Mechanism of resistance to acyclovir in thymidine kinase mutants from Herpes simplex virus type 1: a computational approach

Abstract

Herpes simplex virus type 1 (HSV-1) infections affect about two-thirds of the world population, and the standard treatment consists of acyclovir (ACV) and its analogs, which interact with thymidine kinase (TK) blocking viral replication. Lately, the emergence of ACV-resistant strains has been reported, especially associated with TK mutations. In this context, ACV therapy fails against isolates encoding Y172C and Y53H/R163H TK mutants, but the molecular mechanism of drug resistance remains unclear. Thus, we examined the effects of these mutations on ACV and the cofactor ATP binding through molecular modeling approaches. We showed that Y172C prevents the anchoring of the aromatic ring of ACV through π–π stacking interactions, leading to an inversed binding mode and different interactions. On the other hand, Y53H/R163H remarkably affected the cofactor binding mode which shifted away from its binding site and also influenced the interaction network of ACV. This is likely due to the loss of polar interactions with R163 residue. Unlike what was observed in the wild-type complex, both drug and cofactor binding poses were not well positioned to allow the phosphorylation reaction which explains the resistance observed. Moreover, energy analysis corroborated the experimental data and showed lower theoretical affinity of ACV with mutant enzymes resulted from energetic loss in polar solvation in Y172C and electrostatic terms in Y53H/R163H mutant enzyme. Therefore, our study shed light on the resistance mechanism toward ACV of two mutant TKs identified in clinical HSV-1 strains and may further support the development of new anti-herpetic drugs to treat resistant infections.

Communicated by Ramaswamy H. Sarma     

Specific features of the mature microrna nucleotide sequences can influence the affinity for the human Ago2 and Ago3 proteins

Mature microRNAs (miRNAs) with a length of 20–24 nucleotides are endogenous, small, non-coding RNAs. They program the RNA-induced silencing complex (RISC) to inhibit translation of the mRNAs carrying the sites complementary to these miRNAs. When the RISC contains Ago3, the mRNA translation is inhibited; however, in the case of Ago2, the mRNA can be also cleaved in the center of mRNA/miRNA heteroduplex. Using the earlier developed system ACTIVITY, we have analyzed the published data on the affinity of mature human miRNA sequences for the Ago2 and Ago3 proteins. It has been found that the higher the abundance of YRHB tetranucleotides near the miRNA 3′-end, the higher the miRNA affinity for both proteins (r = 0.613, α < 0.025) and that the miRNA binding to Ago2 increases relative to that Ago3 with the abundance of RHHK tetranucleotides near the miRNA center (r=0.501, α < 0.05). These two patterns allowed us to propose equations for predicting the affinity of mature miRNAs for the Ago2 and Ago3 proteins and to reliably predict the affinity of canonical (α < 0.00025) and noncanonical (α < 0.05) miRNAs for each protein using independent data.     

Groove binding between ferulic acid and calf thymus DNA: spectroscopic methodology combined with chemometrics and molecular docking studies

Abstract

Ferulic acid (FA), a dietary phenolic acid compound, is proved to possess numerous biological activities. Hence, this study was devoted to explore the interaction between FA and calf thymus DNA (ctDNA) by UV???vis absorption, fluorescence, circular dichroism (CD) spectroscopy combined with multivariate curve resolution-alternating least-squares (MCR???ALS) and molecular docking studies. The concentration curves and the pure spectra of compositions (FA, ctDNA and FA???ctDNA complex) were obtained by MCR???ALS approach to verify and monitor the interaction of FA with ctDNA. The groove binding mode between FA and ctDNA was confirmed by the results of melting analysis, viscosity measurements, single-stranded DNA experiments, and competitive studies. The binding constant of FA???ctDNA complex was 4.87?×?10⁴ L mol^?1 at 298?K. The values of enthalpy (ΔH°) and entropy (ΔS°) changes in the interaction were ?16.24?kJ mol^?1 and 35.02?J mol^?1 K^?1, respectively, indicating that the main binding forces were hydrogen bonds and hydrophobic interactions. The result of CD spectra suggested that a decrease in right-handed helicity of ctDNA was induced by FA and the DNA conformational transition from the B-form to the A-form. The results of docking indicated that FA binding with ctDNA in the minor groove. These findings may be conducive to understand the interaction mechanism of FA with ctDNA and the pharmacological effects of FA.

