Inhibition Of HIV-1 Replication Research Articles

Measuring the latent reservoir for HIV-1: Quantification bias in near full-length genome sequencing methods.

Antiretroviral therapy (ART) effectively inhibits HIV-1 replication but is not curative due to the persistence of a latent viral reservoir in resting CD4+ T cells. This reservoir is a major barrier to cure. Sequencing studies have revealed that the population of proviruses persisting in ART-treated individuals is dominated by defective proviruses that cannot give rise to viral rebound due to fatal defects including large deletions and APOBEC3-mediated hypermutation. Near full genome sequencing (nFGS) of individual proviruses is used in reservoir assays to provide an estimate of the fraction of proviruses that are intact. nFGS methods rely on a long-distance outer PCR capturing most (~9 kb) of the genome, followed by nested inner PCRs. The outer PCR is carried out at limit dilution, and interpretation of the results is based on the assumption that all proviruses are quantitatively captured. Here, we evaluate nFGS methods using the intact proviral DNA assay (IPDA), a multiplex digital droplet PCR assay that quantitates intact and defective proviruses with single molecule sensitivity using only short, highly efficient amplicons. We analyzed proviral templates of known sequence to avoid the additional complication of sequence polymorphism. With the IPDA, we quantitated molecular yields at each step of nFGS methods. We demonstrate that nFGS methods are inefficient and miss ~70% of full-length proviruses due to amplification failure at the initial outer PCR step. In contrast, proviruses with large internal deletions encompassing 70% of the genome can be quantitatively amplified under the same conditions. Accurate measurement of the latent reservoir of HIV-1 is essential for evaluating the efficacy of cure strategies, and the bias against full length proviruses in nFGS methods must be considered.

Bictegravir nanomicelles and anionic pullulan loaded vaginal film: Dual mechanistic pre-exposure prophylaxis (PrEP) for HIV

Locally delivered pre-exposure prophylaxis (PrEP) has proven to be a promising strategy to combat Human immunodeficiency virus (HIV) transmission but several findings encountered toxicities or proved to be marginally effective in clinical settings. Therefore, innovative, multifunctional, and safer alternatives are being progressively investigated. Herein, we explored negatively charged carbohydrate, anionic pullulan (AP) as a rapidly soluble film-former and novel anti-HIV agent. Additionally, Bictegravir (BCT), an HIV integrase inhibitor was co-delivered in the form of nanomicelles for sustained antiviral activity. BCT-loaded PLGA-PEG polymeric nanomicelles (BN) were incorporated into PVA/pullulan-based film matrix comprising of 2 % w/v AP (BN-AP film). In cell-based assays, biocompatibility and TEER values for BN-AP films were similar to control while the commercial vaginal contraceptive film (VCF®) showed severe cytotoxicity and drastically reduced the tight junction integrity. Rapid disintegration of BN-AP film with >85 % drug release was observed in simulated vaginal and seminal fluid. Most importantly, AP and BN-AP film significantly inhibited HIV-1 replication with IC50 at as low as 91 μg/mL and 0.708 nM, respectively. Therefore, this study entails successful development of BN-AP film that functioned as an effective, biocompatible dual-acting PrEP formulation.

Cabotegravir Extended-Release Injectable Suspension: A Review in HIV-1 Pre-Exposure Prophylaxis.

