Pharmacological advances in anti-retroviral therapy for human immunodeficiency virus-1 infection: A comprehensive review.

The discovery of anti-retroviral (ARV) drugs over the past 36 years has introduced various classes, including nucleoside/nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitor, fusion, and integrase strand transfer inhibitors inhibitors. The introduction of combined highly active anti-retroviral therapies in 1996 was later proven to combat further ARV drug resistance along with enhancing human immunodeficiency virus (HIV) suppression. As though the development of ARV therapies was continuously expanding, the variation of action caused by ARV drugs, along with its current updates, was not comprehensively discussed, particularly for HIV-1 infection. Thus, a range of HIV-1 ARV medications is covered in this review, including new developments in ARV therapy based on the drug's mechanism of action, the challenges related to HIV-1, and the need for combination therapy. Optimistically, this article will consolidate the overall updates of HIV-1 ARV treatments and conclude the significance of HIV-1-related pharmacotherapy research to combat the global threat of HIV infection.

The Impact of Lens Epithelium-Derived Growth Factor p75 Dimerization on Its Tethering Function.

The transcriptional co-activator lens epithelium-derived growth factor/p75 (LEDGF/p75) plays an important role in the biology of the cell and in several human diseases, including MLL-rearranged acute leukemia, autoimmunity, and HIV-1 infection. In both health and disease, LEDGF/p75 functions as a chromatin tether that interacts with proteins such as MLL1 and HIV-1 integrase via its integrase-binding domain (IBD) and with chromatin through its N-terminal PWWP domain. Recently, dimerization of LEDGF/p75 was shown, mediated by a network of electrostatic contacts between amino acids from the IBD and the C-terminal α6-helix. Here, we investigated the functional impact of LEDGF/p75 variants on the dimerization using biochemical and cellular interaction assays. The data demonstrate that the C-terminal α6-helix folds back in cis on the IBD of monomeric LEDGF/p75. We discovered that the presence of DNA stimulates LEDGF/p75 dimerization. LEDGF/p75 dimerization enhances binding to MLL1 but not to HIV-1 integrase, a finding that was observed in vitro and validated in cell culture. Whereas HIV-1 replication was not dependent on LEDGF/p75 dimerization, colony formation of MLLr-dependent human leukemic THP-1 cells was. In conclusion, our data indicate that intricate changes in the quaternary structure of LEDGF/p75 modulate its tethering function.

GS-9822, a preclinical LEDGIN candidate, displays a block-and-lock phenotype in cell culture.

The ability of HIV to integrate into the host genome and establish latent reservoirs is the main hurdle preventing an HIV cure. LEDGINs are small-molecule integrase inhibitors that target the binding pocket of LEDGF/p75, a cellular cofactor that substantially contributes to HIV integration site selection. They are potent antivirals that inhibit HIV integration and maturation. In addition, they retarget residual integrants away from transcription units and towards a more repressive chromatin environment. As a result, treatment with the LEDGIN CX14442 yielded residual provirus that proved more latent and more refractory to reactivation, supporting the use of LEDGINs as research tools to study HIV latency and a functional cure strategy. In this study we compared GS-9822, a potent, pre-clinical lead compound, with CX14442 with respect to antiviral potency, integration site selection, latency and reactivation. GS-9822 was more potent than CX14442 in most assays. For the first time, the combined effects on viral replication, integrase-LEDGF/p75 interaction, integration sites, epigenetic landscape, immediate latency and latency reversal was demonstrated at nanomolar concentrations achievable in the clinic. GS-9822 profiles as a preclinical candidate for future functional cure research.

BET-Independent Murine Leukemia Virus Integration Is Retargeted In Vivo and Selects Distinct Genomic Elements for Lymphomagenesis.