Communicated by Ramaswamy H. Sarma

     

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex.     

Expanded RNA-binding activities of mammalian Argonaute 2

Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells.     

Identification of novel argonaute-associated proteins

RNA silencing processes are guided by small RNAs known as siRNAs and microRNAs (miRNAs) . They reside in ribonucleoprotein complexes, which guide the cleavage of complementary mRNAs or affect stability and translation of partial complementary mRNAs . Argonaute (Ago) proteins are at the heart of silencing effector complexes and bind the single-stranded siRNA and miRNA . Our biochemical analysis revealed that Ago2 is present in a pre-miRNA processing complex that is able to transfer the miRNA into a target-mRNA cleaving complex. To gain insight into the function and composition of RNA silencing complexes, we purified Ago1- and Ago2-containing complexes from human cells. Several known Ago1- and/or Ago2-associated proteins including Dicer were identified, but also two novel factors, the putative RNA helicase MOV10, and the RNA recognition motif (RRM)-containing protein TNRC6B/KIAA1093. The new proteins localize, similar to Ago proteins, to mRNA-degrading cytoplasmic P bodies, and they are functionally required to mediate miRNA-guided mRNA cleavage.     

Drosophila microRNAs are sorted into functionally distinct argonaute complexes after production by dicer-1

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide distinct classes of RNA-induced silencing complexes (RISCs) to repress mRNA expression in biological processes ranging from development to antiviral defense. In Drosophila, separate but conceptually similar endonucleolytic pathways produce siRNAs and miRNAs. Here, we show that despite their distinct biogenesis, double-stranded miRNAs and siRNAs participate in a common sorting step that partitions them into Ago1- or Ago2-containing effector complexes. These distinct complexes silence their target RNAs by different mechanisms. miRNA-loaded Ago2-RISC mediates RNAi, but only Ago1 is able to repress an mRNA with central mismatches in its miRNA-binding sites. Conversely, Ago1 cannot mediate RNAi, because it is an inefficient nuclease whose catalytic rate is limited by the dissociation of its reaction products. Thus, the two members of the Drosophila Ago subclade of Argonaute proteins are functionally specialized, but specific small RNA classes are not restricted to associate with Ago1 or Ago2.     

Novel functional small RNAs are selectively loaded onto mammalian Ago1

Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1–4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein–Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1–4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.     

Conserved association of Argonaute 1 and 2 proteins with miRNA and siRNA pathways throughout insect evolution,from cockroaches to flies

Background

Argonaute proteins are key in RNA silencing. In Drosophila melanogaster, the five proteins of the Argonaute family participate in the pathways and mechanisms mediated by three types of small RNAs: piRNAs, miRNAs, and siRNAs. Two Argonaute proteins, Argonaute 1 (Ago1) and Argonaute 2 (Ago2), are associated with miRNA and siRNA mechanisms, which are the most thoroughly studied. The available data points to a sorting specialization of Ago1 for miRNAs and Ago2 for siRNAs. However, this has been demonstrated only in D. melanogaster, one of the most modified insects, which emerged some 100 million years ago. Thus, an important question is whether this association of Ago1 with miRNAs and Ago2 with siRNAs occurs generally in insects, or was a specific innovation in higher flies.

Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance

microRNAs (miRNAs) are small non-coding RNAs that regulate mRNA stability and translation through the action of the RNAi-induced silencing complex (RISC). Our current understanding of miRNA function is inferred largely from studies of the effects of miRNAs on steady-state mRNA levels and from seed match conservation and context in putative targets. Here we have taken a more direct approach to these issues by comprehensively assessing the miRNAs and mRNAs that are physically associated with Argonaute 2 (Ago2), which is a core RISC component. We transfected HEK293T cells with epitope-tagged Ago2, immunopurified Ago2 together with any associated miRNAs and mRNAs, and quantitatively determined the levels of these RNAs by microarray analyses. We found that Ago2 immunopurified samples contained a representative repertoire of the cell's miRNAs and a select subset of the cell's total mRNAs. Transfection of the miRNAs miR-1 and miR-124 caused significant changes in the association of scores of mRNAs with Ago2. The mRNAs whose association with Ago2 increased upon miRNA expression were much more likely to contain specific miRNA seed matches and to have their overall mRNA levels decrease in response to the miRNA transfection than expected by chance. Hundreds of mRNAs were recruited to Ago2 by each miRNA via seed sequences in 3'-untranslated regions and coding sequences and a few mRNAs appear to be targeted via seed sequences in 5'-untranslated regions. Microarray analysis of Ago2 immunopurified samples provides a simple, direct method for experimentally identifying the targets of miRNAs and for elucidating roles of miRNAs in cellular regulation.     

