HIV-1 Integrase-DNA Recognition Mechanisms

Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review

Highly Potent Synthetic Polyamides, Bisdistamycins, and Lexitropsins as Inhibitors of Human Immunodeficiency Virus Type 1 Integrase

ABSTRACT Alignment of the available human immunodeficiency virus type 1 (HIV-1) viral DNA termini [U5 and U3 long terminal repeats (LTRs)] shows a high degree of conservation and the presence of a stretch of five or six consecutive adenine and thymine (AT) sequences ϳ10 nucleotides away from each LTR end. A series of AT-selective minor-groove binders, including distamycin and bisdistamycins, bisnetropsins, novel lexitropsins, and the classic monomeric DNA binders Hoechst 33258, 4Ј-diamino-2-phenylindole, pentamidine, berenil, spermine, and spermidine, were tested for their inhibitory activities against HIV-1 integrase (IN). Although netropsin, distamycin, and all other monomeric DNA binders showed weak activities in the range of 50 -200 M, some of the polyamides, bisdistamycins, and lexitropsins were remarkably active at nanomolar concentrations. Bisdistamycins were 200 times less potent when the conserved AAAAT stretch present in the U5 LTR was replaced with GGGGG, consistent with the preferred binding of these drugs to AT sequences. DNase I footprinting of the U5 LTR further demonstrated the selectivity of these bisdistamycins for the conserved AT sequence. The tested compounds were more potent in Mg ϩ2 than in Mn ϩ2 and inhibited IN 50 -212 deletion mutant in disintegration assays and the formation of IN/DNA complexes. The lexitropsins also were active against HIV-2 IN. Some of the synthetic polyamides exhibited significant antiviral activity. Taken together, these data suggest that selective targeting of the U5 and U3 ends of the HIV-1 LTRs can inhibit IN function. Polyamides might represent new leads for the development of antiviral agents against acquired immune deficiency syndrome

Role of raltegravir in the management of HIV-1 infection

The development of multiple agents with potent antiretroviral activity against HIV has ushered in a new age of optimism in the management of patients infected with the virus. However, the viruses’ dynamic ability to develop resistance against these agents necessitates the investigation of novel targets for viral suppression. Raltegravir represents a first-in-class agent targeting the HIV integrase enzyme, which is responsible for integration of virally encoded DNA into the host genome. Over the last 5 years, clinical trials data has demonstrated an increasing role for raltegravir in the management of both treatment-experienced and treatment-naïve HIV-1-infected patients. This review focuses on the evidence supporting raltegravir’s efficacy in an array of clinical settings. Other HIV-1 integrase inhibitors in development are also briefly discussed

Proteolytic cleavage events in the maturation of HIV-1 reverse transcriptase

Each of the HIV-1 pol-encoded enzymes, protease (PR), reverse transcriptase (RT) and integrase (IN) are released during virion maturation and are active only as dimers. Of the three, only RT comprises subunits of different mass. RT in mature infectious virions is a heterodimer of 66 kDa and 51 kDa subunits, even though its gene encodes a 66 kDa protein. The RT p51 subunit is formed by HIV-1 PR-catalyzed cleavage of RT p66, resulting in the removal of a ribonuclease H (RNH) domain. Given the existence of completely active recombinant p66/66 RT homodimers and alternative RT oligomers in other retroviruses, the apparent need for p66/51 RT heterodimers in the HIV-1 virion is unclear. To determine why the generation of active viral RT requires three processing events, we introduced mutations in the p51-RNH and RT-IN protease cleavage sites of an infectious HIV-1 molecular clone. Mutation of the RT-IN cleavage site had no effect on the activity or proteolytic stability of the p98/51 RT product, although infectivity was severely attenuated. This result was similar to findings previously reported for the PR-RT cleavage site. Surprisingly, mutation of the internal p51-RNH cleavage site did not increase RT p66 content, but resulted in attenuated virus containing greatly decreased levels of RT that was primarily RT p51. We further identified a compensatory second-site mutation T477A, found to restore RT activity and processing to p66/51 RT when introduced in the background of p51-RNH cleavage site mutations. These studies demonstrate that cleavage of the internal p51-RNH junction, not the flanking N-terminal or C-terminal junctions is essential for proteolytic stability of functional RT during virion maturation. These findings further emphasize the importance of the RNH domain in regulating proteolytic generation of p66/51 RT. The overall need for the RT heterodimer is attributable to the generation of its subunits. Formation of the 51 kDa subunit or cleavage of the p51-RNH junction is essential for RT stability in the virion, whereas formation of the 66 kDa subunit is important for efficient viral replication