Cabotegravir extended-release (ER) injectable suspension (Apretude™) is the first long-acting injectable option to be approved for HIV-1 pre-exposure prophylaxis (PrEP). As an HIV-1 integrase strand transfer inhibitor, cabotegravir ER injectable suspension prevents DNA integration and inhibits HIV-1 replication. Its slow absorption and long elimination half-life permit infrequent dosing (1month apart for two consecutive months, and every 2months thereafter). Cabotegravir ER injectable suspension is indicated in the USA for PrEP to reduce the risk of sexually acquired HIV-1 infection in at-risk adults and adolescents weighing ≥ 35 kg who have a negative HIV-1 test prior to initiation. In clinical trials, cabotegravir ER injectable suspension had superior efficacy to oral daily emtricitabine/tenofovir disoproxil fumarate (DF) in preventing acquisition of HIV-1 in at-risk transgender women (TGW), cisgender men who have sex with men (MSM), and cisgender women. The drug was generally well tolerated, although further long-term data are needed to fully determine its safety. With its convenient, less-frequent dosing schedule and its long-acting formulation enabling intramuscular administration, cabotegravir ER injectable suspension represents a novel and efficacious alternative to daily oral PrEP.

Detecting Sources of Immune Activation and Viral Rebound in HIV Infection.

Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA+/p24+ cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

Omic Technologies in HIV: Searching Transcriptional Signatures Involved in Long-Term Non-Progressor and HIV Controller Phenotypes.

This article reviews the main discoveries achieved by transcriptomic approaches on HIV controller (HIC) and long-term non-progressor (LTNP) individuals, who are able to suppress HIV replication and maintain high CD4+ T cell levels, respectively, in the absence of antiretroviral therapy. Different studies using high throughput techniques have elucidated multifactorial causes implied in natural control of HIV infection. Genes related to IFN response, calcium metabolism, ribosome biogenesis, among others, are commonly differentially expressed in LTNP/HIC individuals. Additionally, pathways related with activation, survival, proliferation, apoptosis and inflammation, can be deregulated in these individuals. Likewise, recent transcriptomic studies include high-throughput sequencing in specific immune cell subpopulations, finding additional gene expression patterns associated to viral control and/or non-progression in immune cell subsets. Herein, we provide an overview of the main differentially expressed genes and biological routes commonly observed on immune cells involved in HIV infection from HIC and LTNP individuals, analyzing also different technical aspects that could affect the data analysis and the future perspectives and gaps to be addressed in this field.

CD8 + T-cell responses in HIV controllers: potential implications for novel HIV remission strategies.

Purpose of reviewImmunological studies of spontaneous HIV and simian virus (SIV) controllers have identified virus-specific CD8+ T cells as a key immune mechanism of viral control. The purpose of this review is to consider how knowledge about the mechanisms that are associated with CD8+ T cell control of HIV/SIV in natural infection can be harnessed in HIV remission strategies.Recent findingsWe discuss characteristics of CD8+ T-cell responses that may be critical for suppressing HIV replication in spontaneous controllers comprising HIV antigen recognition including specific human leukocyte antigen types, broadly cross-reactive T cell receptors and epitope targeting, enhanced expansion and antiviral functions, and localization of virus-specific T cells near sites of reservoir persistence. We also discuss the need to better understand the timing of CD8+ T-cell responses associated with viral control of HIV/SIV during acute infection and after treatment interruption as well as the mechanisms by which HIV/SIV-specific CD8+ T cells coordinate with other immune responses to achieve control.SummaryWe propose implications as to how this knowledge from natural infection can be applied in the design and evaluation of CD8+ T-cell-based remission strategies and offer questions to consider as these strategies target distinct CD8+ T-cell-dependent mechanisms of viral control.

Adherence to Antiretroviral Therapy in People Living with HIV/AIDS: A Cross-Sectional Study.