Moloney murine leukemia virus (MLV) infects BALB/c mice and induces T-cell lymphoma in mice. Retroviral integration is mediated by the interaction of the MLV integrase (IN) with members of the bromodomain and extraterminal motif (BET) protein family (BRD2, BRD3, and BRD4). The introduction of the W390A mutation into MLV IN abolishes the BET interaction. Here, we compared the replication of W390A MLV to that of wild-type (WT) MLV in adult BALB/c mice to study the role of BET proteins in replication, integration, and tumorigenesis in vivo. Comparing WT and W390A MLV infections revealed similar viral loads in the blood, thymus, and spleen cells. Interestingly, W390A MLV integration was retargeted away from GC-enriched genomic regions. However, both WT MLV- and W390A MLV-infected mice developed T-cell lymphoma after similar latencies represented by an enlarged thymus and spleen and multiorgan tumor infiltration. Integration site sequencing from splenic tumor cells revealed clonal expansion in all WT MLV- and W390A MLV-infected mice. However, the integration profiles of W390A MLV and WT MLV differed significantly. Integrations were enriched in enhancers and promoters, but compared to the WT, W390A MLV integrated less frequently into enhancers and more frequently into oncogene bodies such as Notch1 and Ppp1r16b. We conclude that host factors direct MLV in vivo integration site selection. Although BET proteins target WT MLV integration preferentially toward enhancers and promoters, insertional lymphomagenesis can occur independently from BET, likely due to the intrinsically strong enhancer/promoter of the MLV long terminal repeat (LTR). IMPORTANCE In this study, we have shown that the in vivo replication of murine leukemia virus happens independently of BET proteins, which are key host determinants involved in retroviral integration site selection. This finding opens a new research line in the discovery of alternative viral or host factors that may complement the dominant host factor. In addition, our results show that BET-independent murine leukemia virus uncouples insertional mutagenesis from gene enhancers, although lymphomagenesis still occurs despite the lack of an interaction with BET proteins. Our findings also have implications for the engineering of BET-independent MLV-based vectors for gene therapy, which may not be a safe alternative.

Design, synthesis, in silico studies, and antiproliferative evaluations of novel indolin-2-one derivatives containing 3-hydroxy-4-pyridinone fragment.

Keeping in view the pharmacological properties of indolinones as promising scaffold as kinase inhibitors, herein, a novel series of 3-hydrazonoindolin-2-one derivatives bearing 3-hydroxy-4-pyridinone moiety were synthesized, studied by molecular docking, and fully characterized by spectroscopic techniques. All the prepared compounds were evaluated for their cytotoxicity attributes against a panel of tumor cell lines, including non-small cell lung cancer (A549), breast carcinoma (MCF-7), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). They displayed moderate to promising antiproliferative effects toward A549 and MCF-7 cells but remarkable results against AML and CML. Especially, compound 10k was found to be more potent against AML (EC50 = 0.69 µM) compare to the other halogen-substituted derivatives. FMS-like tyrosine kinase 3 (FLT3) is known to be expressed in AML cancer cells. The molecular docking studies demonstrated that our prepared compounds were potentially bound to AML active site through essential H-bond and other vital interactions with critical binding residues.

Novel CFTR modulator combinations maximise rescue of G85E and N1303K in rectal organoids.

Introduction: Cystic fibrosis (CF) is a severe monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Several types of CFTR modulators (correctors/potentiators) have been developed to overcome protein dysfunction associated with these mutations. CFTR modulator therapy is now available for the major CF-causing mutations; however, 10% of people with CF remain without causal treatments. By combining investigational and market-approved CFTR modulators, we aimed to maximise functional rescue of iva-, luma- and tezacaftor refractory mutants G85E and N1303K. Methods: We used the well-established forskolin-induced swelling (FIS) in primary rectal organoids to assess responses to different CFTR corrector and potentiator types. The FIS analysis was performed with brightfield microscopy, allowing both 1-h and 24-h follow-up. Corrector and potentiator activity of elexacaftor was investigated. Results: For G85E, maximal rescue was observed by a combination of elexacaftor and corr4a. For N1303K, the quadruple combination teza-elexa-ivacaftor with apigenin was required to obtain a rescue similar to that of luma-ivacaftor rescued F508del. Elexacaftor rescued G85E and N1303K by different mechanisms, with chronic corrector effects on G85E and acute potentiation of N1303K only in the presence of ivacaftor. Synergy in N1303K rescue for iva-elexacaftor and apigenin suggests at least three potentiator mechanisms for this mutant. 24-h FIS identified ivacaftor as the main CFTR modulator for N1303K and elexacaftor and apigenin as co-potentiators. Conclusions: Novel combinations of CFTR modulators can further improve functional rescue of G85E and N1303K in rectal organoids, although for N1303K, more effective CFTR modulators are still needed.

Determinants of Retroviral Integration and Implications for Gene Therapeutic MLV-Based Vectors and for a Cure for HIV-1 Infection.

