Polypurine Tract Research Articles

EDITORIAL Implication of Non-coding RNAs in Cancer Biology and Cellular Physiology.

In the recent years, a huge number of human transcripts have been found in the human genome that do not encode for proteins, which have been named non-coding RNAs (npcRNAs) containing secondary structures or short regions highly conserved within mammalian sequences. Long RNAs (antisense RNA, structured RNA, and long interspersed ncRNAs) and small RNAs (miRNAs, siRNAs, snoRNAs) have shown to exert many roles: functioning as regulators of other mRNAs, at transcriptional and post-transcriptional level, controlling protein ubiquitination and degradation, regulating epigenetic marks and affecting chromosome structure. One group of npcRNAs that is well-characterized, at the biochemical level, is represented by miRNAs. This group comprises a large class of small npcRNAs (~22-nucleotide RNAs) acting through base pairing to partially complementary sites in the 3’untranslated regions (3’UTR) of the targeted messenger RNA. Circular RNAs, competing endogenous RNAs (ceRNAs) acting as RNA sponges, natural antisense RNAs (NAT), enhancer RNAs (eRNAs), and RNA decoys are further expanding the wide array of functionalities exerted by ncRNAs. In the last case, as example, Growth Arrest Suppressor 5 (GAS5) forms a structured RNA tha is a decoy for the glucocorticoid receptor (GR), mimicking the DNA structure of the GR element (GRE). Long noncoding RNAs (lncRNAs) have emerged as key players in regulating various fundamental cellular processes. Many of the mechanisms that modify the 3' UTRs, or that affect differential splicing, make use of RNA regulation, antisense RNAs, and may involve RNP complexes. Many human ncRNAs have been characterized in terms of function or expression profiles. HOTAIR, described in detail in the review by Ge Shan, is a structured RNA that assemble several proteins to form an epigenetic regulation complex: it assembles Polycomb Repressive Complex (PRC) proteins and determines the silencing of specific genes. Terminal differentiation induced ncRNA (TINCR) destabilises ALU elements in mRNAs through the RNA binding protein Staufen 1 binding to polypurine tract. Thus, proteins involved in the functioning of ncRNAs are highly varied: RNA binding proteins, ribonucleoprotein complexes, alternative splicing proteins, alternative polyadenylation proteins, chromatin remodeling complexes, and gene activation and repression complexes (PRC) and enzymes positioning or eliminating histone marks. Then, it is clear that the changes determined in several diseases and in cancer are caused non only by mutated genes but also by epigenetic deregulation and by alternative spliced genes and alternative polyA tails that evade microRNA recognition. Concerning miRs, the varied presence of Argonaute family of proteins and the link with diseases are well detailed in more than one review in this special issue. In this thematic issue, Ge Shan's paper provides an overview of several examples in small and long RNAs; Yangchao Chen introduces the recent topic of circular RNAs; Charles Lawrie presents the deregulated pathways involving miRNAs in myeloma malignancies, while George Calin classifies leukemias according to epigenetic deregulation, oncomiRs and loss of antioncomiRs; Massimo Mallardo reviews the mechanism of infectivity in RNA viruses; Marek Sanak describes a unique microRNA differently expressed in neutrophils in healthy individuals and in Granulomatosis with Polyangiitis patients; finally two papers describe the circulating miRNAs protected by exosomes that are released by cancer cells and can be found in bodily fluids. Sonia Melo gives a detailed overview on circulating miRNAs, and Valeria Mezzolla proposes a method to group glioma and differentiate them from other malignancies based on the most representative circulating miRNAs. This field is a promising means to ensure novel approaches to treatment and more effective therapies exploiting ncRNAs.

