigen (HBcrAg) cannot distinguish between HBcAg, HBeAg, and precore (PreC) proteins. OBJECTIVE: Two fully automated assays were developed to specifically detect phosphorylated HBcAg (P-HBcAg, representing non-HBV DNA-containing particles) and non-phosphorylated HBcAg (representing HBV DNA-containing particles) circulating in HBV infected patients. STUDY DESIGN: P-HBcAg and HBcAg levels were analyzed in 124 single timepoint patients with active infections, in three longitudinal specimens from patients with acute HBV infections, and in four chronic hepatitis B (CHB) patients on-therapy (TDF - tenofovir disoproxil fumarate, pegIFN - pegylated interferon, NAPs - nucleic acids polymers). RESULTS: Analyzing acute infections revealed that P-HBcAg and HBcAg levels correlate more closely than HBcrAg to HBV DNA. During antiviral treatment of CHB patients, HBcAg correlates well with HBV DNA and indicates a therapeutic response to the treatment at the beginning of the therapy. In contrast, P-HBcAg tracks more closely to HBV RNA. Importantly, P-HBcAg is detectable several months after HBcAg became undetectable indicating that cccDNA is still transcriptionally active in hepatocytes. CONCLUSIONS: Overall, the ability to specifically distinguish between the various states of HBcAg (phosphorylated and non-phosphorylated) can provide additional insights for disease staging, drug development, and management of HBV therapies.

Pre-analytical considerations in the development of a prototype SARS-CoV-2 antigen ARCHITECT automated immunoassay.

OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.

Evolutionary dynamics of microRNA target sites across vertebrate evolution.

MicroRNAs (miRNAs) control the abundance of the majority of the vertebrate transcriptome. The recognition sequences, or target sites, for bilaterian miRNAs are found predominantly in the 3' untranslated regions (3'UTRs) of mRNAs, and are amongst the most highly conserved motifs within 3'UTRs. However, little is known regarding the evolutionary pressures that lead to loss and gain of such target sites. Here, we quantify the selective pressures that act upon miRNA target sites. Notably, selective pressure extends beyond deeply conserved binding sites to those that have undergone recent substitutions. Our approach reveals that even amongst ancient animal miRNAs, which exert the strongest selective pressures on 3'UTR sequences, there are striking differences in patterns of target site evolution between miRNAs. Considering only ancient animal miRNAs, we find three distinct miRNA groups, each exhibiting characteristic rates of target site gain and loss during mammalian evolution. The first group both loses and gains sites rarely. The second group shows selection only against site loss, with site gains occurring at a neutral rate, whereas the third loses and gains sites at neutral or above expected rates. Furthermore, mutations that alter the strength of existing target sites are disfavored. Applying our approach to individual transcripts reveals variation in the distribution of selective pressure across the transcriptome and between miRNAs, ranging from strong selection acting on a small subset of targets of some miRNAs, to weak selection on many targets for other miRNAs. miR-20 and miR-30, and many other miRNAs, exhibit broad, deeply conserved targeting, while several other comparably ancient miRNAs show a lack of selective constraint, and a small number, including mir-146, exhibit evidence of rapidly evolving target sites. Our approach adds valuable perspective on the evolution of miRNAs and their targets, and can also be applied to characterize other 3'UTR regulatory motifs.

Characterizing mRNA Sequence Motifs in the 3'-UTR Using GFP Reporter Constructs.

GFP reporter constructs are widely used as an expression system for studying the function of regulatory sequence motifs (cis elements) within the 3'-UTRs (3' untranslated regions) of mRNAs. Here we provide details on the characterization of individual sequence motifs, which typically regulate mRNA decay and translation. In addition, we describe methods to identify trans factors required for the function of such elements. To facilitate efficient identification of novel functional 3'-UTR motifs, we describe a screening approach based on dual-color fluorescence reporter constructs. Such screening approaches can be used to test large collections of defined sequence or libraries of random sequences.

