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HIV-1 delivers its genetic material to infect a cell after fusion of the viral and host cell membranes, which takes place after the viral envelope (Env) binds host receptor and co-receptor proteins. Binding of host receptor CD4 to Env results in conformational changes that allow interaction with a host co-receptor (CCR5 or CXCR4). Further conformational rearrangements result in an elongated pre-hairpin intermediate structure in which Env is anchored to the viral membrane by its transmembrane region and to the host cell membrane by its fusion peptide. Although budding virions can be readily imaged by electron tomography (ET) of HIV-1-infected tissues and cultured cells, virions that are fusing (attached to host cells via pre-hairpin intermediates) are not normally visualized, perhaps because the process of membrane fusion is too fast to capture by ET. To image virions during fusion, we used fusion inhibitors to prevent downstream conformational changes in Env that lead to membrane fusion, thereby trapping HIV-1 virions linked to target cells by pre-hairpin intermediates. ET of HIV-1 pseudovirions bound to CD4 + /CCR5 + TZM-bl cells revealed presumptive pre-hairpin intermediates as 2-4 narrow spokes linking a virion to the cell surface. To extend these results to a more physiological setting, we used ET to image tissues and organs derived from humanized bone marrow/liver/thymus mice infected with HIV-1 and then treated with CPT31, a high-affinity D-peptide fusion inhibitor linked to cholesterol. Trapped HIV-1 virions were found in all tissues studied (small intestine, mesenteric lymph nodes, spleen, and bone marrow), and spokes representing pre-hairpin intermediates linking trapped virions to cell surfaces were similar in structure and number to those seen in the previous pseudovirus and cultured cell ET study.IMPORTANCETrapped and untrapped HIV-1 virions, both mature and immature, were distinguished by localizing spokes via 3D tomographic reconstructions of HIV-1 infected and fusion-inhibitor-treated tissues of humanized mice. The findings of trapped HIV-1 virions in all tissues examined demonstrate a wide distribution of the CPT31 inhibitor, a desirable property for a potential therapeutic. In addition, the presence of virions trapped by spokes, particularly in vascular endothelial cells, demonstrates that the fusion inhibitors can be used as markers for potential HIV-1-target cells within tissues, facilitating the mapping of HIV-1 target cells within the complex cellular milieu of infected tissues., Competing Interests: The authors declare no conflict of interest.

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion "closed" conformation, which is targeted by broadly neutralizing antibodies (bnAbs). CD4 binding drives Env into more "open" conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively infected cells that already downmodulated CD4. This suggests that CD4 downmodulation precedes env mRNA expression. Consequently, productively infected cells express "closed" Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1-infected humanized mice with the ADCC-mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy., Importance: Antibody-dependent cellular cytotoxicity (ADCC) represents an effective immune response for clearing virally infected cells, making ADCC-mediating antibodies promising therapeutic candidates for HIV-1 cure strategies. Broadly neutralizing antibodies (bNAbs) target epitopes present on the native "closed" envelope glycoprotein (Env), while non-neutralizing antibodies (nnAbs) recognize epitopes exposed upon Env-CD4 interaction. Here, we provide evidence that env mRNA is predominantly expressed by productively infected cells that have already downmodulated cell-surface CD4. This indicates that CD4 downmodulation by HIV-1 precedes Env expression, making productively infected cells resistant to ADCC mediated by nnAbs but sensitive to those mediated by bnAbs. These findings offer critical insights for the development of immunotherapy-based strategies aimed at targeting and eliminating productively infected cells in people living with HIV., Competing Interests: The authors declare no conflict of interest.

Importance: Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 gene editing to engineer a "mousified" version of one such host protein, cyclin T1 (CCNT1), in human T cells. CCNT1 is essential for efficient HIV-1 transcription, making it an intriguing target for gene-based inactivation of virus replication. We show that isogenic cell lines engineered to encode CCNT1 bearing a single mouse-informed amino acid change (tyrosine in place of cysteine at position 261) exhibit potent, durable, and broad-spectrum resistance to HIV-1 and other pathogenic lentiviruses, and with no discernible impact on host cell biology. These results provide proof of concept for targeting CCNT1 in the context of one or more functional HIV-1 cure strategies., Competing Interests: The authors declare no conflict of interest.

