Human Immunodeficiency Virus Type 1 Integration Research Articles

HIV-1 Gag co-localizes with euchromatin histone marks at the nuclear periphery.

The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.

HIV-1 replication and latency are balanced by mTOR-driven cell metabolism.

Human Immunodeficiency virus type 1 (HIV-1) relies on host cell metabolism for all aspects of viral replication. Efficient HIV-1 entry, reverse transcription, and integration occurs in activated T cells because HIV-1 proteins co-opt host metabolic pathways to fuel the anabolic requirements of virion production. The HIV-1 viral life cycle is especially dependent on mTOR, which drives signaling and metabolic pathways required for viral entry, replication, and latency. As a central regulator of host cell metabolism, mTOR and its downstream effectors help to regulate the expression of enzymes within the glycolytic and pentose phosphate pathways along with other metabolic pathways regulating amino acid uptake, lipid metabolism, and autophagy. In HIV-1 pathogenesis, mTOR, in addition to HIF-1α and Myc signaling pathways, alter host cell metabolism to create an optimal environment for viral replication. Increased glycolysis and pentose phosphate pathway activity are required in the early stages of the viral life cycle, such as providing sufficient dNTPs for reverse transcription. In later stages, fatty acid synthesis is required for creating cholesterol and membrane lipids required for viral budding. Epigenetics of the provirus fueled by metabolism and mTOR signaling likewise controls active and latent infection. Acetyl-CoA and methyl group abundance, supplied by the TCA cycle and amino acid uptake respectively, may regulate latent infection and reactivation. Thus, understanding and exploring new connections between cellular metabolism and HIV-1 pathogenesis may yield new insights into the latent viral reservoirs and fuel novel treatments and cure strategies.

HIV-Quasipore: A Suite of HIV-1-Specific Nanopore Basecallers Designed to Enhance Viral Quasispecies Detection

Accounting for genetic variation is an essential consideration during human immunodeficiency virus type 1 (HIV-1) investigation. Nanopore sequencing preserves proviral integrity by passing long genomic fragments through ionic channels, allowing reads that span the entire genome of different viral quasispecies (vQS). However, this sequencing method has suffered from high error rates, limiting its utility. This was the inspiration behind HIV-Quasipore: an HIV-1-specific Nanopore basecaller suite designed to overcome these error rates through training with gold-standard data. It comprises three deep learning-based R9.4.1 basecallers: fast, high accuracy (HAC), super accuracy (SUP), and two R10.3 deep learning-based basecallers: HAC and SUP. This was accomplished by sequencing the HIV-1 J-Lat 10.6 cell line using Nanopore and high-quality Sanger techniques. Training significantly reduced basecaller error rates across all models (Student’s one-sided t-test; p = 0.0) where median error rates were 0.0189, 0.0018, 0.0008, for R9.4.1 HIV-Quasipore-fast, HAC, SUP, and 0.0007, 0.0011 for R10.3 HIV-Quasipore-HAC, and SUP, respectively. This improved quality reduces the resolution needed to accurately detect a vQS from 22.4 to 2.6% of total positional coverage for R9.4.1 HIV-Quasipore-fast, 6.9 to 0.5% for R9.4.1 HIV-Quasipore-HAC, 4.5 to 0.3% for R9.4.1 HIV-Quasipore-SUP, 8.0 to 0.3% for R10.3 HIV-Quasipore-HAC, and 5.4 to 0.3% for R10.3 HIV-Quasipore-SUP. This was consistently observed across the entire J-Lat 10.6 genome and maintained across longer reads. Reads with greater than 8,000 nucleotides display a median nucleotide identity of 0.9819, 0.9982, and 0.9991, for R9.4.1 HIV-Quasipore-fast, HAC, SUP, and 0.9993, 0.9988 for R10.3 HIV-Quasipore-HAC, and SUP, respectively. To evaluate the robustness of this tool against unseen data, HIV-Quasipore and their corresponding pretrained basecallers were used to sequence the J-Lat 9.2 cell line and a clinical isolate acquired from the Drexel Medicine CARES cohort. When sample reads were compared against their corresponding consensus sequence, all HIV-Quasipore basecallers displayed higher median alignment accuracies than their pretrained counterparts for both the J-Lat 9.2 cell line and clinical isolate. Using Nanopore sequencing can allow investigators to explore topics, such as vQS profile detection, HIV-1 integration site analysis, whole genome amplification, gene coevolution, and CRISPR-induced indel detection, among others. HIV-Quasipore basecallers can be acquired here: https://github.com/DamLabResources/HIV-Quasipore-basecallers.