To complete their replication cycle, retroviruses need to integrate a DNA copy of their RNA genome into a host chromosome. Integration site selection is not random and is driven by multiple viral and cellular host factors specific to different classes of retroviruses. Today, overwhelming evidence from cell culture, animal experiments and clinical data suggests that integration sites are important for retroviral replication, oncogenesis and/or latency. In this review, we will summarize the increasing knowledge of the mechanisms underlying the integration site selection of the gammaretrovirus MLV and the lentivirus HIV-1. We will discuss how host factors of the integration site selection of retroviruses may steer the development of safer viral vectors for gene therapy. Next, we will discuss how altering the integration site preference of HIV-1 using small molecules could lead to a cure for HIV-1 infection.

LEDGF/p75-mediated chemoresistance of mixed-lineage leukemia involves cell survival pathways and super enhancer activators.

MLL is an aggressive subtype of leukemia with a poor prognosis that mostly affects pediatric patients. MLL-rearranged fusion proteins (MLLr) induce aberrant target gene expression resulting in leukemogenesis. MLL and its fusions are tethered to chromatin by LEDGF/p75, a transcriptional co-activator that specifically recognizes H3K36me2/3. LEDGF/p75 is ubiquitously expressed and associated with regulation of gene expression, autoimmune responses, and HIV replication. LEDGF/p75 was proven to be essential for leukemogenesis in MLL. Apart from MLL, LEDGF/p75 has been linked to lung, breast, and prostate cancer. Intriguingly, LEDGF/p75 interacts with Med-1, which co-localizes with BRD4. Both are known as co-activators of super-enhancers. Here, we describe LEDGF/p75-dependent chemoresistance of MLLr cell lines. Investigation of the underlying mechanism revealed a role of LEDGF/p75 in the cell cycle and in survival pathways and showed that LEDGF/p75 protects against apoptosis during chemotherapy. Remarkably, LEDGF/p75 levels also affected expression of BRD4 and Med1. Altogether, our data suggest a role of LEDGF/p75 in cancer survival, stem cell renewal, and activation of nuclear super enhancers.

CRISPR/Cas9-Induced Mutagenesis Corroborates the Role of Transportin-SR2 in HIV-1 Nuclear Import.

To infect nondividing cells, HIV-1 needs to cross the nuclear membrane. The importin transportin-SR2 (TRN-SR2 or transportin-3) has been proposed to mediate HIV-1 nuclear import, but the detailed mechanism remains unresolved. The direct interaction of TRN-SR2 with HIV-1 integrase (IN) has been proposed to drive HIV-1 nuclear import. Alternatively, TRN-SR2 may play an indirect role by mediating nuclear import of cleavage and polyadenylation specificity factor 6 (CPSF6). To unravel the role of TRN-SR2, we designed CRISPR/Cas9 guide RNAs targeting different exons of TNPO3. Although this approach failed to generate full knockouts, monoallelic knockout clones were generated with indel mutations. HIV-1 replication was hampered in those clones at the level of HIV-1 nuclear import without an effect on the cellular distribution of the TRN-SR2 cargoes CPSF6 or alternative splicing factor1/pre-mRNA splicing factor SF2 (ASF/SF2). Recombinant ΔV105 TRN-SR2 expressed in clone 15.15 was 2-fold impaired for interaction with HIV-1 IN and classified as an interaction mutant. Our data support a model whereby TRN-SR2 acts as a cofactor of HIV-1 nuclear import without compromising the nuclear import of cellular cargoes. CRISPR/Cas9-induced mutagenesis can be used as a method to generate interface mutants to characterize host factors of human pathogens. IMPORTANCE Combination antiretroviral therapy (cART) effectively controls HIV-1 by reducing viral loads, but it does not cure the infection. Lifelong treatment with cART is a prerequisite for sustained viral suppression. The rapid emergence of drug-resistant viral strains drives the necessity to discover new therapeutic targets. The nuclear import of HIV-1 is crucial in the HIV-1 replication cycle, but the detailed mechanism remains incompletely understood. This study provides evidence that TRN-SR2 directly mediates HIV-1 nuclear import via the interaction with HIV-1 integrase. The interaction between those proteins is therefore a promising target toward a rational drug design which could lead to new therapeutic strategies due to the bottleneck nature of HIV-1 nuclear import.

LEDGINs, Inhibitors of the Interaction Between HIV-1 Integrase and LEDGF/p75, Are Potent Antivirals with a Potential to Cure HIV Infection.