Despite remarkable progress, a cure for HIV-1 infection remains elusive. Rebound competent latent and transcriptionally active reservoir cells persevere despite antiretroviral therapy and rekindle infection due to inefficient proviral silencing. We propose a novel "block-lock-stop" approach, entailing long term durable silencing of viral expression towards an irreversible transcriptionally inactive latent provirus to achieve long term antiretroviral free control of the virus. A graded transformation of remnant HIV-1 in PLWH from persistent into silent to permanently defective proviruses is proposed, emulating and accelerating the natural path that human endogenous retroviruses (HERVs) take over millions of years. This hypothesis was based on research into delineating the mechanisms of HIV-1 latency, lessons from latency reversing agents and advances of Tat inhibitors, as well as expertise in the biology of HERVs. Insights from elite controllers and the availability of advanced genome engineering technologies for the direct excision of remnant virus set the stage for a rapid path to an HIV-1 cure.

Non-neutralizing antibodies (nnAbs) can eliminate HIV-1-infected cells via antibody-dependent cellular cytotoxicity (ADCC) and were identified as a correlate of protection in the RV144 vaccine trial. Fc-mediated effector functions of nnAbs were recently shown to alter the course of HIV-1 infection in vivo using a vpu-defective virus. Since Vpu is known to downregulate cell-surface CD4, which triggers conformational changes in the viral envelope glycoprotein (Env), we ask whether the lack of Vpu expression was linked to the observed nnAbs activity. We find that restoring Vpu expression greatly reduces nnAb recognition of infected cells, rendering them resistant to ADCC. Moreover, administration of nnAbs in humanized mice reduces viral loads only in animals infected with a vpu-defective but not with a wild-type virus. CD4-mimetics administration, known to "open" Env and expose nnAb epitopes, renders wild-type viruses sensitive to nnAbs Fc-effector functions. This work highlights the importance of Vpu-mediated evasion of humoral responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Small CD4-mimetic compounds (CD4mc) sensitize HIV-1-infected cells to antibody-dependent cellular cytotoxicity (ADCC) by facilitating antibody recognition of epitopes that are otherwise occluded on the unliganded viral envelope (Env). Combining CD4mc with two families of CD4-induced (CD4i) antibodies, which are frequently found in plasma of HIV-1-infected individuals, stabilizes Env in a conformation that is vulnerable to ADCC. We employed new-generation SRG-15 humanized mice, supporting natural killer (NK) cell and Fc-effector functions to demonstrate that brief treatment with CD4mc and CD4i-Abs significantly decreases HIV-1 replication, the virus reservoir and viral rebound after ART interruption. These effects required Fc-effector functions and NK cells, highlighting the importance of ADCC. Viral rebound was also suppressed in HIV-1+-donor cell-derived humanized mice supplemented with autologous HIV-1+-donor-derived plasma and CD4mc. These results indicate that CD4mc could have therapeutic utility in infected individuals for decreasing the size of the HIV-1 reservoir and/or achieving a functional cure., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Non-invasive bioluminescent imaging (NIBLI) of HIV-1 infection dynamics allows for real-time monitoring of viral spread and the localization of infected cell populations in living animals. In this report, we describe full-length replication-competent GFP and Nanoluciferase (Nluc) expressing HIV-1 reporter viruses from two clinical transmitted / founder (T/F) strains: TRJO.c and Q23.BG505. By infecting humanized mice with these HIV-1 T/F reporter viruses, we were able to directly monitor longitudinal viral spread at whole-animal resolution via NIBLI at a sensitivity of as few as 30-50 infected cells. Bioluminescent signal strongly correlated with HIV-1 infection and responded proportionally to virus suppression in vivo in animals treated daily with a combination antiretroviral therapy (cART) regimen. Longitudinal NIBLI following cART withdrawal visualized tissue-sites that harbored virus during infection recrudescence. Notably, we observed rebounding infection in the same lymphoid tissues where infection was first observed prior to ART treatment. Our work demonstrates the utility of our system for studying in vivo viral infection dynamics and identifying infected tissue regions for subsequent analyses., Competing Interests: The authors have declared that no competing interests exist.

Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48 h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The HIV-1 pandemic affecting over 37 million people worldwide continues, with nearly one-half of the infected population on highly active antiretroviral therapy (HAART). Major therapeutic challenges remain because of the emergence of drug-resistant HIV-1 strains, limitations because of safety and toxicity with current HIV-1 drugs, and patient compliance for lifelong, daily treatment regimens. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral polymerase have been a key component of the current HIV-1 combination drug regimens; however, these issues hamper them. Thus, the development of novel more effective NNRTIs as anti-HIV-1 agents with fewer long-term liabilities, efficacy on new drug-resistant HIV-1 strains, and less frequent dosing is crucial. Using a computational and structure-based design strategy to guide lead optimization, a 5 µM virtual screening hit was transformed to a series of very potent nanomolar to picomolar catechol diethers. One representative, compound I, was shown to have nanomolar activity in HIV-1-infected T cells, potency on clinically relevant HIV-1 drug-resistant strains, lack of cytotoxicity and off-target effects, and excellent in vivo pharmacokinetic behavior. In this report, we show the feasibility of compound I as a late-stage preclinical candidate by establishing synergistic antiviral activity with existing HIV-1 drugs and clinical candidates and efficacy in HIV-1-infected humanized [human peripheral blood lymphocyte (Hu-PBL)] mice by completely suppressing viral loads and preventing human CD4 + T-cell loss. Moreover, a long-acting nanoformulation of compound I [compound I nanoparticle (compound I-NP)] in poly(lactide-coglycolide) (PLGA) was developed that shows sustained maintenance of plasma drug concentrations and drug efficacy for almost 3 weeks after a single dose., Competing Interests: The authors declare no conflict of interest.

Dendritic cells can capture and transfer retroviruses in vitro across synaptic cell-cell contacts to uninfected cells, a process called trans-infection. Whether trans-infection contributes to retroviral spread in vivo remains unknown. Here, we visualize how retroviruses disseminate in secondary lymphoid tissues of living mice. We demonstrate that murine leukemia virus (MLV) and human immunodeficiency virus (HIV) are first captured by sinus-lining macrophages. CD169/Siglec-1, an I-type lectin that recognizes gangliosides, captures the virus. MLV-laden macrophages then form long-lived synaptic contacts to trans-infect B-1 cells. Infected B-1 cells subsequently migrate into the lymph node to spread the infection through virological synapses. Robust infection in lymph nodes and spleen requires CD169, suggesting that a combination of fluid-based movement followed by CD169-dependent trans-infection can contribute to viral spread., (Copyright © 2015, American Association for the Advancement of Science.)

Despite nearly three decades of research, a safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) has yet to be achieved. More recently, the discovery of highly potent anti-gp160 broadly neutralizing antibodies (bNAbs) has garnered renewed interest in using antibody-based prophylactic and therapeutic approaches. Here, we encoded bNAbs in first-generation adenoviral (ADV) vectors, which have the distinctive features of a large coding capacity and ease of propagation. A single intramuscular injection of ADV-vectorized bNAbs in humanized mice generated high serum levels of bNAbs that provided protection against multiple repeated challenges with a high dose of HIV-1, prevented depletion of peripheral CD4(+) T cells, and reduced plasma viral loads to below detection limits. Our results suggest that ADV vectors may be a viable option for the prophylactic and perhaps therapeutic use of bNAbs in humans.

Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir, primarily in resting memory CD4(+) T cells. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed and tested both in vitro and in vivo. A key remaining question is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad-spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.

The high genetic diversity and mutability of HIV pose a major problem for RNAi-mediated antiviral therapy. Simultaneous targeting of multiple highly conserved viral sequences has been suggested for durable cross-clade inhibition. Here we validate the approach of co-targeting two conserved sequences in the Tat and Vif genes. When coexpressed as artificial microRNA from a PolII driven miR-155-based vector, the sequences together mediated effective and sustained inhibition of HIV replication without virus breakout. To understand the nature of this efficient control, we analyzed genome sequences of 625 HIV-1 isolates in the Los Alamos Sequence database. Interestingly most natural variants were capable of wobble binding with the Tat/Vif siRNAs. Efficient silencing of reporter luciferase constructs bearing these variants residues verified that the Tat/Vif sequences together tolerated wobble binding and mediated functional RNAi. We propose the rationale of targeting highly conserved HIV sequences where wobble substitutions permit functional RNAi for global HIV repression.

Viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference (RNAi) against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested 3 viral target sites with differing propensity for mutations: a highly variable rev sequence, a gag sequence conserved only among clade B isolates, and a vif sequence highly conserved across clades. Lentiviral expression of all 3 shRNAs inhibited replication of the homologous HIV(IIIB) strain. However, they differed in their ability to protect primary CD4 T cells against multiple isolates within and across HIV clades. The least conserved rev sequence inhibited only 2 of 5 clade B isolates. The gag sequence (conserved within clade B) protected 5 of 5 clade B isolates but not other clade viruses with 2 or 3 mutations in the central region. In contrast, the vif sequence, which was conserved across clades except for single mutations at positions 14 and 17, inhibited viruses from 5 different clades. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas mutations in vif siRNA only modestly decreased silencing. Thus, although 1 or 2 mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.