Synaptosomes secrete and uptake functionally active microRNAs via exocytosis and endocytosis pathways

In this study, we first characterized synaptosome microRNA (miRNA) profiles using microarray and qRT‐PCR. MicroRNAs were detected in isolated synaptic vesicles, and Ago2 immunoprecipitation studies revealed an association between miRNAs and Ago2. Second, we found that miR‐29a, miR‐99a, and miR‐125a were significantly elevated in synaptosome supernatants after depolarization. MiRNA secretion by the synaptosome was Ca²⁺‐dependent and was inhibited by the exocytosis inhibitor, okadaic acid. Furthermore, application of nerve growth factor increased miRNA secretion without altering the spontaneous release of miRNAs. Conversely, kainic acid decreased miRNA secretion and enhanced the spontaneous release of miRNAs. These results indicate that synaptosomes could secrete miRNAs. Finally, synthesized miRNAs were taken up by synaptosomes, and the endocytosis inhibitor Dynasore blocked this process. After incubation with miR‐125a, additional miR‐125a was bound to Ago2 in the synaptosome, and expression of the miR‐125a target gene (PSD95 mRNA) was decreased; these findings suggest that the ingested miRNAs were assembled in the RNA‐induced silencing complex, resulting in the degradation of target mRNAs. To our knowledge, this is the first study that demonstrates the secretion of miRNAs by synaptosomes under physiological stimulation and demonstrates that secreted miRNAs might be functionally active after being taken up by the synaptic fraction via the endocytic pathway.     

The nucleotide sequence features of the mature microRNA seem to be responsible for the affinity to human Ago2 AND Ago3 proteins

Mature miRNA of 20-24 nt in length are the endogenous sncRNA. They programs RISC to regulate functioning of mRNA with complimentary sites for these miRNAs. In case of Ago3 protein present in human RISC miRNAs direct inhibition of translation, whereas in case of Ago2 is in RISC, than mRNA cleavage in the middle of miRNA/mRNA heteroduplex is also possible. Using ACTIVITY system, that we developed earlier, we analyzed published data on miRNA affinity to human Ago2 and Ago3 proteins. We found increase in miRNA affinity to both Ago2 and Ago3 with the increase of the YRHB tetranucleotide abundance near 3'-end of these miRNAs (r = 0.613, alpha < 0.025). We also found that miRNA tendency to bind Ago2 in favor of Ago3 increases with the RHHK tetranucleotide abundance near miRNA center (r = 0.501, alpha < 0.05). Using these two findings we proposed two formulas to predict miRNA affinity to Ago2 and Ago3 proteins based on the YRHB and RHHK abundances within this arbitrary miRNA. Thereby we made reliable predictions of miRNA affinity to these proteins in RISC for both canonical (alpha < 0.00025) and non-canonical (alpha < 0.05) miRNAs in comparison with independent experimental data.     

拟南芥Argonaute1/2/4/5序列分歧以及与特定miRNA的协同表达

Argonaute(Ago)是RNA诱导的基因沉默复合体（RISC）中的一个重要组成部分,在RNA沉默通路中结合小RNA发挥着关键的作用. 本文对12个物种的85个Ago/Ago-like基因序列进行了系统发育进化分析. 结果表明,植物Ago可被分为3大分支,且基因在物种产生分歧之前就已经发生了重复. 3个分支之间高同义和非同义突变频率揭示了Ago蛋白家族功能的高度保守性.采用实时荧光定量PCR对拟南芥Ago1/2/4/5在不同发育阶段和胁迫条件下的表达情况进行了分析. 结果显示,Ago1在拟南芥幼苗和成熟组织中表达显著;与23℃（最适温度）相比,16℃（亚适温度）条件下,Ago1表达量显著下调,Ago5表达量则显著上调;在脱水胁迫下,Ago5表达量下调,Ago4表达量上调. 另外,根据5′端碱基类型不同,188 954个幼苗miRNAs和2 047 843个成熟组织miRNAs被分类. 结果显示,5′端为U的miRNAs在植物中的含量最多,这与Ago1表达量最高是协同的;16℃条件下,5′ 端为C的miRNAs和Ago5表达协同上调. 这些结果都表明,Ago蛋白及其偏好的miRNAs是协同表达的.     