Early Emergence and Long-Term Persistence of HIV-Infected T-Cell Clones in Children.

Little is known about the emergence and persistence of human immunodeficiency virus (HIV)-infected T-cell clones in perinatally infected children. We analyzed peripheral blood mononuclear cells (PBMCs) for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8 and 17.4 months of age and with viremia suppressed for 6 to 9 years. We obtained 8,662 HIV type 1 (HIV-1) integration sites from pre-ART samples and 1,861 sites from on-ART samples. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6 to 9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMCs infected ex vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B Our analyses indicate that, despite marked differences in T-cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.IMPORTANCE HIV-1 integrates its genome into the DNA of host cells. Consequently, HIV-1 genomes are copied with the host cell DNA during cellular division. Pediatric immune systems differ significantly from adults, consisting primarily of naive T cells, which have low expression of the HIV-1 coreceptor CCR5. This difference may result in variances in the number or size of infected cell clones that persist in children on ART. Here, we provide the most extensive analysis of the integration landscape of HIV-1 in children. We found that, despite the largely naive cell populations in neonatal immune systems, patterns of HIV-1 integration and the size of infected cell clones are as large and widespread as those in adults. Furthermore, selection for integration events in proto-oncogenes were observed in children despite early ART. If such cell clones persist for the life span of these individuals, there may be long-term consequences that have yet to be realized.

CPSF6-Dependent Targeting of Speckle-Associated Domains Distinguishes Primate from Nonprimate Lentiviral Integration.

Lentiviral DNA integration favors transcriptionally active chromatin. We previously showed that the interaction of human immunodeficiency virus type 1 (HIV-1) capsid with cleavage and polyadenylation specificity factor 6 (CPSF6) localizes viral preintegration complexes (PICs) to nuclear speckles for integration into transcriptionally active speckle-associated domains (SPADs). In the absence of the capsid-CPSF6 interaction, PICs uncharacteristically accumulate at the nuclear periphery and target heterochromatic lamina-associated domains (LADs) for integration. The integrase-binding protein lens epithelium-derived growth factor (LEDGF)/p75 in contrast to CPSF6 predominantly functions to direct HIV-1 integration to interior regions of transcription units. Though CPSF6 and LEDGF/p75 can reportedly interact with the capsid and integrase proteins of both primate and nonprimate lentiviruses, the extents to which these different viruses target SPADs versus LADs, as well as their dependencies on CPSF6 and LEDGF/p75 for integration targeting, are largely unknown. Here, we mapped 5,489,157 primate and nonprimate lentiviral integration sites in HEK293T and Jurkat T cells as well as derivative cells that were knocked out or knocked down for host factor expression. Despite marked preferences of all lentiviruses to target genes for integration, nonprimate lentiviruses only marginally favored SPADs, with corresponding upticks in LAD-proximal integration. While LEDGF/p75 knockout disrupted the intragenic integration profiles of all lentiviruses similarly, CPSF6 depletion specifically counteracted SPAD integration targeting by primate lentiviruses. CPSF6 correspondingly failed to appreciably interact with nonprimate lentiviral capsids. We conclude that primate lentiviral capsid proteins evolved to interact with CPSF6 to optimize PIC localization for integration into transcriptionally active SPADs.IMPORTANCE Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on the factors that guide provirus formation and associated chromatin environments that regulate transcriptional reactivation, and studies of orthologous infectious agents such as nonprimate lentiviruses can inform basic principles of HIV biology. HIV-1 utilizes the integrase-binding protein LEDGF/p75 and the capsid interactor CPSF6 to target speckle-associated domains (SPADs) for integration. However, the extent to which these two host proteins regulate integration of other lentiviruses is largely unknown. Here, we mapped millions of retroviral integration sites in cell lines that were depleted for LEDGF/p75 and/or CPSF6. Our results reveal that primate lentiviruses uniquely target SPADs for integration in a CPSF6-dependent manner.