Retrotransposon based TRAP marker displays diversity among onion (Allium cepa L.) genotypes

Ty1-copia like elements are distributed throughout the onion (Allium cepa) genome, and are actively transcribed. Sequence analysis by LTR finder identified that the copia element in onion is bound by long terminal repeats (LTR) of length 121bp. Primer binding site and poly purine tract are located on copia element at 193–211bp and 4593–4607bp respectively. To exploit the variation at the insertional locus and to explore its further use as a marker, a method using a primer located on the LTR and an arbitrary primer was developed for detecting variation in onion genotypes. This method resulted in the detection of polymorphism in 22 onion genotypes. Thirty-three polymorphic amplicons were detected in these genotypes. The genetic distance between the varieties varied from 0.83 to 0.99. Phylogenetic analysis revealed that the Indian short day onion clustered separately from the exotic varieties indicating that the origin of Indian varieties is distinct. Further, variation observed among Indian varieties was less than that of exotic varieties. The marker was detected across eight species of genus Allium and the genetic distance between the species varied from 0.34 to 0.88. Polymorphism detection was better across the species than within A. cepa. This retrotransposon based method is likely to supplement the marker resources for Allium spp.

SiRNA-induced mutation in HIV-1 polypurine tract region and its influence on viral fitness.

Converting single-stranded viral RNA into double stranded DNA for integration is an essential step in HIV-1 replication. Initial polymerization of minus-strand DNA is primed from a host derived tRNA, whereas subsequent plus-strand synthesis requires viral primers derived from the 3′ and central polypurine tracts (3′ and cPPTs). The 5′ and 3′ termini of these conserved RNA sequence elements are precisely cleaved by RT-associated RNase H to generate specific primers that are used to initiate plus-strand DNA synthesis. In this study, siRNA wad used to produce a replicative HIV-1 variant contained G(-1)A and T(-16)A substitutions within/adjacent to the 3′PPT sequence. Introducing either or both mutations into the 3′PPT region or only the G(-1)A substitution in the cPPT region of NL4-3 produced infectious virus with decreased fitness relative to the wild-type virus. In contrast, introducing the T(-16)A or both mutations into the cPPT rendered the virus(es) incapable of replication, most likely due to the F185L integrase mutation produced by this nucleotide substitution. Finally, the effects of G(-1)A and T(-16)A mutations on cleavage of the 3′PPT were examined using an in vitro RNase H cleavage assay. Substrate containing both mutations was mis-cleaved to a greater extent than either wild-type substrate or substrate containing the T(-16)A mutation alone, which is consistent with the observed effects of the equivalent nucleotide substitutions on the replication fitness of NL4-3 virus. In conclusion, siRNA targeting of the HIV-1 3′PPT region can substantially suppress virus replication, and this selective pressure can be used to generate infectious virus containing mutations within or near the HIV-1 PPT. Moreover, in-depth analysis of the resistance mutations demonstrates that although virus containing a G(-1)A mutation within the 3′PPT is capable of replication, this nucleotide substitution shifts the 3′-terminal cleavage site in the 3′PPT by one nucleotide (nt) and significantly reduces viral fitness.

Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis.

HIV-1 reverse transcriptase (RT) connection subdomain mutations at positions 348, 369 and 376 have been associated with resistance to non-nucleoside RT inhibitors (NNRTIs). N348I may interfere with the initiation of (+)-strand DNA synthesis by reducing polypurine tract (PPT) removal in the presence of nevirapine. The effect of NNRTIs on the RNase H-mediated cleavage of PPT-containing template-primers has been studied with wild-type HIV-1 RT and mutants N348I, T369I, T369V, T376S and N348I/T369I. In the presence of NNRTIs, all RTs were able to stimulate PPT cleavage after primer elongation. The enhancing effects of nevirapine and efavirenz were reduced in RTs carrying mutation N348I, and specially N348I/T369I. However, those mutations had no effect on rilpivirine-mediated cleavage. Prior to elongation, the PPT remains resilient to cleavage, although efavirenz and rilpivirine facilitate RNase H-mediated trimming of its 3′-end. The integrity of the 3′-end is essential for the initiation of (+)-strand DNA synthesis. In the presence of dNTPs, rilpivirine was the most effective inhibitor of (+)-strand DNA synthesis blocking nucleotide incorporation and preventing usage of available PPT primers. The N348I/T369I RT showed reduced ability to generate short RNA products revealing a cleavage window defect. Its lower RNase H activity could be attributed to enhanced rigidity compared to the wild-type enzyme.

Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors

The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors. Here, we describe the development of the first tetracycline-inducible stable IDLV packaging cell line comprising the D64E integrase mutant and the VSV-G envelope protein. A conditional self-inactivating (cSIN) vector and a novel polypurine tract (PPT)-deleted vector were incorporated into the newly developed stable packaging cell line by transduction and stable transfection, respectively. High-titer (~107 infectious units (IU)/ml) cSIN vectors were routinely generated. Furthermore, screening of single-cell clones stably transfected with PPT-deleted vector DNA resulted in the identification of highly efficient producer cell lines generating IDLV titers higher than 108 IU/mL, which upon concentration increased to 1010 IU/ml. IDLVs generated by stable producer lines efficiently transduce CNS tissues of rodents. Overall, the availability of high-titer IDLV lentivirus packaging cell line described here will significantly facilitate IDLV-based basic science research, as well as preclinical and clinical applications.

Structural studies of a double-stranded RNA from trypanosome RNA editing by small-angle X-ray scattering.

We used small-angle X-ray scattering (SAXS) to evaluate the solution structure of a double-stranded RNA with 32 base pairs. We wanted to compare the solution structure to the crystal structure to assess the impact of the crystal lattice on the overall conformation of the RNA. The RNA was designed to self-anneal and form a head-to-head fusion of two identical mRNA/oligo(U) tail domains (the U-helix) from a trypanosome RNA editing substrate formed by the annealing of a guide RNA to a pre-edited mRNA. This substrate is from the U insertion/deletion RNA editing system of trypanosomes. Each strand in the fusion RNA had 16 purines from the pre-mRNA followed by 16 uracils (Us) from the U-tail at the 3' end of the guide RNA. The strands were designed to form a double helix with blunt ends, but each strand had the potential to form hairpins and single-stranded RNA helices. Hairpins could form by the 3' oligouridylate tract folding back to hybridize with the 5' oligopurine tract and forming an intervening loop. Single-stranded helices could form by the stacking of bases in the polypurine tract. Some of the 16 Us 3' to the polypurine tract may have been unstacked and in random coils. Our SAXS studies showed that the RNA formed a mix of single-stranded structures in the absence of MgCl2. In the presence of MgCl2 at concentrations similar to those in the crystal, the solution structure was consistent with the double-stranded, blunt-ended structure, in agreement with the crystal structure. Here we describe the preparation of RNA samples, data collection with an in-house SAXS instrument designed for biological samples, and the processing and modeling of the scattering data.

Delivery of antisense peptide nucleic acids to cells by conjugation with small arginine-rich cell-penetrating peptide (R/W)9.

Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor β-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.

Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry

Retrotransposons are a class of mobile genetic elements that replicate by converting their single-stranded RNA intermediate to double-stranded DNA through the combined DNA polymerase and ribonuclease H (RNase H) activities of the element-encoded reverse transcriptase (RT). However, while a wealth of structural information is available for lentiviral and gammaretroviral RTs, equivalent studies on counterpart enzymes of long terminal repeat (LTR)-containing retrotransposons, from which they are evolutionarily derived, is lacking. In this study, we report the first crystal structure of a complex of RT from the Saccharomyces cerevisiae LTR-retrotransposon Ty3 in the presence of its polypurine tract-containing RNA-DNA hybrid. In contrast to its retroviral counterparts, Ty3 RT adopts an asymmetric homodimeric architecture, whose assembly is substrate-dependent. More strikingly, our structure and biochemical data suggest that the RNase H and DNA polymerase activities are contributed by individual subunits of the homodimer.

Unraveling the Gymnastics of Reverse Transcription Through Single Molecule Spectroscopy