Overlapping functions of argonaute proteins in patterning and morphogenesis of Drosophila embryos

Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs) of unknown function. Here we show that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, we further demonstrate that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. Our findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning.     

Unprecedented hydrophobic stabilizations from a reverse wobble T·T mispair in DNA minidumbbell

Abstract

Minidumbbell (MDB) is a newly found non-B DNA structure formed by short single-strand sequences. Up to now, three MDBs have been reported to form at neutral pH by sequences containing two repeats of TTTA, CCTG and CTTG. Among them, the thermodynamically less stable TTTA and CCTG MDBs have been proposed to be the structural intermediates that cause TTTA and CCTG repeat expansions during DNA replication in Staphylococcus aureus pathogen and myotonic dystrophy type 2 patients, respectively. Although the CTTG MDB has a melting temperature of at least 13?°C higher than those of the other two, no CTTG repeat expansion has ever been reported in any genomes. In this study, we successfully determined the solution structure of the CTTG MDB and observed for the first time the formation of a reverse wobble T·T mispair with two symmetric hydrogen bonds. More importantly, we identified unprecedented hydrophobic interactions between the two methyl groups of this T·T mispair and the four 2′-methylene groups of their nearby loop-closing base pair residues. These stabilizations account for the substantial increase in the MDB thermodynamic stability which may govern the occurrence of repeat expansions.

Communicated by Ramaswamy H. Sarma

     

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.     

Hypoxia potentiates microRNA-mediated gene silencing through posttranslational modification of Argonaute2

Hypoxia contributes to the pathogenesis of various human diseases, including pulmonary artery hypertension (PAH), stroke, myocardial or cerebral infarction, and cancer. For example, acute hypoxia causes selective pulmonary artery (PA) constriction and elevation of pulmonary artery pressure. Chronic hypoxia induces structural and functional changes to the pulmonary vasculature, which resembles the phenotype of human PAH and is commonly used as an animal model of this disease. The mechanisms that lead to hypoxia-induced phenotypic changes have not been fully elucidated. Here, we show that hypoxia increases type I collagen prolyl-4-hydroxylase [C-P4H(I)], which leads to prolyl-hydroxylation and accumulation of Argonaute2 (Ago2), a critical component of the RNA-induced silencing complex (RISC). Hydroxylation of Ago2 is required for the association of Ago2 with heat shock protein 90 (Hsp90), which is necessary for the loading of microRNAs (miRNAs) into the RISC, and translocation to stress granules (SGs). We demonstrate that hydroxylation of Ago2 increases the level of miRNAs and increases the endonuclease activity of Ago2. In summary, this study identifies hypoxia as a mediator of the miRNA-dependent gene silencing pathway through posttranslational modification of Ago2, which might be responsible for cell survival or pathological responses under low oxygen stress.     

NMDAR‐dependent Argonaute 2 phosphorylation regulates miRNA activity and dendritic spine plasticity

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins to form the RNA‐induced silencing complex (RISC), underpinning a powerful mechanism for fine‐tuning protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)‐dependent synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic transmission. However, it is unknown how NMDAR stimulation stimulates RISC activity to rapidly repress translation of synaptic proteins. We show that NMDAR stimulation transiently increases Akt‐dependent phosphorylation of Ago2 at S387, which causes an increase in binding to GW182 and a rapid increase in translational repression of LIMK1 via miR‐134. Furthermore, NMDAR‐dependent down‐regulation of endogenous LIMK1 translation in dendrites and dendritic spine shrinkage requires phospho‐regulation of Ago2 at S387. AMPAR trafficking and hippocampal LTD do not involve S387 phosphorylation, defining this mechanism as a specific pathway for structural plasticity. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA‐mediated translational repression to control dendritic spine morphology.