Alpha-enolase in viral target cells suppresses the human immunodeficiency virus type 1 integration

BackgroundA protein exhibiting more than one biochemical function is termed a moonlighting protein. Glycolytic enzymes are typical moonlighting proteins, and these enzymes control the infection of various viruses. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and alpha-enolase (ENO1) are incorporated into human immunodeficiency virus type 1 (HIV-1) particles from viral producer cells and suppress viral reverse transcription independently each other. However, it remains unclear whether these proteins expressed in viral target cells affect the early phase of HIV-1 replication.ResultsHere we show that the GAPDH expression level in viral target cells does not affect the early phase of HIV-1 replication, but ENO1 has a capacity to suppress viral integration in viral target cells. In contrast to GAPDH, suppression of ENO1 expression by RNA interference in the target cells increased viral infectivity, but had no effect on the expression levels of the HIV-1 receptors CD4, CCR5 and CXCR4 and on the level of HIV-1 entry. Quantitative analysis of HIV-1 reverse transcription products showed that the number of copies of the late products (R/gag) and two-long-terminal-repeat circular forms of viral cDNAs did not change but that of the integrated (Alu-gag) form increased. In contrast, overexpression of ENO1 in viral target cells decreased viral infectivity owing to the low viral integration efficiency. Results of subcellular fractionation experiments suggest that the HIV integration at the nucleus was negatively regulated by ENO1 localized in the nucleus. In addition, the overexpression of ENO1 in both viral producer cells and target cells most markedly suppressed the viral replication.ConclusionsThese results indicate that ENO1 in the viral target cells prevents HIV-1 integration. Importantly, ENO1, but not GAPDH, has the bifunctional inhibitory activity against HIV-1 replication. The results provide and new insights into the function of ENO1 as a moonlighting protein in HIV-1 infection.

Nuclear architecture dictates HIV-1 integration site selection.

Long-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This region contains a series of cellular genes, which are preferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associated domains and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellular Nup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome. These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle.

Zinc finger protein designed to target 2-long terminal repeat junctions interferes with human immunodeficiency virus integration.

Integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host chromosome is a vital step in the HIV life cycle. The highly conserved cytosine-adenine (CA) dinucleotide sequence immediately upstream of the cleavage site is crucial for integrase (IN) activity. As this viral enzyme has an important role early in the HIV-1 replication cycle, interference with the IN substrate has become an attractive strategy for therapeutic intervention. We demonstrated that a designed zinc finger protein (ZFP) fused to green fluorescent protein (GFP) targets the 2-long terminal repeat (2-LTR) circle junctions of HIV-1 DNA with nanomolar affinity. We report now that 2LTRZFP-GFP stably transduced into 293T cells interfered with the expression of vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral red fluorescent protein (RFP), as shown by the suppression of RFP expression. We also used a third-generation lentiviral vector and pCEP4 expression vector to deliver the 2LTRZFP-GFP transgene into human T-lymphocytic cells, and a stable cell line for long-term expression studies was selected for HIV-1 challenge. HIV-1 integration and replication were inhibited as measured by Alu-gag real-time PCR and p24 antigen assay. In addition, the molecular activity of 2LTRZFP-GFP was evaluated in peripheral blood mononuclear cells. The results were confirmed by Alu-gag real-time PCR for integration interference. We suggest that the expression of 2LTRZFP-GFP limited viral integration on intracellular immunization, and that it has potential for use in HIV gene therapy in the future.