Design and synthesis of HIV-1 integrase inhibitors

In recent years, HIV-1 integrase (IN) has emerged as an attractive target for the treatment of human immunodeficiency virus type 1 (HIV-1), the causative pathogen of acquired immuno-deficiency syndrome (AIDS). Several classes of IN inhibitors are known but many of these compounds are toxic, do not show antiviral activity or display decreased potency. Therefore, new classes of potent IN inhibitors are desperately needed. The b-diketo (b-DK) class of compounds has emerged as one of the most successful classes of IN inhibitors. Although several b-DK inhibitors with potent antiviral activity are known, compounds containing b-DK motifs have limitations in drug development. The overall objective of this dissertation was to design and synthesize a novel series of IN inhibitors that retain the favorable characteristics of the b-DK scaffolds but are devoid of the undruggable properties. The design of the target molecules was established from crystal structure-based correlation and structure-activity relationship studies, which led to scaffolds containing three specific functional groups. Each molecule was designed to contain the core functional motif (a,b-diketoamide), optimal aryl groups (3-benzylphenyl or substituted 3-benzylphenyl) and a terminal group (proton donor or acceptor or amphoteric functional groups) in a planar or near planar configuration. Several oxalamate containing compounds were successfully designed and synthesized; and many of these synthetic analogs were sent to be screened for inhibitory activity against HIV-1 IN. The synthetic analogs described herein may elicit alluring antiviral activity, serve as potential lead molecules for future optimizations and ultimately elucidate mechanistic insight into HIV-1 IN inhibition

Exploring the Early Events in the HIV-1 Life Cycle: From Post-Entry Restriction to Nuclear Import

Early events in the viral replication cycle of the human immunodeficiency virus (HIV) determine the virus\u27 ability to sustain a persistent infection in susceptible host species. However, many of the molecular machinations through which the virus progresses during its earliest moments in the cell\u27s interior remain enigmatic. Similar to other virus families, HIV virions are packed with a specific assortment of viral and cellular proteins that allow them to carry out their unique program for replication inside of target cells. One such protein that is present in incoming virions, HIV-1 integrase, is an enzyme that facilitates the fusion of the viral genome and the host genomic DNA in the nucleus, thereby making HIV a permanent fixture in infected individuals. We have discovered that, in addition to its role in catalyzing integration, the HIV-1 integrase plays key part in transporting the viral DNA through the cytoplasm to the nucleus during a process known as viral nuclear import. Viral nuclear import is a key early step in the viral life cycle directly preceding integration that has to date been exploited as a target for antiretroviral therapy. Our recent studies indicate that a small stretch of sequence within the carboxyl terminus of the HIV-1 integrase protein contains a nuclear localization signal (NLS) that is indispensable for viral nuclear import and therefore absolutely required for productive HIV infection. Importantly, we have identified several mutations (NLS mutants) within this region that can disrupt nuclear import and therefore completely stall HIV infection. In addition, alignment of all the known subtypes of HIV within the region specified by our NLS mutants indicates that the virus seldom changes its sequence in this part of its genome, suggesting that this domain is a critical factor that it must preserve for optimal infection. Thus, a major focus of this thesis is to clarify the role that the HIV integrase plays in nuclear import. In turn, these analyses may provide a rational basis for the design of new experimental drug screening systems dedicated to finding inhibitors that block HIV-1 nuclear import and curtail HIV infection. A secondary aim of this work is to understand the mechanism of species-specific restriction of lentivirus infection in primates mediated by endogenous host cell restriction factors such as Lvl/TRIM5-a. Lastly, a potential role for non-pathogenic viral DNA episomal circles formed from integration-defective HIV virions as a novel platform for gene therapy based vaccines is discussed

