Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell

The kissing-loop complex that initiates dimerization of genomic RNA is crucial for Human Immunodeficiency Virus Type 1 (HIV-1) replication. We showed that owing to its strong similitude with the bacterial ribosomal A site it can be targeted by aminoglycosides. Here, we present its crystal structure in complex with neamine, ribostamycin, neomycin and lividomycin. These structures explain the specificity for 4,5-disubstituted 2-deoxystreptamine (DOS) derivatives and for subtype A and subtype F kissing-loop complexes, and provide a strong basis for rational drug design. As a consequence of the different topologies of the kissing-loop complex and the A site, these aminoglycosides establish more contacts with HIV-1 RNA than with 16S RNA. Together with biochemical experiments, they showed that while rings I, II and III confer binding specificity, rings IV and V are important for affinity. Binding of neomycin, paromomycin and lividomycin strongly stabilized the kissing-loop complex by bridging the two HIV-1 RNA molecules. Furthermore, in situ footprinting showed that the dimerization initiation site (DIS) of HIV-1 genomic RNA could be targeted by these aminoglycosides in infected cells and virions, demonstrating its accessibility

Footprinting, circular dichroism and UV melting studies on neomycin B binding to the packaging region of human immunodeficiency virus type-1 RNA

We have studied the binding of neomycin to a 171mer RNA (ψ-RNA) from the packaging region of the LAI strain of human immunodeficiency virus type-1, HIV-1 (LAI). The RNase I footprinting studies reveal that the primary binding site for the drug is in stem–loop 1, which contains the dimer initiation site of HIV-1. Loading this site with neomycin causes a structural change in the RNA, allowing nucleotides in the neighboring stem–loop 2 to participate in the drug site. Drug binding to secondary sites induces structural changes in other stem–loops of the RNA. Footprinting plots, showing cutting at a site as a function of drug concentration, were analyzed using a two-state model to obtain relative site-specific binding constants. Circular dichroism measurements show that neomycin binding to ψ-RNA changes the intensity of the strong negative CD band at 208 nm, confirming that neomycin induces structural changes. Melting studies of the RNA showed melting transitions in the absence of drug at 28.2, 37.2, 47.4, 55.5 and 60.8°C. Only the first two were affected by drug binding, the reason for this being explained by our analysis

Hiv-1 Rna Dimerization At Single Molecule Level

The Dimerization Initiation Sequence (DIS) is a conserved hairpin-loop motif on the 5\u27 UTR of the HIV-1 genome. It plays an important role in genome dimerization through formation of a kissing complex intermediate between two homologous DIS sequences. This bimolecular kissing complex ultimately leads to the formation of an extended RNA duplex. Understanding the kinetics of this interaction is key to exploiting DIS as a possible drug target against HIV. We wish to report a novel study that makes an important contribution to understanding the dimerization mechanism of HIV-1 RNA in vitro. Our work has employed single-molecule fluorescence resonance energy transfer to monitor the dimerization of minimal HIV-1 RNA sequence containing DIS. Most significantly, we observed a previously uncharacterized folding intermediate that plays a critical role in the dimerization mechanism. Our data clearly show that dimerization involves three distinct steps in dynamic equilibrium and regulated by Mg2+ ions. Two of the steps correspond to previously proposed structures: the kissing complex and the extended duplex. Surprisingly, our data reveal a previously unobserved obligatory folding intermediate, consistent with a bent kissing complex conformation, similar to the TAR complex. Mutations of the highly conserved purines flanking the DIS loop destabilize this intermediate, indicating that these purines may play an important role in the HIV-1 RNA dimerization in vivo

Investigating Kissing to Duplex Dimer Transition Mechanism of HIV-1 SL1 by NMR.

As a common feature to retroviruses, human immunodeficiency virus (HIV) packages two identical copies of its single stranded RNA genome (gRNA) that hold together at its 5’-end. gRNA dimerization is initiated by stem loop 1 (SL1), which consists of a highly conserved asymmetric internal loop and a GC-rich self-interacting palindromic apical loop that can drive dimerization by forming a meta-stable kissing dimer. During maturation, the kissing dimer undergoes a transition catalyzed by the viral nucleocapsid protein (NCp) into a thermodynamically more stable duplex. Both dimerization and structural isomerization between the kissing and duplex dimer are critical for viral replication and packaging. While SL1 and NCp have been the focus of many studies, the mechanism of the NCp dependent SL1 dimerization and isomerization remains poorly understood. This dissertation describes the characterization of SL1 structural dynamics, its Mg2+ and NCp binding properties, and the time-course of the kissing to duplex transition using high resolution nuclear magnetic resonance (NMR) spectroscopy. Initial studies were conducted on the conformational properties of the internal loop of SL1. Subsequently, we characterized the corresponding properties in kissing and duplex SL1 dimers along with their interaction with NCp and followed site-specifically the timecourse of the kissing to duplex transition using time-resolved NMR. We observe three types of motions that may promote conversion of the kissing dimer into its duplex form: (i) diffusion-limited nanosecond collective helix motions about the G-AGG internal loop that may help bring strands from distinct monomeric units into a proper register; (ii) a secondary structural switch occurring at μs-ms timescales which leads to partial melting of the upper-helix; and (iii) looping-in-and-out hinge motions of adenine residues in the apical loop that may help bring strands from different monomers into close spatial proximity. All three classes of motions are significantly dampened by Mg2+ which likely serves to make the transition strongly dependent on NCp. The NCp interacts with the internal loop and the apical loop of kissing dimer. Our results suggest that NCp stabilizes an alternative SL1 conformation, likely involving a quadruplex geometry, prior to transitioning in a single step into the duplex dimer.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/61728/1/yanxsun_1.pd