Correlation of Recombinant Integrase Activity and Functional Preintegration Complex Formation during Acute Infection by Replication-Defective Integrase Mutant Human Immunodeficiency Virus

Previous studies characterized two types of replication-defective human immunodeficiency virus type 1 (HIV-1) integrase mutants: class I, which are specifically blocked at the integration step, and class II, which harbor additional virion production and/or reverse transcription defects. Class I mutant enzymes supported little if any metal ion-dependent 3'-processing and DNA strand transfer activities in vitro, whereas class II enzymes displayed partial or full catalytic function in studies with simplified assay designs, suggesting that defective interaction(s) with heterologous integrase binding proteins might underlie the class II mutant viral phenotype. To address this hypothesis, class I and II mutant enzymes were interrogated under expanded sets of in vitro conditions. The majority failed to catalyze the concerted integration of two viral DNA ends into target DNA, highlighting defective integrase function as the root cause of most class II in addition to all class I mutant virus infection defects. One mutant protein, K264E, in contrast, could support the wild-type level of concerted integration activity. After accounting for its inherent reverse transcription defect, HIV-1(K264E) moreover formed preintegration complexes that supported the efficient integration of endogenous viral DNA in vitro and normal levels and sequences of 2-long terminal repeat-containing circle junctions during acute infection. K264E integrase furthermore efficiently interacted in vitro with two heterologous binding partners, LEDGF/p75 and reverse transcriptase. Our results underscore the physiological relevance of concerted integration assays for tests of integrase mutant function and suggest that the K264E mutation disrupts an interaction with an intranuclear integrase binding partner that is important for HIV-1 integration.

Enhanced HIV-1 replication in ex vivo ectocervical tissues from post-menopausal women correlates with increased inflammatory responses

AbstractKnowledge about early innate immune responses at the mucosal surfaces of the female genital tract is important in understanding the pathogenesis of heterosexual transmission of human immunodeficiency virus type-1 (HIV-1). As estradiol decreases inflammatory responses, we postulated that an estradiol-deficient state such as post-menopause could enhance expression of inflammatory factors that stimulate HIV-1 replication. We compare HIV-1 integration, transcription, and viral p24 release levels among ectocervical tissues obtained from pre- and post-menopausal donors. We detected enhanced HIV-1 p24 release levels in post- compared with pre-menopausal tissues (P<0.0001), but saw no difference in HIV-1 integration. Overall, 100% of post-menopausal tissues exhibited levels of HIV-1 transcription above background compared with only 60% of pre-menopausal tissues. Increased HIV-1 transcription was associated with enhanced interleukin (IL)-1β, IL-6, monocyte chemotactic protein-1, growth-regulated oncogene-α, and interferon-γ-inducible protein-10 expression. Neutralization and nuclear factor-κB-targeting small-interfering RNA experiments both decreased HIV-1 transcription, suggesting that the early inflammatory response may facilitate HIV-1 replication in ex vivo ectocervical tissues from post-menopausal women.

Changes in the topology of gene expression networks by human immunodeficiency virus type 1 (HIV-1) integration in macrophages