Adherence to antiretroviral therapy (ART) is essential to suppress HIV replication, preserve immune competence, and ensure quality of life for people living with HIV/AIDS. This is a cross-sectional study to assess adherence to ART in HIV-infected adults and its associated factors in São Carlos, SP, Brazil, from June 2018 to January 2019. Standardized interviews were conducted covering demographic, clinical, and laboratory characteristics and instruments to assess compliance to treatment (CEAT-VIH), HIV/AIDS-targeted quality of life (HAT-QoL), and self-efficacy expectations of adherence (SEA-ART). Each variable was analyzed for association with adherence to ART, by refilling at least 90% of the prescribed doses in the 6 months before the interview date. The study consisted of 220 participants, with a mean age of 43 years, 60.5% male, and 24.5% men who have sex with men. Previous consumption of alcohol or illicit drugs was reported by 44.1% of participants and current or previous smoking by 34.1%. The most common regimen was two nucleoside reverse transcriptase inhibitors combined with one non-nucleoside reverse transcriptase inhibitor (37.3%). The adherence to ART was 62%, and the factors associated with it were living alone [adjusted odds ratio (aOR) 2.79], not having an active sexual life (aOR 0.43), not being a smoker (aOR 0.36), having a CD4 count ≥350 cells/mm3 (aOR 2.50), and having a SEA-ART Score >100 (aOR 1.94). The fear of disclosing HIV status could make adherence to treatment difficult. This could be the reason that living alone and not having an active sexual life have been associated with better adherence. Encouraging healthy lifestyle habits and promoting self-efficacy tools can also improve adherence.

HPLC-Based Purification and Isolation of Potent Anti-HIV and Latency Reversing Daphnane Diterpenes from the Medicinal Plant Gnidia sericocephala (Thymelaeaceae).

Despite the success of combination antiretroviral therapy (cART), HIV persists in low- and middle-income countries (LMIC) due to emerging drug resistance and insufficient drug accessibility. Furthermore, cART does not target latently-infected CD4+ T cells, which represent a major barrier to HIV eradication. The “shock and kill” therapeutic approach aims to reactivate provirus expression in latently-infected cells in the presence of cART and target virus-expressing cells for elimination. An attractive therapeutic prototype in LMICs would therefore be capable of simultaneously inhibiting viral replication and inducing latency reversal. Here we report that Gnidia sericocephala, which is used by traditional health practitioners in South Africa for HIV/AIDS management to supplement cART, contains at least four daphnane-type compounds (yuanhuacine A (1), yuanhuacine as part of a mixture (2), yuanhuajine (3), and gniditrin (4)) that inhibit viral replication and/or reverse HIV latency. For example, 1 and 2 inhibit HIV replication in peripheral blood mononuclear cells (PBMC) by >80% at 0.08 µg/mL, while 1 further inhibits a subtype C virus in PBMC with a half-maximal effective concentration (EC50) of 0.03 µM without cytotoxicity. Both 1 and 2 also reverse HIV latency in vitro consistent with protein kinase C activation but at 16.7-fold lower concentrations than the control prostratin. Both 1 and 2 also reverse latency in primary CD4+ T cells from cART-suppressed donors with HIV similar to prostratin but at 6.7-fold lower concentrations. These results highlight G. sericocephala and components 1 and 2 as anti-HIV agents for improving cART efficacy and supporting HIV cure efforts in resource-limited regions.

Identification of 2-(4-N,N-Dimethylaminophenyl)-5-methyl-1-phenethyl-1H-benzimidazole targeting HIV-1 CA capsid protein and inhibiting HIV-1 replication in cellulo

The capsid (CA) subunit of the HIV-1 Gag polyprotein is involved in several steps of the viral cycle, from the assembly of new viral particles to the protection of the viral genome until it enters into the nucleus of newly infected cells. As such, it represents an interesting therapeutic target to tackle HIV infection. In this study, we screened hundreds of compounds with a low cost of synthesis for their ability to interfere with Gag assembly in vitro. Representatives of the most promising families of compounds were then tested for their ability to inhibit HIV-1 replication in cellulo. From these molecules, a hit compound from the benzimidazole family with high metabolic stability and low toxicity, 2-(4-N,N-dimethylaminophenyl)-5-methyl-1-phenethyl-1H-benzimidazole (696), appeared to block HIV-1 replication with an IC50 of 3 µM. Quantitative PCR experiments demonstrated that 696 does not block HIV-1 infection before the end of reverse transcription, and molecular docking confirmed that 696 is likely to bind at the interface between two monomers of CA and interfere with capsid oligomerization. Altogether, 696 represents a promising lead molecule for the development of a new series of HIV-1 inhibitors.