AIDS Research and Human RetrovirusesVol. 30, No. 3 Images in HIV ResearchFree AccessUnraveling the Gymnastics of Reverse Transcription Through Single Molecule SpectroscopyElio A. Abbondanzieri and Stuart F.J. Le GriceElio A. AbbondanzieriDepartment of Bionanoscience, Delft University of Technology, Delft, The Netherlands.Search for more papers by this author and Stuart F.J. Le GriceRT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, Maryland.Search for more papers by this authorPublished Online:3 Mar 2014https://doi.org/10.1089/aid.2014.0023AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Synthesizing integration-competent, double-stranded DNA from the single-stranded human immunodeficiency virus (HIV) RNA genome might more aptly be described as molecular gymnastics (Fig. 1). Key elements of this routine include intramolecular and intermolecular transfer of the replication complex between genome termini, strand displacement synthesis, selecting plus-strand RNA primers from within the RNA/DNA replication intermediate, their excision from nascent DNA, and phased A-tract-induced cessation of DNA synthesis near the center of the genome. While site-directed mutagenesis identifies residues of HIV-1 reverse transcriptase (RT) that mediate these functions, and X-ray crystallography gives elegant, but static snapshots of nucleoprotein complexes, they cannot assess conformational dynamics of this remarkably versatile enzyme. To better understand how HIV-1 RT interacts with nucleic acid required designing a single molecule strategy based on Foerster resonance energy transfer (FRET), placing fluorophores into the enzyme and its nucleic acid substrates.FIG. 1. Multistep acrobatics and reverse transcription.Studies with duplex DNA predictably recapitulated an enzyme with its DNA polymerase catalytic center straddled over the primer terminus. However, an unexpected observation of Abbondanzieri et al.1 was that this orientation reversed when the DNA primer was replaced with RNA. Later studies examining polypurine tract recognition not only confirmed that HIV-1 RT could assume different orientations, but also was sufficiently agile to “flip” between these during a single binding event. Such reversal in RT orientation could help explain how its N-terminal DNA polymerase and C-terminal ribonuclease H (RNase H) domains can recognize the PPT primer terminus at different steps of plus-strand synthesis.Another concept we accept when studying HIV-1 RT is that it is a distributive polymerase, i.e., it frequently dissociates from its nucleic acid substrate. How then, upon rebinding, does the enzyme locate the primer terminus to reengage in DNA synthesis? Subsequent studies of Liu et al. illustrated the capacity of RT to slide on the nucleic acid duplex, a property that could be enhanced in the presence of nonnucleoside RT inhibitors or restricted by forming a ternary complex. However, the story does not end here. Since there appears to be no orientation (or directional) bias during sliding, the polymerization target site could be accessed by the retroviral polymerase in two different orientations. Single molecule spectroscopy not only confirmed this, but elegantly demonstrated that an enzyme accessing the primer terminus in an “RNase H mode” could spontaneously flip into the polymerization mode, thereby linking the properties of flipping and sliding.2A state-of-the-art technology, high-profile publications, but what is the significance? For many years, we have presumed that nonnucleoside inhibition of HIV-1 RT occurred through a “grinding of the gears” and an inability to support DNA synthesis. However, examining the interaction of HIV-1 RT with its PPT by single molecule spectroscopy revealed that the nonnucleoside reverse transcriptase inhibitor (NNRTI) nevirapine promotes a reversal of enzyme orientation, positioning the RNase H domain over the primer terminus, thereby inhibiting synthesis of plus-strand DNA. These and related studies on tRNA-primed initiation of minus-strand DNA synthesis3 show that, in a different sense, HIV fitness requires nimble and flexible maneuvering of its DNA polymerase!References1 Abbondanzieri EA, Bokinsky G, Rausch JW, Zhang J, Le Grice SFJ, and Zhuang X: Dynamic binding orientations determine the enzymatic activity of HIV reverse transcriptase. Nature 2008;453:184–189. Crossref, Medline, Google Scholar2 Liu S, Abbondanzieri EA, Rausch JW, Le Grice SFJ, and Zhuang X: Slide into action dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates. Science 2008;322:1092–1097. Crossref, Medline, Google Scholar3 Liu S, Harada BT, Miller JT, Le Grice SFJ, and Zhuang X: Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription. Nat Struct Mol Biol 2010;17:1453–1460. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 30Issue 3Mar 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Elio A. Abbondanzieri and Stuart F.J. Le Grice.Unraveling the Gymnastics of Reverse Transcription Through Single Molecule Spectroscopy.AIDS Research and Human Retroviruses.Mar 2014.209-210.http://doi.org/10.1089/aid.2014.0023Published in Volume: 30 Issue 3: March 3, 2014PDF download

