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Background. Antiretroviral therapy (ART)-mediated immune reconstitution fails to restore the capacity of the immune system to spontaneously control human immunodeficiency virus (HIV) replication. Methods. A total of 23 HIV type 1 (HIV-1)-infected, virologically suppressed subjects receiving ART (CD4⁺ T-cell count, >450 cells/μL) were randomly assigned to have 180 μg/week (for arm A) or 90 μg/week (for arm B) of pegylated (Peg) interferon alfa-2a added to their current ART regimen. After 5 weeks, ART was interrupted, and Peg-interferon alfa-2a was continued for up to 12 weeks (the primary end point), with an option to continue to 24 weeks. End points included virologie failure (viral load, ≥400 copies/mL) and adverse events. Residual viral load and HIV-1 DNA integration were also assessed. Results. At week 12 of Peg-interferon alfa-2a monotherapy, viral suppression was observed in 9 of 20 subjects (45%), a significantly greater proportion than expected (arm A, P =. 0088; arm B, P =. 0010; combined arms, P< .0001). Over 24 weeks, both arms had lower proportions of subjects who had viral load, compared with the proportion of subjects in a historical control group (arm A, P =. 0046; arm B, P =.0011). Subjects who had a sustained viral load of <400 copies/mL had decreased levels of integrated HIV DNA (P =.0313) but increased residual viral loads (P =.0078), compared with subjects who experienced end-point failure. Conclusions. Peg-interferon alfa-2a immunotherapy resulted in control of HIV replication and decreased HIV-1 integration, supporting a role for immunomediated approaches in HIV suppression and/or eradication.

The persistence of HIV-1 in a latent state within long-lived immune cells remains a major barrier to a cure for HIV-1 infection. The “block-and-lock” strategy aims to silence the HIV-1 provirus permanently using latency promoting agents (LPAs). LEDGINs, a well-known class of LPAs, inhibit the interaction between viral integrase and LEDGF/p75, reducing viral integration and retargeting the provirus to regions resistant to reactivation. However, proximity to enhancers may still permit residual transcription. Given BRD4’s central role in the enhancer biology, we now test two BRD4 modulators, JQ1 and ZL0580. Mechanistic studies reveal that JQ1 and ZL0580 have contrasting effects on Tat-dependent HIV-1 transcription, resulting in JQ1 promoting viral reactivation and ZL0580 inducing transcriptional silencing. Combining ZL0580 with LEDGINs has an additive effect in blocking HIV-1 transcription and reactivation, in both cell lines and primary cells. These findings demonstrate the potential of ZL0580 to enhance the block-and-lock cure strategy. In this work, we combine LEDGINs with BRD4 modulator ZL0580 to achieve a functional HIV cure. This approach silences the virus by retargeting viral integration into silent chromatin and blocking enhancers, offering a powerful combination strategy to block-and-lock HIV.

The HIV intasome is a large nucleoprotein assembly that mediates the integration of a DNA copy of the viral genome into host chromatin. Intasomes are targeted by the latest generation of antiretroviral drugs, integrase strand-transfer inhibitors (INSTIs). Challenges associated with lentiviral intasome biochemistry have hindered high-resolution structural studies of how INSTIs bind to their native drug target. Here, we present high-resolution cryo–electron microscopy structures of HIV intasomes bound to the latest generation of INSTIs. These structures highlight how small changes in the integrase active site can have notable implications for drug binding and design and provide mechanistic insights into why a leading INSTI retains efficacy against a broad spectrum of drug-resistant variants. The data have implications for expanding effective treatments available for HIV-infected individuals.

HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4(+) T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4(+) T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4(+) T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research.

Interaction between HIV-1 integrase and the cellular cofactor LEDGF/p75 is important for viral integration. Newly designed small molecules that block this interaction inhibit HIV replication, illustrating the potential of viral–host protein-protein interaction inhibitors. Lens epithelium–derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase that promotes viral integration by tethering the preintegration complex to the chromatin. By virtue of its crucial role in the early steps of HIV replication, the interaction between LEDGF/p75 and integrase represents an attractive target for antiviral therapy. We have rationally designed a series of 2-(quinolin-3-yl)acetic acid derivatives (LEDGINs) that act as potent inhibitors of the LEDGF/p75-integrase interaction and HIV-1 replication at submicromolar concentration by blocking the integration step. A 1.84-Å resolution crystal structure corroborates the binding of the inhibitor in the LEDGF/p75-binding pocket of integrase. Together with the lack of cross-resistance with two clinical integrase inhibitors, these findings define the 2-(quinolin-3-yl)acetic acid derivatives as the first genuine allosteric HIV-1 integrase inhibitors. Our work demonstrates the feasibility of rational design of small molecules inhibiting the protein-protein interaction between a viral protein and a cellular host factor.