A permanent cure remains the greatest challenge in the field of HIV research. In order to reach this goal, a profound understanding of the molecular mechanisms controlling HIV integration and transcription is needed. Here we provide an overview of recent advances in the field. Lens epithelium-derived growth factor p75 (LEDGF/p75), a transcriptional coactivator, tethers and targets the HIV integrase into transcriptionally active regions of the chromatin through an interaction with the epigenetic mark H3K36me2/3. This finding prompted us to propose a "block-and-lock" strategy to retarget HIV integration into deep latency. A decade ago, we pioneered protein-protein interaction inhibitors for HIV and discovered LEDGINs. LEDGINs are small molecule inhibitors of the interaction between the integrase binding domain (IBD) of LEDGF/p75 and HIV integrase. They modify integration site selection and therefore might be molecules with a "block-and-lock" mechanism of action. Here we will describe how LEDGINs may become part in the future functional cure strategies.

Role of Transportin-SR2 in HIV-1 Nuclear Import.

The HIV replication cycle depends on the interaction of viral proteins with proteins of the host. Unraveling host-pathogen interactions during the infection is of great importance for understanding the pathogenesis and the development of antiviral therapies. To date HIV uncoating and nuclear import are the most debated steps of the HIV-1 replication cycle. Despite numerous studies during past decades, there is still much controversy with respect to the identity and the role of viral and host factors involved in these processes. In this review, we provide a comprehensive overview on the role of transportin-SR2 as a host cell factor during active nuclear transport.

Towards a Functional Cure of HIV-1: Insight Into the Chromatin Landscape of the Provirus.

Despite potent combination antiretroviral therapy, HIV-1 infection persists due to irreversible integration of the virus in long-living cells of the immune system. The main focus of HIV-1 cure strategies has been on HIV-1 eradication, yet without great success so far. Therefore, HIV-1 remission or a functional cure, whereby the virus is silenced rather than eradicated, is considered as an alternative strategy. Elite controllers, individuals who spontaneously control HIV-1, may point us the way toward a functional HIV-1 cure. In order to achieve such a cure, a profound understanding of the mechanisms controlling HIV-1 expression and silencing is needed. In recent years, evidence has grown that the site of integration as well as the chromatin landscape surrounding the integration site affects the transcriptional state of the provirus. Still, at present, the impact of integration site selection on the establishment and maintenance of the HIV-1 reservoirs remains poorly understood. The discovery of LEDGF/p75 as a binding partner of HIV-1 integrase has led to a better understanding of integration site selection. LEDGF/p75 is one of the important determinants of integration site selection and targets integration toward active genes. In this review, we will provide an overview of the most important determinants of integration site selection. Secondly, we will discuss the chromatin landscape at the integration site and its implications on HIV-1 gene expression and silencing. Finally, we will discuss how interventions that affect integration site selection or modifications of the chromatin could yield a functional cure of HIV-1 infection.

Unlike Its Paralog LEDGF/p75, HRP-2 Is Dispensable for MLL-R Leukemogenesis but Important for Leukemic Cell Survival.

HDGF-related protein 2 (HRP-2) is a member of the Hepatoma-Derived Growth Factor-related protein family that harbors the structured PWWP and Integrase Binding Domain, known to associate with methylated histone tails or cellular and viral proteins, respectively. Interestingly, HRP-2 is a paralog of Lens Epithelium Derived Growth Factor p75 (LEDGF/p75), which is essential for MLL-rearranged (MLL-r) leukemia but dispensable for hematopoiesis. Sequel to these findings, we investigated the role of HRP-2 in hematopoiesis and MLL-r leukemia. Protein interactions were investigated by co-immunoprecipitation and validated using recombinant proteins in NMR. A systemic knockout mouse model was used to study normal hematopoiesis and MLL-ENL transformation upon the different HRP-2 genotypes. The role of HRP-2 in MLL-r and other leukemic, human cell lines was evaluated by lentiviral-mediated miRNA targeting HRP-2. We demonstrate that MLL and HRP-2 interact through a conserved interface, although this interaction proved less dependent on menin than the MLL-LEDGF/p75 interaction. The systemic HRP-2 knockout mice only revealed an increase in neutrophils in the peripheral blood, whereas the depletion of HRP-2 in leukemic cell lines and transformed primary murine cells resulted in reduced colony formation independently of MLL-rearrangements. In contrast, primary murine HRP-2 knockout cells were efficiently transformed by the MLL-ENL fusion, indicating that HRP-2, unlike LEDGF/p75, is dispensable for the transformation of MLL-ENL leukemogenesis but important for leukemic cell survival.

Molecular Mechanism of LEDGF/p75 Dimerization.

