Characterizing Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Integrase Interaction
Characterizing Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Integrase Interaction

Author: Shewit Tekeste

Publisher:

Published: 2014

Total Pages: 83

ISBN-13:

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Human immunodeficiency virus type 1 (HIV-1) replication requires the reverse transcription of its RNA genome into double-stranded DNA copies within the cytoplasm before integration into the host chromosome. Reverse transcriptase (RT) and integrase (IN) are the viral enzymes responsible for catalyzing the essential steps of reverse transcription and integration, respectively. While numerous studies have led to a greater understanding of the functional roles that RT and IN individually play in HIV-1 replication, little is known about the functional role of RT-IN complex formation in vivo. We hypothesize that RT-IN interaction has functional significance in HIV-1 reverse transcription and replication kinetics. We have mapped the putative binding domain of RT on IN to nine residues on the IN C-terminal domain (CTD). We tested the significance of RT-IN interaction on reverse transcription and viral replication, and identified the step at which viral replication of these IN mutants become defective. We observed impairment of viral cDNA synthesis in viruses harboring IN mutations at the putative RT-binding surface, supporting our hypothesis that the RT-IN interaction during the reverse transcription step is biologically relevant. We have developed a pharmacological approach to study and screen for inhibitors against the RT-IN interaction. Lastly, we have also initiated biochemical studies to determine the IN binding domain domain on RT to contribute to the full understanding of the binding mechanism.

From the Notorious to the Novel
From the Notorious to the Novel

Author: Mia Biondi

Publisher:

Published: 2013

Total Pages:

ISBN-13:

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"34 million individuals worldwide are currently infected with the human immunodeficiency virus (HIV). Following the isolation of HIV, the first direct acting antiviral was approved. This particular compound targeted the reverse transcriptase (RT) enzyme, and resulted in a decrease in viral load in patients receiving therapy. Shortly after, it was demonstrated that the use of a single inhibitor quickly leads to the selection of drug resistance mutations, and subsequently to viral rebound. To combat this, combination therapy was introduced which targets multiple proteins, including RT and other enzymes, such as HIV integrase (IN). RT reverse transcribes the single-stranded RNA genome into double-stranded DNA, followed by the insertion of this product into the host genome by IN. A detailed understanding of the mechanism of action of the drug target, the inhibitor itself, as well as how resistance-associated mutations act, is necessary to improve current therapies. Typically, drug resistance mutations occur near the binding site of the inhibitor. However, the first part of this thesis examines a mutation in HIV-1 RT, N348I, which confers, and is distant from the binding site of, both nucleoside reverse transcriptase inhibitors, and non-nucleoside reverse transcriptase inhibitors (NNRTIs). We describe the specific stage during HIV-1 reverse transcription where N348I exerts its effect on the first-generation NNRTI, neverapine, but not efavirenz. RT is targeted by multiple classes of drugs, while only one inhibitor, raltegravir (RAL), is currently approved to target HIV-1 IN. The development of IN inhibitors has been hindered by the lack of structural information with respect to the enzyme, IN-DNA interactions, as well as the mechanism of action of RAL. We pinpoint several interactions between HIV-1 IN and its DNA substrate, which are important for enzymatic activity. We also elucidate the contributions of these interactions in the context of inhibition by RAL, with respect to the wild-type enzyme, as well as the IN drug resistance mutation N155H. Following the acquisition of primary resistance mutations in HIV-1 IN, such as N155H, secondary mutations often appear. These mutations compensate for decreased viral fitness, and can potentially amplify resistance. The final study presented herein, examines whether one such mutation recently identified in RAL-experienced patients at position N117, confers resistance to RAL." --

Biochemical and Biophysical Characterization of the Interaction Between Human Immunodeficiency Virus Type 1 Integrase and Capsid
Biochemical and Biophysical Characterization of the Interaction Between Human Immunodeficiency Virus Type 1 Integrase and Capsid

Author: Xiaowen Xu

Publisher:

Published: 2017

Total Pages: 71

ISBN-13:

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Upon infection of host cells by human immunodeficiency virus type 1 (HIV-1), the viral membrane fuses with the cell plasma membrane and the viral core is released into the cytoplasm, where uncoating of viral capsid (CA) core takes place. Numerous studies have shown that optimal core stability is a key determinant in the uncoating. However, the underlying factors and mechanisms governing uncoating are poorly understood. We have previously shown that HIV-1 integrase (IN) is involved in uncoating of the viral core and required for optimal core stability. In this study, we have demonstrated that IN interacts with in vitro assembled CA tubes and preferentially binds to CA hexamers. Our biochemical and biophysical analyses have further determined that the reaching dimer of IN is required for interacting with CA hexamer. Moreover, we show that both NTD and CTD of IN are involved in interacting with CA assemblies, while IN NTD contributes to the preferential recognition towards CA hexamers. This project provides useful information to understand crucial but yet poorly characterized processes during HIV-1 life cycle. By characterizing the IN-CA interaction, we may provide a mechanical basis for the requirement of IN during HIV-1 uncoating. The IN-CA interaction also indicates that IN may play a role during late stage of HIV-1 replication, as recent studies in the field suggested that IN may be involved in virion maturation. Finally, this finding highlights the potential for exploiting the CA and IN interaction as a new therapeutic target.