A position-specific 3'UTR sequence that accelerates mRNA decay.

The 3' untranslated regions (3'UTRs) of mammalian mRNAs direct an extensive range of alternative post-transcriptional outcomes, including regulation of mRNA decay and translation, contributing significantly to overall gene regulation. However, our knowledge of the underlying sequences and mechanisms is incomplete. We identified a novel 3'UTR sequence motif in mammals that targets mRNAs for transcript degradation. The motif is found in hundreds of mRNAs and is enriched in transcripts encoding regulatory proteins, such as transcription and signaling factors. Degradation of mRNAs containing the motif is mediated by the CCR4-NOT deadenylation complex. We identified hnRNPs A1 and A2/B1 as trans factors that directly bind to the motif, indicating a novel role for these proteins in deadenylation. Interestingly, a genome-wide analysis of the impact of this new regulatory pathway showed that the most active motifs are located within the 5' and 3'-terminal portions of 3'UTRs, whereas elements in the center tend to be inactive. The highly position-specific function of the motif adds a new layer of regulation to gene expression mediated by 3'UTRs.

A widespread sequence-specific mRNA decay pathway mediated by hnRNPs A1 and A2/B1.

3'-untranslated regions (UTRs) specify post-transcriptional fates of mammalian messenger RNAs (mRNAs), yet knowledge of the underlying sequences and mechanisms is largely incomplete. Here, we identify two related novel 3' UTR motifs in mammals that specify transcript degradation. These motifs are interchangeable and active only within 3' UTRs, where they are often preferentially conserved; furthermore, they are found in hundreds of transcripts, many encoding regulatory proteins. We found that degradation occurs via mRNA deadenylation, mediated by the CCR4-NOT complex. We purified trans factors that recognize the motifs and identified heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 and A2/B1, which are required for transcript degradation, acting in a previously unknown manner. We used RNA sequencing (RNA-seq) to confirm hnRNP A1 and A2/B1 motif-dependent roles genome-wide, profiling cells depleted of these factors singly and in combination. Interestingly, the motifs are most active within the distal portion of 3' UTRs, suggesting that their role in gene regulation can be modulated by alternative processing, resulting in shorter 3' UTRs.

Patient-adapted, specific activation of HIV-1 by customized TAL effectors (TALEs), a proof of principle study.

The major obstacle to cure infections with human immunodeficiency virus (HIV-1) is integrated proviral genomes, which are not eliminated by antiretroviral therapies (ART). Treatment approaches with latency-reversing agents (LRAs) aim at inducing provirus expression to tag latently-infected cells for clearance through viral cytopathic effects or cytotoxic T cell (CTL) responses. However, the currently tested LRAs reveal evident drawbacks as gene expression is globally induced and viral outgrowth is insecure. Here, we present transcription activator-like effector (TALE) proteins as potent tools to activate HIV-1 specifically. The large variety of circulating HIV-1 strains and, accordingly, integrated proviruses was addressed by the programmable DNA-specificity of TALEs. Using customized engineered TALEs, a substantial transcription activation and viral outgrowth was achieved with cells obtained from different HIV-1 patients. Our data suggest that TALEs may be useful tools in future strategies aimed at removing HIV-1 reservoirs.

Antiviral interferon-beta signaling induced by designed transcription activator-like effectors (TALE).

Here we show that designed transcription activator-like effectors (TALEs) that bind to defined areas of the interferon beta promoter are capable to induce IFN-beta expression and signaling in human cells. Importantly, TALE-mediated IFN-beta signaling occurs independently of pathogen pattern recognition but effectively prohibits viral RNA replication as demonstrated with a hepatitis C virus replicon. TALEs were thus indicated to be valuable tools in various applications addressing, for example, virus-host interactions.

Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D (1)H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3'(-)SL by (1)H NMR, the base pair at the stem's edge was identified as the most stable component of the structure. (1)H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3'(-)SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process.