Dimerization of many eukaryotic transcription regulatory factors is critical for their function. Regulatory role of an epigenetic reader lens epithelium-derived growth factor/p75 (LEDGF/p75) requires at least two copies of this protein to overcome the nucleosome-induced barrier to transcription elongation. Moreover, various LEDGF/p75 binding partners are enriched for dimeric features, further underscoring the functional regulatory role of LEDGF/p75 dimerization. Here, we dissected the minimal dimerization region in the C-terminal part of LEDGF/p75 and, using paramagnetic NMR spectroscopy, identified the key molecular contacts that helped to refine the solution structure of the dimer. The LEDGF/p75 dimeric assembly is stabilized by domain swapping within the integrase binding domain and additional electrostatic "stapling" of the negatively charged α helix formed in the intrinsically disordered C-terminal region. We validated the dimerization mechanism using structure-inspired dimerization defective LEDGF/p75 variants and chemical crosslinking coupled to mass spectrometry. We also show how dimerization might affect the LEDGF/p75 interactome.

Correction: LEDGF/p75-Independent HIV-1 Replication Demonstrates a Role for HRP-2 and Remains Sensitive to Inhibition by LEDGINs.

[This corrects the article DOI: 10.1371/journal.ppat.1002558.].

Correction to: HIV-1 IN/Pol recruits LEDGF/p75 into viral particles.

An amendment to this paper has been published and can be accessed via the original article.

Correction to: LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions.

An amendment to this paper has been published and can be accessed via the original article.

Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects.

Background. The most common CFTR mutation, F508del, presents with multiple cellular defects. However, the possible multiple defects caused by many rarer CFTR mutations are not well studied. We investigated four rare CFTR mutations E60K, G85E, E92K and A455E against well-characterized mutations, F508del and G551D, and their responses to corrector VX-809 and/or potentiator VX-770. Methods. Using complementary assays in HEK293T stable cell lines, we determined maturation by Western blotting, trafficking by flow cytometry using extracellular 3HA-tagged CFTR, and function by halide-sensitive YFP quenching. In the forskolin-induced swelling assay in intestinal organoids, we validated the effect of tagged versus endogenous CFTR. Results. Treatment with VX-809 significantly restored maturation, PM localization and function of both E60K and E92K. Mechanistically, VX-809 not only raised the total amount of CFTR, but significantly increased the traffic efficiency, which was not the case for A455E. G85E was refractory to VX-809 and VX-770 treatment. Conclusions. Since no single model or assay allows deciphering all defects at once, we propose a combination of phenotypic assays to collect rapid and early insights into the multiple defects of CFTR variants.

Capsid-Labelled HIV To Investigate the Role of Capsid during Nuclear Import and Integration.

The HIV-1 capsid protein performs multiple roles in virus replication both during assembly and particle release and during virus trafficking into the nucleus. In order to decipher the roles of capsid protein during early replication, a reliable method to follow its intracellular distribution is required. To complement existing approaches to track HIV-1 capsid during early infection, we developed an HIV-1 imaging strategy, relying on viruses incorporating enhanced green fluorescent protein (eGFP)-tagged capsid (CA-eGFP) protein and mCherry-tagged integrase (IN-mCherry). Wild-type infectivity and sensitivity to inhibition by PF74 point to the functionality of CA-eGFP-containing complexes. Low numbers of CA-eGFP molecules were located inside the viral core and imported into the nucleus without significant loss in intensity. Less than 5% of particles carrying both CA-eGFP and IN-mCherry retained both labelled proteins after nuclear entry, implying a major uncoating event at the nuclear envelope dissociating IN and CA. Still, 20% of all CA-eGFP-containing complexes were detected in the nucleus. Unlike for IN-mCherry complexes, addition of the integrase inhibitor raltegravir had no effect on CA-eGFP-containing complexes, suggesting that these may be not (yet) competent for integration. Our imaging strategy offers alternative visualization of viral capsid trafficking and helps clarify its potential role during integration.IMPORTANCE HIV-1 capsid protein (CA) builds a conical shell protecting viral genomic RNA inside the virus particles. Upon entry into host cells, this shell disassembles in a process of uncoating, which is coordinated with reverse transcription of viral RNA into DNA. After uncoating, a portion of CA remains associated with the viral DNA and mediates its nuclear import and, potentially, integration into host DNA. In this study, we tagged CA with eGFP to follow its trafficking in host cells and address potential CA roles in the nucleus. We found that while functional viruses import the tagged CA into the nucleus, this capsid protein is not part of integration-competent complexes. The roles of nuclear CA thus remain to be established.