Constituents of Croton megalocarpus with Potential Anti-HIV Activity.

Reported herein is an anti-HIV monochlorinated compound, 1β-acetoxy-3β-chloro-5α,6α-dihydroxycrotocascarin L (1), of the rare crotofolane diterpenoid class. Compound 1, a suspected artifact of extraction, along with the previously undescribed 11β-acetoxycrotocascarin L (2) and a known compound, crotocascarin K (3), were isolated from the bark of Croton megalocarpus, a Kenyan oil-producing seed crop. Compounds 1 and 3 inhibited HIV-1 replication with IC50 values of 28 and 5.5 nM, respectively. Furthermore, both compounds lacked cytotoxicity toward MT-4 cells and FM-55-M1 cells at concentrations of up to 50 μM. Compounds 1 and 3 were both found to inhibit HIV-1 protease.

Schlafen 5 suppresses human immunodeficiency virus type 1 transcription by commandeering cellular epigenetic machinery.

Schlafen-5 (SLFN5) is an interferon-induced protein of the Schlafen family, which are involved in immune responses and oncogenesis. To date, little is known regarding its anti-HIV-1 function. Here, the authors report that overexpression of SLFN5 inhibits HIV-1 replication and reduces viral mRNA levels, whereas depletion of endogenous SLFN5 promotes HIV-1 replication. Moreover, they show that SLFN5 markedly decreases the transcriptional activity of HIV-1 long terminal repeat (LTR) via binding to two sequences in the U5-R region, which consequently represses the recruitment of RNA polymerase II to the transcription initiation site. Mutagenesis studies show the importance of nuclear localization and the N-terminal 1–570 amino acids fragment in the inhibition of HIV-1. Further mechanistic studies demonstrate that SLFN5 interacts with components of the PRC2 complex, G9a and Histone H3, thereby promoting H3K27me2 and H3K27me3 modification leading to silencing HIV-1 transcription. In concert with this, they find that SLFN5 blocks the activation of latent HIV-1. Altogether, their findings demonstrate that SLFN5 is a transcriptional repressor of HIV-1 through epigenetic modulation and a potential determinant of HIV-1 latency.

Evaluation of a flavonoid library for inhibition of interaction of HIV-1 integrase with human LEDGF/p75 towards a structure–activity relationship

Background: Proteinśprotein interaction (PPI) between lens epithelium-derived growth factor (LEDGF/p75) and human immunodeficiency virus (HIV) integrase (IN) becomes an attractive target for anti-HIV drug development. Methods: The blockade of this interaction by small molecules could potentially inhibit HIV-1 replication. In this study, a panel of 99 structurally related flavonoids were was tested, concerning their ability to inhibit IN-LEDGF/p75 interaction, using a homogeneous time time-resolved fluorescence (HTRF) assay. Results: From the obtained results, it was possible to observe that the flavonoid with hydroxyl group in C3-, C4-, C5- and C7-position on the A-ring, C4′- and C5′-position of the B-ring, a carbonyl group of the C-ring, was more active against IN-LEDGF/p75 interaction, through competitive inhibition. Moreover, the binding modes of representative compounds, including myricetin, luteolin, dihydrorobinetin, naringenin, epicatechin, genistein and helichrysetin, were analyzedanalysed by molecular docking. Biolayer interferometry assay confirmed that these representative compounds disrupted the PPI by binding to IN with KD values ranging from 1.0 to 3.6 µM. Conclusion: This study presents the first to quantitative comparation of the effect of flavonoids with different structural subclasses on IN-LEDGF/p75 interaction. Our findings provide new insights into the development of inhibitors targeting IN-LEDGF/p75 interaction using flavonoids. Key Messages HIV-1 integrase (IN)-LEDGF/p75 interaction is an attractive target for antiviral drug development. For the first time, the structure-activity relationship of flavonoids belonging to seven flavonoidic subclasses on IN-LEDGF/p75 interaction was determined. This study comprehends an HTRF-based screening system, biolayer interferometry and an in silico molecular docking analysis.