One key step of human immunodeficiency virus type 1 (HIV-1) infection is the integration of its viral cDNA. This process is mediated through complex networks of host–virus interactions that alter several normal cell functions of the host. To study the complexity of disturbances in cell gene expression networks by HIV-1 integration, we constructed a network of human macrophage genes located close to chromatin regions rich in proviruses. To perform the network analysis, we selected 28 genes previously identified as the target of cDNA integration and their transcriptional profiles were obtained from GEO Profiles (NCBI).A total of 2770 interactions among the 28 genes located around the HIV-1 proviruses in human macrophages formed a highly dense main network connected to five sub-networks. The overall network was significantly enriched by genes associated with signal transduction, cellular communication and regulatory processes. To simulate the effects of HIV-1 integration in infected macrophages, five genes with the most number of interaction in the normal network were turned off by putting in zero the correspondent expression values. The HIV-1 infected network showed changes in its topology and alteration in the macrophage functions reflected in a re-programming of biosynthetic and general metabolic process.Understanding the complex virus-host interactions that occur during HIV-1 integration, may provided valuable genomic information to develop new antiviral treatments focusing on the management of some specific gene expression networks associated with viral integration. This is the first gene network which describes the human macrophages genes interactions related with HIV-1 integration.

ChemInform Abstract: Inhibitors of the Interactions Between HIV‐1 IN and the Cofactor LEDGF/p75

AbstractReview: 58 refs.

APOBEC3G complexes decrease human immunodeficiency virus type 1 production.

APOBEC3G (A3G) is packaged into human immunodeficiency virus type 1 (HIV-1) virions unless HIV-1 virion infectivity factor (Vif) counteracts it. Virion A3G restricts HIV-1 reverse transcription and integration in target cells. Some A3G in producer cells colocalizes with specific cytoplasmic structures, in what are called "A3G complexes" here. Functional effects of producer cell A3G complexes on HIV-1 replication were studied. HeLa cells were cotransfected with HIV-1 constructs producing pseudoviruses, as well as either wild-type (WT) A3G or a mutant A3G (C97A, Y124A, W127A, or D128K A3G). Pseudovirus particle production was decreased from cells expressing any of the A3Gs that formed complexes by 24 h after transfection, relative to cells with C97A A3G that did not form detectable A3G complexes by 24 h or A3G-negative cells. The intracellular HIV-1 Gag half-life was shorter in cells containing A3G complexes than in those lacking complexes. HIV-1 virion output was decreased in a single round of replication from a T cell line containing A3G complexes (CEM cells) after infection with Vif-negative HIV-1, compared to Vif-positive HIV-1 that depleted A3G. Levels of production of Vif-negative and Vif-positive virus were similar from cells not containing A3G (CEM-SS cells). Knockdown of the mRNA processing body (P-body) component RCK/p54, eliminated A3G complex formation, and increased HIV-1 production. We conclude that endogenous A3G complexes in producer cells decrease HIV-1 production if not degraded by Vif.

Inhibitors of the Interactions between HIV‐1 IN and the Cofactor LEDGF/p75

The replication cycle of human immunodeficiency virus type 1 (HIV-1) is a complex multistep process that depends on both viral and host cell factors. The nuclear protein lens epithelium-derived growth factor (LEDGF/p75) is a multidomain protein, present in host cells, which plays an important role in the integration process. LEDGF/p75 not only binds HIV-1 integrase (IN) at its IN binding domain (IBD) but also contains several motifs that function in DNA and chromatin binding. The demonstrated importance of the association between IN and LEDGF/p75 in HIV-1 integration suggests the possibility that this protein-protein interaction (PPI) could be exploited as an antiviral target. We describe herein the progress to date in developing inhibitors of this promising target.

Novel regulation of HIV-1 replication and pathogenicity: Rev inhibition of integration

Following fusion of the human immunodeficiency virus type-1 (HIV-1) with host cells' membrane and reverse transcription of the viral RNA, the resulted cDNA is integrated into the host genome by the viral integrase enzyme (IN). Quantitative estimations have revealed that only 1-2 copies are integrated per infected cell, although many copies of the viral RNA are reverse-transcribed. The molecular mechanism that restricts the integration degree has not, so far, been elucidated. Following integration, expressed partially spliced and unspliced transcripts are exported from the nuclei by the viral Rev protein. Here, we show that in virally infected cells, the Rev interacts with the IN forming a Rev-IN complex and consequently limits the number of integration events. Disruption of the Rev-IN complex by selected IN-derived peptides or infection by a Rev-deficient virus stimulate integration resulting in large numbers of integration event/cell. Conversely, infection of Rev-expression cells blocks integration and inhibits virus production. Increased integration appears to correlate with increased cell death of infected cultures. Our results thus demonstrate a new regulatory function of Rev and probably establish a link between Rev restriction of HIV-1 integration and protection of HIV-1-infected cells from premature cell death.