Insights into the Motif Preference of APOBEC3 Enzymes

We used a multivariate data analysis approach to identify motifs associated with HIV hypermutation by different APOBEC3 enzymes. The analysis showed that APOBEC3G targets G mainly within GG, TG, TGG, GGG, TGGG and also GGGT. The G nucleotides flanked by a C at the 3′ end (in +1 and +2 positions) were indicated as disfavoured targets by APOBEC3G. The G nucleotides within GGGG were found to be targeted at a frequency much less than what is expected. We found that the infrequent G-to-A mutation within GGGG is not limited to the inaccessibility, to APOBEC3, of poly Gs in the central and 3′polypurine tracts (PPTs) which remain double stranded during the HIV reverse transcription. GGGG motifs outside the PPTs were also disfavoured. The motifs GGAG and GAGG were also found to be disfavoured targets for APOBEC3. The motif-dependent mutation of G within the HIV genome by members of the APOBEC3 family other than APOBEC3G was limited to GA→AA changes. The results did not show evidence of other types of context dependent G-to-A changes in the HIV genome.

A simple and efficient method to isolate LTR sequences of plant retrotransposon.

Retrotransposons (RTNs) have important roles in the formation of plant genome size, structure, and evolution. Ubiquitous distributions, abundant copy numbers, high heterogeneities, and insertional polymorphisms of RTNs have made them as excellent sources for molecular markers development. However, the wide application of RTNs-based molecular markers is restricted by the scarcity of the LTR (long terminal repeat) sequences information. A new, simple, and efficient method to isolate LTR sequences of RTNs was presented based on the degenerate RNase H nested primers and PPT (polypurine tract) primer of RTNs in tree peony. This method combined the characteristics and advantages of high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), annealing control primer (ACP) system, and suppression PCR method. Nineteen LTR sequences were isolated using this new method in tree peony and the applicability of the LTR sequences based markers was validated by further SSAP analysis. The results showed that the new method is simple, of low-cost, and highly efficient, which is just conducted by three rounds of PCR and does not need any restriction enzymes and adapters, much less the hybridizations. This new method is rapid, economical, and cost- and time-saving, which could be easily used to isolate LTR sequences of RTNs.

Calreticulin novel mutations in type 2 diabetes mellitus

Imbalance in Ca2+ concentration and dysfunction of the chaperone system are linked with type 2 diabetes mellitus (T2DM). Two-dimensional protein profiling of pancreatic beta cells in T2DM subjects has shown that the Ca2+ binding chaperone, calreticulin (CALR), plays a role in the pathophysiology of this disease. In a case/control study, we performed mutation screening of the promoter region, 9 exons, and exon/intron boundaries of CALR by PCR-SSCP and sequencing in 120 patients with T2DM and 530 controls. Two novel mutations with an estimated frequency of 0.0005 were detected in T2DM patients, which were absent in the control pool (Mid P exact T transversion in intron 2 conserved polypurine tract at IVSII-142. The second mutation was a 9-bp deletion in the highly conserved exon 9 encompassing amino acids 397–399. To our knowledge, the current study reports for the first time, that CALR gene mutations that occur with T2DM. Studying larger groups of patients with T2DM for the CALR gene as well as other genes in the chaperone system is warranted to further elucidate the role of low frequency mutations in the causation of this complex disorder.

Isolation and characterization of novelTy1-copia-like retrotransposons from lily

Species of the genus Lilium are well known for their large genomes. Although expansion of noncoding repeated DNA is believed to account for this genome size, retroelement del Ty3-gypsy is the only one described so far in the genus Lilium. We isolated Ty1-copia elements from Lilium longiflorum and named them LIREs (lily retrotransposons). The long terminal repeats, primer binding site, and polypurine tract sequences are highly similar among the LIRE elements, indicating that they are in the same lineage. Although the protein-coding regions were highly decayed, the sequence motifs of the integrase, reverse transcriptase, and RNase H domains were identifiable as belonging to the order of Ty1-copia elements. Phylogenetic analysis and primer binding site sequences revealed that these elements belonged to the Ale lineage among the six lineages of plant Ty1-copia elements. Base substitutions in the long terminal repeats estimated that the integration times of the LIRE Ty1-copia elements were between 0.7 and 5.5 mya. In situ hybridization showed that the LIRE elements were present in all the chromosomes of L. longiflorum and L. lancifolium, but absent in centromeres, telomeres, and 45S rRNA sites in both species. The LIRE elements were present very abundantly in species of the genus Lilium, but absent in other genera of the family Liliaceae, implying that the LIRE elements might have contributed to the expansion of the genome in the genus Lilium.