Human Pegivirus Type 1: A Common Human Virus That Is Beneficial in Immune-Mediated Disease?

Two groups identified a novel human flavivirus in the mid-1990s. One group named the virus hepatitis G virus (HGV) and the other named it GB Virus type C (GBV-C). Sequence analyses found these two isolates to be the same virus, and subsequent studies found that the virus does not cause hepatitis despite sharing genome organization with hepatitis C virus. Although HGV/GBV-C infection is common and may cause persistent infection in humans, the virus does not appear to directly cause any other known disease state. Thus, the virus was renamed “human pegivirus 1” (HPgV-1) for “persistent G” virus. HPgV-1 is found primarily in lymphocytes and not hepatocytes, and several studies found HPgV-1 infection associated with prolonged survival in people living with HIV. Co-infection of human lymphocytes with HPgV-1 and HIV inhibits HIV replication. Although three viral proteins directly inhibit HIV replication in vitro, the major effects of HPgV-1 leading to reduced HIV-related mortality appear to result from a global reduction in immune activation. HPgV-1 specifically interferes with T cell receptor signaling (TCR) by reducing proximal activation of the lymphocyte specific Src kinase LCK. Although TCR signaling is reduced, T cell activation is not abolished and with sufficient stimulus, T cell functions are enabled. Consequently, HPgV-1 is not associated with immune suppression. The HPgV-1 immunomodulatory effects are associated with beneficial outcomes in other diseases including Ebola virus infection and possibly graft-versus-host-disease following stem cell transplantation. Better understanding of HPgV-1 immune escape and mechanisms of inflammation may identify novel therapies for immune-based diseases.

NK Cells in Protection from HIV Infection.

Some people, known as HIV-exposed seronegative (HESN) individuals, remain uninfected despite high levels of exposure to HIV. Understanding the mechanisms underlying their apparent resistance to HIV infection may inform strategies designed to protect against HIV infection. Natural Killer (NK) cells are innate immune cells whose activation state depends on the integration of activating and inhibitory signals arising from cell surface receptors interacting with their ligands on neighboring cells. Inhibitory NK cell receptors use a subset of major histocompatibility (MHC) class I antigens as ligands. This interaction educates NK cells, priming them to respond to cells with reduced MHC class I antigen expression levels as occurs on HIV-infected cells. NK cells can interact with both autologous HIV-infected cells and allogeneic cells bearing MHC antigens seen as non self by educated NK cells. NK cells are rapidly activated upon interacting with HIV-infected or allogenic cells to elicit anti-viral activity that blocks HIV spread to new target cells, suppresses HIV replication, and kills HIV-infected cells before HIV reservoirs can be seeded and infection can be established. In this manuscript, we will review the epidemiological and functional evidence for a role for NK cells in protection from HIV infection.

Dopamine May Exacerbate Neuroinflammation in the Context of HIV by Driving Inflammatory Phenotypes in Macrophages