Transcriptional competence of the integrated HIV-1 provirus at the nuclear periphery

Spatial distribution of genes within the nucleus contributes to transcriptional control, allowing optimal gene expression as well as constitutive or regulated gene repression. Human immunodeficiency virus type 1 (HIV-1) integrates into host chromatin to transcribe and replicate its genome. Lymphocytes harbouring a quiescent but inducible provirus are a challenge to viral eradication in infected patients undergoing antiviral therapy. Therefore, our understanding of the contribution of sub-nuclear positioning to viral transcription may also have far-reaching implications in the pathology of the infection. To gain an insight into the conformation of chromatin at the site of HIV-1 integration, we investigated lymphocytes carrying a single latent provirus. In the silenced state, the provirus was consistently found at the nuclear periphery, associated in trans with a pericentromeric region of chromosome 12 in a significant number of quiescent cells. After induction of the transcription, this association was lost, although the location of the transcribing provirus remained peripheral. These results, extended to several other cell clones, unveil a novel mechanism of transcriptional silencing involved in HIV-1 post-transcriptional latency and reinforce the notion that gene transcription may also occur at the nuclear periphery.

Raltegravir: The First HIV Type 1 Integrase Inhibitor

Raltegravir is the first approved human immunodeficiency virus type 1 (HIV-1) integrase inhibitor; it targets the strand transfer step of HIV-1 integration. Clinical trials have demonstrated that raltegravir-containing regimens have potent antiretroviral activity and are well tolerated in HIV-1-infected individuals. In antiretroviral treatment-experienced persons with drug-resistant HIV infection, raltegravir-containing treatment with an optimized background regimen was superior to an optimized background regimen alone. In treatment-naive persons, raltegravir was not inferior to efavirenz when the drugs were administered with tenofovir and lamivudine or emtricitabine. Raltegravir is metabolized by glucuronidation, not hepatically; thus, the potential for drug-drug interactions is decreased. Drug resistance, conferred by substitutions in the gene coding for the HIV-1 integrase enzyme, develops relatively frequently after virologic failure. As an antiretroviral drug with a novel mechanism of action, raltegravir is an important advancement in HIV-1 treatment options.

Preclinical Evaluation of GS-9160, a Novel Inhibitor of Human Immunodeficiency Virus Type 1 Integrase

GS-9160 is a novel and potent inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN) that specifically targets the process of strand transfer. It is an authentic inhibitor of HIV-1 integration, since treatment of infected cells results in an elevation of two-long terminal repeat circles and a decrease of integration junctions. GS-9160 has potent and selective antiviral activity in primary human T lymphocytes producing a 50% effective concentration (EC(50)) of approximately 2 nM, with a selectivity index (50% cytotoxic concentration/EC(50)) of approximately 2,000. The antiviral potency of GS-9160 decreased by 6- to 10-fold in the presence of human serum. The antiviral activity of GS-9160 is synergistic in combination with representatives from three different classes of antiviral drugs, namely HIV-1 protease inhibitors, nonnucleoside reverse transcriptase inhibitors, and nucleotide reverse transcriptase inhibitors. Viral resistance selections performed with GS-9160 yielded a novel pattern of mutations within the catalytic core domain of IN; E92V emerged initially, followed by L74M. While E92V as a single mutant conferred 12-fold resistance against GS-9160, L74M had no effect as a single mutant. Together, these mutations conferred 67-fold resistance to GS-9160, indicating that L74M may potentiate the resistance caused by E92V. The pharmacokinetic profile of GS-9160 in healthy human volunteers revealed that once-daily dosing was not likely to achieve antiviral efficacy; hence, the clinical development of this compound was discontinued.