Despite highly active antiretroviral therapy, active replication persists and drives immune activation in some individuals with HIV. This activation is higher if therapy is intensified by additional antiretroviral drugs ( pages 373–374 ). Highly active antiretroviral therapy (HAART) results in potent and durable suppression of HIV-1 viremia. However, HIV-1 replication resumes if therapy is interrupted 1 , 2 . Although it is generally believed that active replication has been halted in individuals on HAART, immune activation and inflammation continue at abnormal levels 3 , suggesting continued, low-level viral replication. To assess whether active replication might be driving immune activation in HAART, we examined the impact of treatment intensification with the integrase inhibitor raltegravir on viral complementary DNA and immune activation parameters. In the presence of raltegravir, linear HIV-1 cDNA is prevented from integrating into chromatin and is subsequently converted to episomal cDNAs 4 , 5 . Raltegravir intensification of a three-drug suppressive HAART regimen resulted in a specific and transient increase in episomal DNAs in a large percentage of HAART-suppressed subjects. Furthermore, in subjects with these episomal DNAs, immune activation was higher at baseline and was subsequently normalized after raltegravir intensification. These results suggest that, despite suppressive HAART, active replication persists in some infected individuals and drives immune activation. The ability of raltegravir intensification to perturb the reservoir that supports active replication has implications for therapeutic strategies aimed at achieving viral eradication.

HIV-1 cores enter the nucleus and undergo capsid disassembly (uncoating) near their integration site. Although most viral cores are localized to nuclear speckles (NSs), the spatial relationship between the uncoating site and integration site remains unclear. Here, using fluorescently labeled HIV-1 cores and NS markers, we show that uncoating predominantly occurs within NSs. Treatment of infected cells with capsid inhibitors PF-3450074 (PF74) or lenacapavir (LEN) after nuclear entry induced rapid disruption of interactions between capsid and cleavage and polyadenylation specificity factor 6 (CPSF6) followed by exit of HIV-1 cores from NSs, indicating that CPSF6 binding is required to retain the viral cores in the NSs. Treatment with PF74 or LEN led to core disruption and appearance of transcriptionally active proviruses further from the NSs compared to viral cores that uncoated in the NSs in untreated cells. This spatial shift correlated with reduction in integration into gene-rich, transcriptionally active speckle-associated chromatin domains, the preferred sites of HIV-1 integration, and increased integration into gene-sparse lamina-associated domains located away from the nuclear envelope. These findings demonstrate that the HIV-1 uncoating site is a key determinant of integration targeting, and that capsid inhibitors can misdirect integration by relocalizing uncoating to outside of NSs. HIV-1 releases its DNA when its protective capsid shell disassembles (uncoats) inside membraneless subnuclear organelles called nuclear speckles. Drugs that force uncoating to occur elsewhere promote viral DNA insertion into different genomic sites.

HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly.

Excision of integrated HIV proviruses derived from most HIV-1 strains is achieved with a recombinase evolved in vitro . Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo , including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.

The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules. Major advances in the treatment of HIV have been made possible by carefully studying the virus and its interaction with the host cell. The virus consists of two strands of RNA—representing the genetic information of the virus—contained in a protein coat called capsid. Scientists have learned that the virus' RNA is used to create viral DNA in the cytoplasm of an infected cell, in a process called reverse transcription. This viral DNA then enters the cell's nucleus and becomes incorporated into the cell's DNA, and the cell unwittingly begins to help the virus reproduce. It is less clear what happens to the capsid after the virus enters a cell. Some researchers have suggested that it is lost shortly after entry or during reverse transcription. However, some recent studies have found that damaging the capsid hampers reverse transcription and significantly impairs the entry of viral DNA into the cell's nucleus. This suggests that the capsid might continue to protect the viral genome when the RNA is converted into DNA. To learn more about what happens during reverse transcription and when the viral DNA enters the nucleus, it is important to watch individual events as they occur. Until recently, it had been hard to do this without changing the DNA or RNA in ways that might affect their properties. Recently, a technique called click-labeling has been developed that can add a fluorescent label to DNA or RNA without potentially damaging this genetic material. This label allows the movement of the DNA or RNA to be followed when the cell is viewed under a microscope. Peng et al. used this new technique to watch reverse transcription, how viral DNA enters the cell nucleus and what happens to the capsid when HIV invades different kinds of cells. When the virus entered a type of cell often used in laboratory research called HeLa cells, the capsid protected the viral genetic material when it was in the cell's cytoplasm but disappeared before or shortly after the viral DNA entered the cell's nucleus. However, HeLa cells are not natural targets of HIV; when Peng et al. looked at the behavior of the capsid in the immune cells that the virus normally invades, the capsid was present in both the cytoplasm and the nucleus of these cells. Peng et al. also observed what happens in HIV-infected cells treated with a chemical called PF74 that interferes with the capsid. This revealed that low concentrations of PF74 make it hard for the viral DNA to enter the nucleus, probably by blocking the interaction of the capsid with a protein from the host cell. At high concentrations, the drug prevented reverse transcription. The approach used by Peng et al. allows direct visualization of how HIV replicates and how this DNA is imported into the nucleus of cells naturally targeted by the virus. This will aid our understanding of how the virus selects where in the host genome it should insert its DNA, which is important for establishing a permanent infection in the cell.