AUF1 p45 promotes West Nile virus replication by an RNA chaperone activity that supports cyclization of the viral genome.

UNLABELLED: A central aspect of current virology is to define the function of cellular proteins (host factors) that support the viral multiplication process. This study aimed at characterizing cellular proteins that assist the RNA replication process of the prevalent human pathogen West Nile virus (WNV). Using in vitro and cell-based approaches, we defined the p45 isoform of AU-rich element RNA-binding protein 1 (AUF1) as a host factor that enables efficient WNV replication. It was demonstrated that AUF1 p45 has an RNA chaperone activity, which aids the structural rearrangement and cyclization of the WNV RNA that is required by the viral replicase to initiate RNA replication. The obtained data suggest the RNA chaperone activity of AUF1 p45 is an important determinant of the WNV life cycle. IMPORTANCE: In this study, we identified a cellular protein, AUF1 (also known as heterogeneous ribonucleoprotein D [hnRNPD]), acting as a helper (host factor) of the multiplication process of the important human pathogen West Nile virus. Several different variants of AUF1 exist in the cell, and one variant, AUF1 p45, was shown to support viral replication most significantly. Interestingly, we obtained a set of experimental data indicating that a main function of AUF1 p45 is to modify and thus prepare the West Nile virus genome in such a way that the viral enzyme that generates progeny genomes is empowered to do this considerably more efficiently than in the absence of the host factor. The capability of AUF1 p45 to rearrange the West Nile virus genome was thus identified to be an important aspect of a West Nile virus infection.

The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes.

The DEAD-box helicase DDX3 has suggested functions in innate immunity, mRNA translocation and translation, and it participates in the propagation of assorted viruses. Exploring initially the role of DDX3 in the life cycle of hepatitis C virus, we observed the protein to be involved in translation directed by different viral internal ribosomal entry sites. Extension of these studies revealed a general supportive role of DDX3 in translation initiation. DDX3 was found to interact in an RNA-independent manner with defined components of the translational pre-initiation complex and to specifically associate with newly assembling 80S ribosomes. DDX3 knock down and in vitro reconstitution experiments revealed a significant function of the protein in the formation of 80S translation initiation complexes. Our study implies that DDX3 assists the 60S subunit joining process to assemble functional 80S ribosomes.

Transcriptional activators of human genes with programmable DNA-specificity.

TAL (transcription activator-like) effectors are translocated by Xanthomonas bacteria into plant cells where they activate transcription of target genes. DNA target sequence recognition occurs in a unique mode involving a central domain of tandem repeats. Each repeat recognizes a single base pair in a contiguous DNA sequence and a pair of adjacent hypervariable amino acid residues per repeat specifies which base is bound. Rearranging the repeats allows the design of novel TAL proteins with predictable DNA-recognition specificities. TAL protein-based transcriptional activation in plant cells is mediated by a C-terminal activation domain (AD). Here, we created synthetic TAL proteins with designed repeat compositions using a novel modular cloning strategy termed "Golden TAL Technology". Newly programmed TAL proteins were not only functional in plant cells, but also in human cells and activated targeted expression of exogenous as well as endogenous genes. Transcriptional activation in different human cell lines was markedly improved by replacing the TAL-AD with the VP16-AD of herpes simplex virus. The creation of TAL proteins with potentially any desired DNA-recognition specificity allows their versatile use in biotechnology.