Identification and Characterization of PWWP Domain Residues Critical for LEDGF/p75 Chromatin Binding and Human Immunodeficiency Virus Type 1 Infectivity

Lens epithelium-derived growth factor (LEDGF)/p75 functions as a bimodal tether during lentiviral DNA integration: its C-terminal integrase-binding domain interacts with the viral preintegration complex, whereas the N-terminal PWWP domain can bind to cellular chromatin. The molecular basis for the integrase-LEDGF/p75 interaction is understood, while the mechanism of chromatin binding is unknown. The PWWP domain is homologous to other protein interaction modules that together comprise the Tudor clan. Based on primary amino acid sequence and three-dimensional structural similarities, 24 residues of the LEDGF/p75 PWWP domain were mutagenized to garner essential details of its function during human immunodeficiency virus type 1 (HIV-1) infection. Mutating either Trp-21 or Ala-51, which line the inner wall of a hydrophobic cavity that is common to Tudor clan members, disrupts chromatin binding and virus infectivity. Consistent with a role for chromatin-associated LEDGF/p75 in stimulating integrase activity during infection, recombinant W21A protein is preferentially defective for enhancing integration into chromatinized target DNA in vitro. The A51P mutation corresponds to the S270P change in DNA methyltransferase 3B that causes human immunodeficiency, centromeric instability, and facial anomaly syndrome, revealing a critical role for this amino acid position in the chromatin binding functions of varied PWWP domains. Our results furthermore highlight the requirement for a conserved Glu in the hydrophobic core that mediates interactions between other Tudor clan members and their substrates. This initial systematic mutagenesis of a PWWP domain identifies amino acid residues critical for chromatin binding function and the consequences of their changes on HIV-1 integration and infection.

Human Immunodeficiency Virus Type 1 (HIV-1) Integration: a Potential Target for Microbicides To Prevent Cell-Free or Cell-Associated HIV-1 Infection

Conceptually, blocking human immunodeficiency virus type 1 (HIV-1) integration is the last possibility for preventing irreversible cellular infection. Using cocultures of monocyte-derived dendritic cells and CD4(+) T cells, which represent primary targets in sexual transmission, we demonstrated that blocking integration with integrase strand transfer inhibitors (InSTIs), particularly L-870812, could consistently block cell-free and cell-associated HIV-1 infection. In a pretreatment setting in which the compound was present before and during infection and was afterwards gradually diluted during the culture period, the naphthyridine carboxamide L-870812 blocked infection with the cell-free and cell-associated HIV-1 Ba-L strain at concentrations of, respectively, 1,000 and 10,000 nM. The potency of L-870812 was similar to that of the nucleotide reverse transcriptase inhibitor R-9-(2-phosphonylmethoxypropyl) adenine (PMPA) but one or two orders of magnitude lower than those of the nonnucleoside reverse transcriptase inhibitors UC781 and TMC120. In contrast, the diketo acid RDS derivative InSTIs showed clear-cut but weaker antiviral activity than L-870812. Moreover, L-870812 completely blocked subtype C and CRFO2_AG primary isolates, which are prevalent in the African heterosexual epidemic. Furthermore, the addition of micromolar concentrations of L-870812 even 24 h after infection could still block both cell-free and cell-associated Ba-L, opening the prospect of postexposure prophylaxis. Finally, an evaluation of the combined activity of L-870812 with either T20, zidovudine, PMPA, UC781, or TMC120 against replication-deficient HIV-1 Ba-L (env) pseudovirus suggested synergistic activity for all combinations. Importantly, compounds selected for the study by using the coculture model were devoid of acute or delayed cytotoxic effects at HIV-blocking concentrations. Therefore, these findings provide evidence supporting consideration of HIV-1 integration as a target for microbicide development.