Modes and mechanisms of hfq mediated stress regulation in bacteria

To survive bacteria must be able to respond to its ever-changing environmental conditions. sRNAs have been implicated in a variety of stress-response pathways that help bacterial systems modulate gene expression. The RNA binding protein Hfq facilities this process by, helping sRNA to base pair with its target mRNAs to initiate gene regulation. A common feature of Hfq-mediated gene regulation is the network-based organization where a single sRNA can control multiple messages to promote integrated response to stress. Current mechanistic models that are present to describe Hfq functions cannot explain the complexity at which Hfq performs gene regulation. In this work we have used a variety of biophysical, biochemical and biological approaches to understand the nature of Hfq interactions with target mRNAs

Roles of protons and structure in electron-transfer reactions of DNA and RNA

The electron-transfer chemistry of DNA is a well-studied phenomenon; however, the mechanism of electron transfer is still unclear. Similar base oxidation pathways are thought to occur in both DNA and RNA; yet the electron-transfer chemistry of RNA has been studied in far less detail than that of DNA. Here, we study the role protons have on the electron-transfer chemistry of DNA to help identify its mechanism. We also examine how transition metal complexes can better characterize the structure of nucleotides, how those structures influence electron-transfer chemistry, and the specificities of oxidation. The transition metal complexes that are being studied include the following ruthenium complexes, which have different binding modes and electronic properties: Ru(bpy)3 2+ (bpy = 2,2'-bipyridine), Ru(tpy)(bpy)O2+ (tpy = 2,2',2''-terpyridine), Ru(bpz)3 2+ (bpz = 2,2'-bipyrazyl), and Ru(bpy)2(dppz)2+ (dppz = dipyrido [3,2-a:2',3'-c] phenazine). For proton-DNA studies, we will be using a simple DNA strand consisting of fifteen base pairs for comparison of the electrochemical techniques of cyclic voltammetry and digital simulation with the flash-quench technique. In order to better compare the electron-transfer chemistry that is occurring in both DNA and RNA, we will be using sequences based off of the human ferritin iron responsive elements (IREs) RNA. The human ferritin IRE RNA is a well-studied hairpin loop RNA that has a primary role in the regulation of ferritin and iron in the cell. Our lab has also developed a mutated ferritin IRE (MIRE), which has a more rigid structure but still contains the hairpin loop feature. The DNA used for comparison is based off of the template used for the transcription of IRE RNA and MIRE RNA. After the ruthenium complexes' oxidation of DNA is confirmed and specified, these complexes' oxidation will be tested on RNA. Here, we show that transition metal complexes can oxidize RNA very similarly to DNA. We also show that some of these complexes, depending on their electronic properties, can footprint small molecules or proteins bound to RNA, which is useful for drug targeting-RNA studies. These oxidation studies of various nucleotides are also important due to their implications in aging, cancer, atherosclerosis, and neurological disorders

Funktionelle Charakterisierung von Peptidliganden für das komplexe HIV-1-RNA-Verpackungssignal PSI