Novel insights from structural analysis of lentiviral and gammaretroviral reverse transcriptases in complex with RNA/DNA hybrids

Structures of HIV-1 reverse transcriptase (RT) have been reported in several forms, but only one contains an RNA/DNA hybrid, the conformation of which has been controversial. We have been successful in obtaining three structures of HIV-1 RT complexed with a non-nucleoside RT inhibitor (NNRTI) and an RNA/DNA hybrid [1]. In the presence of an NNRTI, our RNA/DNA structure differs from all prior nucleic acid bound to RT including the previously-reported RNA/DNA hybrid derived froom the polypurine tract. The enzyme structure observed in our cocrystals also differs from all previous RT-DNA complexes. As a result, the hybrid has ready access to the ribonuclease H (RNase H) active site. These observations collectively reinforce previous proposals that an RT-nucleic acid complex may be required to adopt independent structural states competent for DNA synthesis and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites map to the unique RT-hybrid interface that undergoes conformational changes between two catalytic states. Structural features of the nucleoprotein complex, including drug resistance mutations, have been verified by site-directed mutagenesis, and will be presented. Although the single-subunit RT of Moloney murine leukemia virus (Mo-MLV) has been extensively characterized biochemically, structural information is lacking that describes the substrate binding mechanism for this RT species. We also present data on the first crystal structure of a complex between an RNA/DNA hybrid and the 72 kDa single-subunit RT from the related xenotropic murine leukemia virus-related virus (XMRV) [2]. A comparison of this structure with its HIV-1 counterpart shows that substrate binding around the DNA polymerase active site is conserved but differs between the two enzymes in their thumb and connection subdomains. Small-angle X-ray scattering (SAXS) was used to model full-length XMRV RT, demonstrating its flexible RNase H domain becomes ordered in the presence of substrate, a key difference between monomeric and dimeric RTs.

Mechanism of HIV-1 RNA Dimerization in the Central Region of the Genome and Significance for Viral Evolution

The genome of HIV-1 consists of two identical or nearly identical RNA molecules. The RNA genomes are held in the same, parallel orientation by interactions at the dimer initiation site (DIS). Previous studies showed that in addition to interactions at DIS, sequences located 100 nucleotides downstream from the 5' splice site can dimerize in vitro through an intermolecular G-quartet structure. Here we report that the highly conserved G-rich sequence in the middle portion of the HIV-1 genome near the central polypurine tract (cPPT) dimerizes spontaneously under high ionic strength in the absence of protein. The antisense RNA does not dimerize, strongly indicating that RNA dimerization does not exclusively involve A:U and G:C base pairing. The cation-dependent reverse transcriptase pausing profile, CD spectra profile, and cation-dependent association and thermal dissociation characteristics indicate G-quartet structures. Different forms of G-quartets are formed including monomers and, significantly, intermolecular dimers. Our results indicate that RNA genome dimerization and parallel alignment initiated through interactions at DIS may be greatly expanded and stabilized by formation of an intermolecular G-quartet at a distant site near the cPPT. It is likely that formation of G-quartet structure near the cPPT in vivo keeps the RNA genomes in proximity over a long range, promoting genetic recombination in numerous hot spots.

Molecular dynamics simulation of DNA duplex, analog of PPT (polypurine tract), its conformation and hydration: a theoretical study

Molecular Dynamics Simulation Study of DNA duplex is reported here in this communication. The duplex is a decamer, which consists of two non-self complementary sequences 5′(CAAAGAAAAG) and 5′(CTTTTCTTTG). This decamer is a DNA analog of the portion of the polypurine tract, which is a RNA/DNA hybrid that serves as a primer for the synthesis of the non-digestible DNA strand by human immunodeficiency virus reverse transcriptase. The result obtained is compared with the crystal structure of this decamer duplex. The calculations described here using AMBER throw light on the effect of counter ions on the binding of water molecules, within the grooves of the duplex. This study may provide important implications for understanding the functional, energetic, and specificity of the interactions of DNA/RNA with regulatory proteins, pharmaceutical agents, and other ligands.