Globally, there are around 38 million people living with HIV (PLWH). Treatment with anti‐retroviral therapy (ART) can suppress HIV replication and improve the length and quality of life, but chronic infection and ART treatment has resulted in the development of a variety of new co‐morbidities. Among these are the constellation of neuropathological, behavioral, cognitive and motor symptoms collectively known as neuroHIV. These symptoms are found in approximately 50% of the infected population, and multiple lines of evidence indicate that neuroHIV is primarily mediated by chronic CNS infection and inflammatory response in CNS myeloid populations such as macrophages and microglia. In many PLWH, these inflammatory conditions can be exacerbated by substance use disorders, which are highly comorbid with HIV infection and disproportionately common in HIV‐infected populations. Substance use disorders of all types are a major risk factor for the development of neuroHIV, suggesting a common mechanism by which substances of abuse interact with HIV to promote inflammation. All addictive substances increase extracellular dopamine in the CNS and our previous data show that dopamine promotes an inflammatory phenotype in human macrophages. Thus, we hypothesize that exposure to dopamine concentrations (10‐6M) induced by stimulant use will more robustly increase inflammation in HIV‐infected human monocyte‐derived macrophages (MDM) than in uninfected MDM. To test this, uninfected and HIV‐infected human MDM were treated with dopamine and changes in inflammatory profiles were assessed by measuring NF‐κB nuclear translocation using high content imaging and quantifying cytokine production by AlphaLISA analysis. Dopamine significantly increased the production of the inflammatory cytokines IL‐6 and IL‐1β, and these were specifically mediated via dopamine receptors and the activation of NF‐κB. These findings correlate with expression of dopaminergic receptors and nod‐like receptor (NLR) inflammasome transcripts and were altered in the presence of HIV infection. We also found that baseline macrophage activation could be attributed to culture medium type. Overall, these studies provide a broader understanding of the immunological role of dopamine, particularly in the development of neuroHIV and may suggest a potential mechanism for further pharmacological investigation to target HIV infection.

Circulating Exosomal Proteins are linked to Neuropathogenesis in SIV‐infected Rhesus Macaque: A Proteomic Approach

BackgroundCombined antiretroviral therapy suppresses HIV replication, but 30‐60% of patients suffer from HIV‐1 associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in HIV CNS infection. Using proteomics, we investigated possible circulating exosomal protein links to neuropathogenesis in SIV‐infected rhesus macaque (RM).MethodsExosomes were isolated from sera of SIV‐infected (SIV‐Exo) and uninfected (CTL‐Exo) RM (N = 3/group) by QIAGEN exoEasy kit and characterized by the qNano‐IZON system. Proteomic analysis of the isolated exosomes was performed using liquid chromatography/mass spectrometry (LC‐MS/MS).ResultsqNano‐IZON analysis indicated that isolated EVs were predominantly exosomes (particle size < 150 nm). In the LC‐MS/MS study, 5,654 proteins were quantified, with 236 proteins (~ 4%) significantly differentially expressed (DE) between CTL‐Exo and SIV‐Exo. Two or more unique peptides were detected in 85% (4777/5654) of quantified proteins, and in 89% (211/236) of significant DE proteins, indicating the depth of analysis. We quantified most of the exosome‐associated proteins (tetraspanins, enzymes, lipid rafts, cytoskeletal, and endosome‐specific proteins) reported in previous studies. The heat‐maps and hierarchical clustering indicated that proteins involved in latent viral reactivation (heat shock transcription factor 1), inflammation (complement factor H, antioxidants, glycoproteins), unfolded protein response (UPR) (proteasome activators, cochaperones), neuropathology (amyloid beta [Aβ] precursor, chromogranin‐A and ‐B), and signaling (cytoskeleton regulators, cyclin‐H, mTOR complex 2, CD74) were expressed at significantly higher levels in SIV‐Exo than CTL‐Exo. However, proteins involved in mitochondrial (Mt) fission (Mt‐fission 1, ‐fission factor, and fission regulator 1), and ATP production (Mt Complex‐I, ‐IV, and ‐V), that play a critical role in the brain energy supply, were significantly decreased in SIV‐Exo. Moreover, exosomal‐proteins involved in autophagy‐mediated degradation (autophagy related 9A, ‐2B, lysosomal associated membrane protein 2), endosomal recycling (sorting nexin 4) and exocytosis (synaptogyrin), sprouting angiogenesis (jumonij domain‐containing 6), and cytoskeleton organization (calponin) were also expressed at significantly lower levels in SIV‐Exo than CTL‐Exo.ConclusionsOur novel findings suggest that circulating exosomal proteins are associated with viral reactivation, inflammation, UPR, mitochondrial dysfunction, defective autophagy, and Aβ and Tau pathology that may elucidate the etiology of HAND, and possibly provide novel therapeutic targets.