HIV-1 Integrase
HIV-1 Integrase

Author: Nouri Neamati

Publisher: John Wiley & Sons

Published: 2011-08-10

Total Pages: 710

ISBN-13: 1118015363

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This book comprehensively covers the mechanisms of action and inhibitor design for HIV-1 integrase. It serves as a resource for scientists facing challenging drug design issues and researchers in antiviral drug discovery. Despite numerous review articles and isolated book chapters dealing with HIV-1 integrase, there has not been a single source for those working to devise anti-AIDS drugs against this promising target. But this book fills that gap and offers a valuable introduction to the field for the interdisciplinary scientists who will need to work together to design drugs that target HIV-1 integrase.

Human Immunodeficiency Virus Reverse Transcriptase
Human Immunodeficiency Virus Reverse Transcriptase

Author: Stuart LeGrice

Publisher: Springer Science & Business Media

Published: 2013-07-23

Total Pages: 358

ISBN-13: 1461472911

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The Reverse Transcriptase (RT) of Human Immunodeficiency Virus Type 1 (HIV-1) arguably ranks amongst one of the most extensively studied retroviral enzymes. Heterologous expression and purification of HIV-1 RT in the early eighties, approval of the first nucleoside analogue RT inhibitor (NRTI) in 1987, discovery of resistance to RT inhibitors, approval of the first non-nucleoside analogue RT inhibitor (NNRTI) in 1996 and the various crystal structures of RT with and without bound substrate(s) and/or inhibitors represent only a few of the important milestones that describe the a bench-to-bedside success in the continuing effort to combat HIV-1 infection and its consequences. Nucleoside and nonnucleoside RT inhibitors remain important components in frequently used drug regimens to treat the infection. RT inhibitors also play important roles in recently validated strategies to prevent transmission of the virus. The relevance of HIV-1 RT as a drug target has simultaneously triggered interest in basic research studies aimed at providing a more detailed understanding of interactions between proteins, nucleic acids, and small molecule ligands in general terms. In light of the ever-growing knowledge on structure and function of HIV-1 RT, this enzyme serves as a valuable “model system” in efforts to develop novel experimental tools and to explain biochemical processes. This monograph is designed to provide an overview of important aspects in past and current HIV-1 RT research, with focus on mechanistic aspects and translation of knowledge into drug discovery and development. The first section includes chapters with emphasis placed on the coordination of the RT-associated DNA polymerase and ribonuclease H (RNase H) activities. The second covers mechanisms of action and future perspectives associated with NRTIs and NNRTIs, while the third section includes chapters focusing on novel strategies to target the RT enzyme. Chapters of the final part are intended to discuss mechanisms involved in HIV variability and the development of drug resistance. We hope that these contributions will stimulate interest, and encourage research aimed at the development of novel RT inhibitors. The lack of bona fide RNase H inhibitors with potent antiviral activity provides an example for challenges and opportunities in the field.

Characterizing Interactions of HIV-1 Integrase with Viral DNA and the Cellular Cofactor LEDGF
Characterizing Interactions of HIV-1 Integrase with Viral DNA and the Cellular Cofactor LEDGF

Author: Christopher J. McKee

Publisher:

Published: 2010

Total Pages: 115

ISBN-13:

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Abstract: A hallmark of retroviral replication is the permanent integration of the viral genome into the host cell genome. This integration is mediated by the viral enzyme integrase (IN) which is bound to the ends of the viral DNA in the context of a large nucleoprotein complex known as the pre-integration complex (PIC). Using the two catalytic activities of IN, the viral DNA ends are first processed then used for a strand transfer reaction that simultaneously breaks the host DNA backbone and ligates the viral genome into it. For HIV-1, the site of integration into the host genome is non-random and occurs most often in active transcription units. This bias is likely explained by the recruitment of pre-integration complexes to chromatin via interactions between IN and the cellular protein Lens Epithelium-Derived Growth Factor (LEDGF). Failure of either enzymatic reaction or failure of the PIC to engage chromatin is a replicative dead end for the virus. Integrase has long been considered an attractive therapeutic target due to its essential role in replication and its lack of a cellular counterpart. The first-in-class HIV IN inhibitors block integration by binding specifically to the IN-viral DNA complex, displacing the viral DNA ends from the active site and effectively preventing strand transfer. The specificity of this inhibition led to the rapid emergence of HIV-1 phenotypes resistant to all available strand transfer inhibitors and highlighted the need for new classes of IN inhibitor. The development of these new inhibitors will be driven by an improved understanding of IN structure and of the sequence of pre-integration events. In spite of tremendous efforts, no high-resolution structural data is available for the full-length HIV-1 IN. The following text describes my efforts to characterize three critical molecular interactions that determine the fate of HIV-1 integration: IN with viral DNA, IN with the cellular cofactor LEDGF, and LEDGF with chromatin. Using innovative mass spectrometric footprinting techniques I have mapped and validated biologically essential contacts at IN-viral DNA, IN-IN, and IN-LEDGF interfaces. These experiments provided structural details that revealed previously undescribed changes in integrase conformation and subunit dynamics upon binding viral DNA and LEDGF, respectively. Finally, I have explored epigenetic modifications in chromatin that are recognized by LEDGF. Specific binding of LEDGF to regions featuring these modifications could explain, at least in part, the targeting of active transcription units for HIV-1 integration in infected cells.