Im Laufe der vergangenen Jahre hat die Identifizierung von Peptidleitstrukturen in der Wirkstoffentwicklung zunehmend an Bedeutung gewonnen. Die Phage Display Technologie ist eine Methode, welche zur Selektion von inhibitorischen Peptiden weit verbreitet ist. Prinzipiell eignet sich dieser Ansatz auch für die Suche nach neuen Leitstrukturen für die Therapie der HIV-Infektion, welche in hochspezifische und -regulierte Schritte im HIV-Replikationszyklus eingreifen sollen. Bei der Verpackung viraler RNA in neu entstehende Virionen handelt es sich um einen Prozess, welcher auf der gezielten Erkennung der dreidimensionalen Struktur der PSI-Region am 5'-Ende ungespleißter, viraler RNA durch die NCp7-Domäne des Gagp55-Vorläuferproteins basiert. Darüber hinaus partizipiert das NCp7-Protein noch an der Reversen Transkription der HIV-RNA sowie an der Integration proviraler DNA und spielt somit eine zentrale Rolle im HIV-1 Replikationszyklus. In vorangegangenen Arbeiten konnten wir mittels der Phage Display Technologie Peptidliganden für die HIV-1 PSI-RNA selektieren, welche die PSI-RNA-NCp7-Interaktion hemmten und in Folge dessen die Verpackung viraler RNA verhindern sollten. Die Bindung der identifizierten tryptophanreichen Peptide an die PSI-RNA konnte zwar zum Teil in vitro mit NCp7 kompetitiert werden, jedoch wiesen die Peptide eine relativ geringe Affinität für die PSI-RNA auf. Im Vordergrund der vorliegenden Arbeit stand nach Optimierung der Affinität eine umfassende funktionelle Charakterisierung der Peptide hinsichtlich ihrer antiviralen Aktivität in vitro. Zunächst gelang es mittels Spot-Synthese-Membranen die Affinität der PSI-RNA-bindenden Peptide um etwa das 30-fache zu verbessern. Der KD-Wert des optimierten HKWPWW-Peptids lag bei 1,1 µM für ein Teilelement der PSI-RNA, das allein über Verpackungsaktivitäten verfügt. Die folgende Analyse der Bindungseigenschaften des HKWPWW-Peptids an die PSI-RNA über NMR und Fluoreszenz-Spektroskopie offenbarte, dass das Peptid über die hydrophoben Aminosäuren an eine charakteristische Schleifenregion in der Sekundärstruktur der PSI-RNA bindet, ähnlich wie der natürliche Ligand NCp7. Gestützt auf diese Ergebnisse, wurde im Hauptteil des Projekts untersucht, ob das HKWPWW-Peptid in der Lage ist, die Verpackung viraler RNA in HI-Virionen zu hemmen. Hierfür erfolgte die Etablierung diverser Testsysteme, welche die intrazelluläre Expression des Peptids ermöglichten. Die Expression von HKWPWW in Fusion mit RFP in Pseudoviren-produzierenden Zellen über transiente Transfektion führte in der höchsten getesteten DNA-Konzentration (2,5 µg) zu einer 95%igen Reduktion des infektiösen Titers. Dieser inhibitorische Effekt war spezifisch für lentivirale Pseudoviren, da die Produktion gammaretroviraler Pseudoviren nicht durch die Anwesenheit des Peptids beeinflusst wurde. Mittels einer stabilen HKWPWW-exprimierenden T-Zelllinie gelang es nachzuweisen, dass das Peptid sogar in der Lage ist, replikationskompetentes HIV über einen Zeitraum von fünf Tagen zu hemmen. Die Synthese des HKWPWW-Peptids in Fusion mit einer Proteintransduktionsdomäne ermöglichte die direkte Behandlung von HIV-infizierten Zellen und führte zu einer verminderten Freisetzung infektiöser HI-Viren in die Zellkulturüberstände. Dabei lagen die IC50- und IC90-Werte des HKWPWW-Peptids nach zweimaliger Peptidzugabe bei 5, 7 bzw. 28,6 µM. Eine in der Literatur oftmals beschriebene Beobachtung ist, dass bei einer reinen Hemmung der HIV-Verpackung Viren entstehen, welche keine virale RNA enthalten. Das Phänomen war in Anwesenheit des HKWPWW-Peptids wenig ausgeprägt wie Korrelationen von p24-Antigen-ELISA und die Quantifizierung viraler RNA in Viruspartikeln zeigten. Diese Gegebenheit sowie das Wissen über die mannigfaltigen Funktionen des NCp7-Proteins im HIV-Replikationszyklus ließen vermuten, dass HKWPWW noch zusätzlich andere Schritte im HIV-Replikationszyklus hemmen könnte. Unterstützt wurde diese Annahme dadurch, dass HKWPWW Ähnlichkeiten zu der hydrophoben Plattform von NCp7 aufweist, welche essentiell für die Verpackung viraler RNA sowie die Reverse Transkription ist. Damit in Einklang steht, das neben einer Bindung an die PSI-RNA auch eine schwächere Interaktion des HKWPWW-Peptids mit den viralen TAR- und PBS-Strukturen nachgewiesen werden konnte. Die auch beobachtete Hemmung der frühen HIV-Replikationsschritte durch HKWPWW könnte somit mit einer möglichen Hemmung der Transkription viraler Gene, der Reversen Transkription oder Integration erklärt werden. Jedoch zeigte die elektronenmikroskopische Analyse, dass nicht nur weniger Viren in Anwesenheit des HKWPWW-Peptids entstehen, sondern dass diese zum Teil einen weniger kondensierten Kern aufweisen. Dies kann als ein Anhaltspunkt angesehen werden, dass HKWPWW tatsächlich auch auf der Ebene der RNA-Verpackung bzw. der viralen Partikelentstehung einen hemmenden Effekt ausübt. Somit resultiert die beobachtete antivirale Aktivität des HKWPWW-Peptids vermutlich aus kombinierten inhibitorischen Effekten auf mehreren Ebenen der HIV-Replikation