Novel Naturally Occurring Dipeptides and Single-Stranded Oligonucleotide Act as Entry Inhibitors and Exhibit a Strong Synergistic Anti-HIV-1 Profile.

IntroductionThe availability of new classes of antiretroviral drugs is critical for treatment-experienced patients due to drug resistance to and unwanted side effects from current drugs. Our aim was therefore to evaluate the anti-HIV-1 activity of a new set of antivirals, dipeptides (WG-am or VQ-am) combined with a single-stranded oligonucleotide (ssON). The dipeptides were identified as naturally occurring and enriched in feces and systemic circulation in HIV-1-infected elite controllers and were proposed to act as entry inhibitors by binding to HIV-1 gp120. The ssON is DNA 35-mer, stabilized by phosphorothioate modifications, which acts on the endocytic step by binding to cell host receptors and inhibiting viruses through interference with binding to nucleolin.MethodsChou–Talalay’s Combination Index method for quantifying synergism was used to evaluate the drug combinations. Patient-derived chimeric viruses encoding the gp120 (env region) were produced by transient transfection and used to evaluate the antiviral profile of the combinations by drug susceptibility assays.ResultsWe found that the combination WG-am:ssON or VQ-am:ssON had low combination index values, suggesting strong antiviral synergism. Of the two combinations, WG-am:ssON (1 mM:1 μM) had high efficacy against all prototype or patient-derived HIV-1 isolates tested, independent of subtype including the HIV-1-A6 sub-subtype. In addition, the antiviral effect was independent of co-receptor usage in patient-derived strains.ConclusionWG-am and ssON alone significantly inhibited HIV-1 replication regardless of viral subtype and co-receptor usage, and the combination WG-am:ssON (1 mM:1 μM) was even more effective due to synergism.

Design of artificial α-helical peptides targeting both gp41 deep pocket and subpocket as potent HIV-1 fusion inhibitors

Both the deep pocket region and its neighboring subpocket site on the N-trimer of HIV-1 gp41 protein can serve as targets for the development of HIV-1 entry inhibitors. Pocket-binding domain (PBD)-containing peptides with the potential to inhibit HIV-1 fusion through targeting the deep pocket have been extensively exploited. However, using an artificial peptide strategy, we herein report the design of α-helical lipopeptides with non-native protein sequences as HIV-1 fusion inhibitors that can occupy both gp41 deep cavity and subpocket sites. The most active compound, PP24C, inhibited HIV-1 replication, including T20-resistant HIV-1 mutants, at low nanomolar level. Biophysical approaches revealed that both the artificial α-helical peptide P35A4 and its cholesterol-tagged peptide PP24C could bind to T21 peptide used as a target surrogate comprising both pockets. Our study offers a new template for the design of artificial anti-HIV-1 therapeutics and highlights the novel concept of peptide secondary structure-based virus fusion inhibitors.

Hide and seek: for HIV-infected CD4+ T cells, playing well comes with maturity.

Antiretroviral therapy suppresses HIV replication but leaves a population of infected CD4+ T cells with integrated proviruses. While most of these proviruses contain defects, such as deletions, some intact proviruses persist and can reinitiate viral replication. In this issue of the JCI, Duette, Hiener, and colleagues performed a tour de force proviral landscape analysis on clinical samples collected over many years with in vitro functional assays. The researchers showed that effector memory CD4+ T cells provide partial sanctuary to intact proviruses from CD8+ T cells and this was associated with superior Nef-mediated MHC-I downregulation relative to less mature CD4+ T cell populations. This finding implicates differential immunoevasion as a cell-intrinsic property, influencing proviral persistence, and highlights Nef as a therapeutic target.

Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication.

Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.