Mechanisms of activity and inhibition of the hepatitis C virus RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase NS5B is a key enzyme of the replication of hepatitis C virus (HCV) and a major therapeutic target. Applying a novel continuous assay with highly purified protein and a fluorescent RNA-template we provide for the first time a comprehensive mechanistic description of the enzymatic reaction. Using fluorescence spectroscopy, the kinetics of NS5B was confirmed to consist of two half-reactions, namely substrate binding and turnover. Determining the binding constants of the substrates and the rate constants of individual reaction steps, NS5B was shown to bind the template single-stranded RNA with high affinity (nanomolar range) and in a stepwise process that reflects the substrate positioning. As demonstrated by CD, NTP(s) binding caused a tertiary structural change of the enzyme into an active conformation. The second half-reaction was dissected into a sequential polymerization and a subsequent, rate-limiting product release reaction. Taking advantage of these tools, we analyzed the mechanism of action of the NS5B inhibitor HCV-796, which was shown to interfere with the formation of double-stranded RNA by blocking the second half-reaction.

Evolution of morphine biosynthesis in opium poppy.

Benzylisoquinoline alkaloids (BIAs) are a group of nitrogen-containing plant secondary metabolites comprised of an estimated 2500 identified structures. In BIA metabolism, (S)-reticuline is a key branch-point intermediate that can be directed into several alkaloid subtypes with different structural skeleton configurations. The morphinan alkaloids are one subclass of BIAs produced in only a few plant species, most notably and abundantly in the opium poppy (Papaver somniferum). Comparative transcriptome analysis of opium poppy and several other Papaver species that do not accumulate morphinan alkaloids showed that known genes encoding BIA biosynthetic enzymes are expressed at higher levels in P. somniferum. Three unknown cDNAs that are co-ordinately expressed with several BIA biosynthetic genes were identified as enzymes in the pathway. One of these enzymes, salutaridine reductase (SalR), which is specific for the production of morphinan alkaloids, was isolated and heterologously overexpressed in its active form not only from P. somniferum, but also from Papaver species that do not produce morphinan alkaloids. SalR is a member of a class of short chain dehydrogenase/reductases (SDRs) that are active as monomers and possess an extended amino acid sequence compared with classical SDRs. Homology modelling and substrate docking revealed the substrate binding site for SalR. The amino acids residues conferring salutaridine binding were compared to several members of the SDR family from different plant species, which non-specifically reduce (-)-menthone to (+)-neomenthol. Previously, it was shown that some of these proteins are involved in plant defence. The recruitment of specific monomeric SDRs from monomeric SDRs involved in plant defence is discussed.

Removal of substrate inhibition and increase in maximal velocity in the short chain dehydrogenase/reductase salutaridine reductase involved in morphine biosynthesis.

Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel alpha-helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking also revealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalR was created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.

Molecular modeling and site-directed mutagenesis reveal the benzylisoquinoline binding site of the short-chain dehydrogenase/reductase salutaridine reductase.

Recently, the NADPH-dependent short-chain dehydrogenase/reductase (SDR) salutaridine reductase (E.C. 1.1.1.248) implicated in morphine biosynthesis was cloned from Papaver somniferum. In this report, a homology model of the Papaver bracteatum homolog was created based on the x-ray structure of human carbonyl reductase 1. The model shows the typical alpha/beta-folding pattern of SDRs, including the four additional helices alphaF'-1 to alphaF'-4 assumed to prevent the dimerization of the monomeric short-chain dehydrogenases/reductases. Site-directed mutagenesis of asparagine-152, serine-180, tyrosine-236, and lysine-240 resulted in enzyme variants with strongly reduced performance or inactive enzymes, showing the involvement of these residues in the proton transfer system for the reduction of salutaridine. The strong preference for NADPH over NADH could be abolished by replacement of arginine residues 44 and 48 by glutamic acid, confirming the interaction between the arginines and the 2'-phosphate group. Docking of salutaridine into the active site revealed nine amino acids presumably responsible for the high substrate specificity of salutaridine reductase. Some of these residues are arranged in the right position by an additional alphaE' helix, which is not present in SDRs analyzed so far. Enzyme kinetic data from mutagenic replacement emphasize the critical role of these residues in salutaridine binding and provide the first data on the molecular interaction of benzylisoquinoline alkaloids with enzymes.