Probing the expanded architecture of the lentvirial intasome

Integration of viral DNA into a host chromosome, by action of the viral enzyme integrase (IN), is an essential step of the retroviral lifecycle. To fulfil its function, IN assembles into a multimer on the viral DNA ends, forming a highly stable nucleoprotein complex known as the intasome. The intasome architecture varies between the retroviral genera, and the maedi-visna virus (MVV) intasome contains a homo-hexadecamer (tetramer-of-tetramers) of IN. The conserved intasome core, observed in all structurally characterized retroviral intasomes, is formed between a pair of MVV IN tetramers, each providing one active site, and is completed by the insertions of the synaptic C-terminal domains (CTDs) donated by a pair of flanking IN tetramers. It was argued that this configuration is necessitated by the propensity of lentiviral INs to form tetramers in solution and the α-helical structure of the linkers connecting the catalytic core domain (CCD) and the CTD. Within the MVV IN hexadecamer, a pair of CTD tetrads bridge the IN tetramers by forming intra- and inter-tetramer interactions. Using site directed mutagenesis, the importance of these distinctive structural features was probed. The mutations disrupting the CTD-CTD interfaces or destabilizing the α-helical configuration of the CCD-CTD linkers perturb the ability of MVV IN to form multimers, assemble into stable intasomes and strongly affect its strand transfer activity in vitro. Moreover, these mutations strongly compromised infectivity of single-cycle MVV vector in cells. Lentiviral integration distinctively favours actively transcribing genes, which is facilitated by the interaction of IN with LEDGF/p75, a chromatin-bound adaptor protein. The presence of LEDGF/p75 was essential to observe MVV IN strand transfer activity or intasome assembly in vitro. To determine the importance of LEDGF/p75 for integration in the context of viral integration in cells, infectivity of MVV-derived vector in LEDGF-knockout cells was tested. Although ablation of the host factor in human and sheep cells did not lead to a reproducible reduction of infectivity, it led to a notable shift in integration pattern, away from the usual gene bodies and transcription units. Collectively, these observations indicate that the hexadecameric architecture is critical for the MVV IN function and suggest that LEDGF/p75 is relevant in targeting its integration towards favored regions of host genome

Synteza wybranych związków zawierających układ azanaftalenowy jako potencjalnych inhibitorów integrazy HiV oraz środków antyproliferacyjnych

Decydujące znaczenie w cyklu replikacyjnym wirusa HIV mają trzy białka enzymatyczne: proteaza, odwrotna transkryptaza oraz integraza. Lata badań prowadzonych w wielu ośrodkach na całym świecie zaowocowały wprowadzeniem do terapii kilkunastu leków, które są inhibitorami dwóch pierwszych enzymów. Niestety do dziś nie zarejestrowano leku aktywnego wobec integrazy. Kilka potencjalnych inhibitorów pozostaje w fazie badań klinicznych. Jednym z kierunków badań realizowanych w Zakładzie Chemii Organicznej Instytutu Chemii Uniwersytetu Śląskiego jest poszukiwanie związków aktywnych wobec integrazy wirusa HIV. Testowane są też inne kierunki aktywności biologicznej syntezowanych połączeń, w szczególności aktywność antyproliferacyjna względem komórek nowotworowych oraz fungistatyczność. Celem niniejszej pracy była synteza wybranych związków zawierających układ azanaftalenowy , w poszukiwaniu nowych potencjalnych inhibitorów integrazy wirusa HIV, szczególnie układy chinolinowy i chinazolinowy oraz pochodne styrylowe i amidowe. Kilka z otrzymanych związków zostało przetestowanych w kierunku aktywności antyproliferacyjnej