Examining the Role of the HIV-1 Reverse Transcriptase p51 Subunit in Positioning and Hydrolysis of RNA/DNA Hybrids

Recent crystallographic analysis of p66/p51 human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) complexed with a non-polypurine tract RNA/DNA hybrid has illuminated novel and important contacts between structural elements at the C terminus of the noncatalytic p51 subunit and the nucleic acid duplex in the vicinity of the ribonuclease H (RNase H) active site. In particular, a short peptide spanning residues Phe-416-Pro-421 was shown to interact with the DNA strand, cross the minor groove of the helix, and then form Van der Waals contacts with the RNA strand adjacent to the scissile phosphate. At the base of the adjoining α-helix M', Tyr-427 forms a hydrogen bond with Asn-348, the latter of which, when mutated to Ile, is implicated in resistance to both nucleoside and non-nucleoside RT inhibitors. Based on our structural data, we analyzed the role of the p51 C terminus by evaluating selectively mutated p66/p51 heterodimers carrying (i) p51 truncations that encroach on α-M', (ii) alterations that interrupt the Asn-348-Tyr-427 interaction, and (iii) alanine substitutions throughout the region Phe-416-Pro-421. Collectively, our data support the notion that the p51 C terminus makes an important contribution toward hybrid binding and orienting the RNA strand for catalysis at the RNase H active site.

Oligo(2′-O-methylribonucleotides) and their derivatives: IV. Conjugates of oligo(2′-O-methylribonucleotides) with minor groove binders and intercalators: Synthesis, properties, and application

Conjugates of pyrimidine triplex forming 3'-protected oligo(2'-O-methylribonucleotides) with minor groove binders (MGB) and triplex specific intercalator benzoindoloquinoline (BIQ) at 5'-terminus were synthesized. The conjugates formed stable complexes with target dsDNA by simultaneous binding both in its minor and major grooves and BIQ intercalation. The dissociation constants and thermal stability of the conjugate complexes with model dsDNA corresponding to polypurine tract (PPT) of genes nef and pol from HIV proviral genome were determined. Conjugation of oligo(2'-O-methylribonucleotides) with MGB and intercalator increased the stability of the triple complexes with dsDNA at pH 7.2 and 37 degrees C. Intercalator introduction accelerates the process of complex formation. Dose-dependent arrest of the in vitro transcription was demonstrated when a 780 b.p. DNA fragment containing the polypurine tract was transcribed under the control of T7 promoter in the presence of different concentrations of conjugates of oligo(2'-O-methylribonucleotides) containing MGB and BIQ intercalator.

Highly diverse chromoviruses of Beta vulgaris are classified by chromodomains and chromosomal integration

BackgroundChromoviruses are one of the three genera of Ty3-gypsy long terminal repeat (LTR) retrotransposons, and are present in high copy numbers in plant genomes. They are widely distributed within the plant kingdom, with representatives even in lower plants such as green and red algae. Their hallmark is the presence of a chromodomain at the C-terminus of the integrase. The chromodomain exhibits structural characteristics similar to proteins of the heterochromatin protein 1 (HP1) family, which mediate the binding of each chromovirus type to specific histone variants. A specific integration via the chromodomain has been shown for only a few chromoviruses. However, a detailed study of different chromoviral clades populating a single plant genome has not yet been carried out.ResultsWe conducted a comprehensive survey of chromoviruses within the Beta vulgaris (sugar beet) genome, and found a highly diverse chromovirus population, with significant differences in element size, primarily caused by their flanking LTRs. In total, we identified and annotated full-length members of 16 families belonging to the four plant chromoviral clades: CRM, Tekay, Reina, and Galadriel. The families within each clade are structurally highly conserved; in particular, the position of the chromodomain coding region relative to the polypurine tract is clade-specific. Two distinct groups of chromodomains were identified. The group II chromodomain was present in three chromoviral clades, whereas families of the CRM clade contained a more divergent motif. Physical mapping using representatives of all four clades identified a clade-specific integration pattern. For some chromoviral families, we detected the presence of expressed sequence tags, indicating transcriptional activity.ConclusionsWe present a detailed study of chromoviruses, belonging to the four major clades, which populate a single plant genome. Our results illustrate the diversity and family structure of B. vulgaris chromoviruses, and emphasize the role of chromodomains in the targeted integration of these viruses. We suggest that the diverse sets of plant chromoviruses with their different localization patterns might help to facilitate plant-genome organization in a structural and functional manner.