The chemokine receptor variant CCR5delta32 is linked to HIV-1 resistance and other conditions. Its evolutionary history and allele frequency (10%-16%) in European populations have been extensively debated. We provide a detailed perspective of the evolutionary history of the deletion through time and space. We discovered that the CCR5delta32 allele arose on a pre-existing haplotype consisting of 84 variants. Using this information, we developed a haplotype-aware probabilistic model to screen 934 low-coverage ancient genomes and traced the origin of the CCR5delta32 deletion to at least 6,700 years before the present (BP) in the Western Eurasian Steppe region. Furthermore, we present strong evidence for positive selection acting upon the CCR5delta32 haplotype between 8,000 and 2,000 years BP in Western Eurasia and show that the presence of the haplotype in Latin America can be explained by post-Columbian genetic exchanges. Finally, we point to complex CCR5delta32 genotype-haplotype-phenotype relationships, which demand consideration when targeting the CCR5 receptor for therapeutic strategies.

The capacity of host cells to sustain or restrict virus infection is influenced by their proteome. Understanding the compendium of proteins defining cellular permissiveness is key to many questions in fundamental virology. Here, we apply a multi-omic approach to determine the proteins that are associated with highly permissive, intermediate, and hostile cellular states. We observed two groups of differentially regulated genes: (i) with robust changes in mRNA and protein levels and (ii) with protein/RNA discordances. While many of the latter are classified as interferon-stimulated genes (ISGs), most exhibit no antiviral effects in overexpression screens. This suggests that IFN-dependent protein changes can be better indicators of antiviral function than mRNA levels. Phosphoproteomics revealed an additional regulatory layer involving non-signaling proteins with altered phosphorylation. Indeed, we confirmed that several permissiveness-associated proteins with changes in abundance or phosphorylation regulate infection fitness. Altogether, our study provides a comprehensive and systematic map of the cellular alterations driving virus susceptibility.

Binding to cellular receptors initiates viral replication and dictates sites in the host infected by the virus. As illustrated by mammalian orthoreovirus (reovirus), viruses can bind several types of receptors using distinct capsid components to facilitate the viral entry steps of attachment, internalization, and disassembly. The outer of the two concentric capsids of reovirus virions is formed by four viral proteins, three of which bind receptors. These capsid-receptor interactions mediate stepwise entry of reovirus, dictate viral tropism in infected animals, and expand the viral host range. Engagement of independent receptors by different capsid proteins is a property of many pathogenic viruses and illustrates common themes of receptor use in viral entry and disease.

HIV-1 envelope glycoproteins (Envs) mediate viral entry and are sole target of neutralizing antibodies. Thus, HIV-1 Envs must maintain a delicate balance between evading neutralizing antibodies while still preserving viral compatibility to mediate entry into target cells. Here, we studied the viral entry effeciency, fitness, and replication of an incompletely closed, transmitted/founder HIV-1 Envs (CH040), which are highly resistant to most bnAbs. CH040 Envs mediated HIV-1 entry to target cells as efficient as other primary Envs, suggesting that antibody resistance and efficient viral entry can develop independently. Expression of CH040 Envs was comparable to other Envs and most CH040 variants that were rationally engineered to increase bnAb resistance showed no significant decrease in their ability to mediate HIV-1 entry. We detected robust in vitro spread of SHIV CH040 in pig-tailed macaque lymphocytes that was comparable to efficient spread of other SHIVs. Our study provides insights into the relationship between bnAb resistance and efficient HIV-1 entry.

People living with HIV (PLWH) experience HIV-associated neurocognitive disorders (HAND), even though combination antiretroviral therapy (cART) suppresses HIV replication. HIV-1 transactivator of transcription (HIV-1 Tat) contributes to the development of HAND through neuroinflammatory and neurotoxic mechanisms. C-C chemokine 5 receptor (CCR5) is important in immune cell targeting and is a co-receptor for HIV viral entry into CD4+ cells. Notably, CCR5 has been implicated in cognition unrelated to HIV infection. Inhibition of CCR5 has been shown to improve learning and memory. To test whether CCR5 is involved in cognitive changes in HAND, we used a non-infectious, transgenic model in which HIV-1 Tat is inducibly expressed. Well-powered cohorts of male and female mice were placed on a diet containing doxycycline to induce Tat expression for 8-wks. Males showed Tat-mediated deficits in the Barnes maze test of spatial learning and memory; females showed no impairments. Deficits in the males were fully reversed by the CCR5 antagonist, maraviroc (MVC). Tat-mediated deficits were not found in novel object recognition or contextual fear conditioning in either sex. Based on earlier work, we hypothesized that MVC might increase brain-derived neurotrophic factor (BDNF), which is essential in maintaining synaptodendritic function. MVC did increase the mBDNF to proBDNF ratio in males, perhaps contributing to improved cognition.

Long-lasting HIV remission has been reported in a small group of people with HIV (PWH) following allogenic hematopoietic stem cell transplants (HSCT) for the treatment of hematologic malignancies. While the mechanisms of HIV remission following release from antiretroviral therapy (ART) were not initially known, subsequent findings from clinical cases and preclinical nonhuman primate studies have implicated mechanisms of clearance. Here, we review the six currently published human cases of long-term ART-free HIV remission.

Since the first report of ART-free HIV remission following HSCT, five subsequent cases of HSCT-induced sustained HIV remission have been published. While the pre- and posttransplant treatment conditions vary greatly, all but one received cells from donors homozygous for a 32 bp deletion in the gene that encodes CCR5 ( ccr5Δ32 ), the major HIV coreceptor. Studies in nonhuman primates and the newest published individual suggest that while CCR5 deficiency can protect donor cells from infection early posttransplant, it is not required for long term remission, as ablation of the viral reservoir is likely due to allogeneic immunity mediating a graft-versus-reservoir response.

Studies of HSCT in PLWH and simian immunodeficiency virus (SIV)-infected monkeys show that those with durable remission are likely cured, demonstrated by complete ablation of the replication-competent HIV reservoir, gradual loss of anti-HIV immunity, and greater than 5 years of aviremia.

CXCR4, a chemokine receptor known as Fusin or CD184, spans the outer membrane of various human cells, including leukocytes. This receptor is essential for HIV infection as well as for many vital cellular processes and is implicated to be associated with multiple pathologies, including cancers. This study employs various computational tools to investigate the molecular effects of disease-vulnerable germ-line missense and non-coding SNPs of the CXCR4 gene. In this investigation, the tools SIFT, PROVEAN, PolyPhen-2, PANTHER, SNAP 2.0, PhD-SNP, and SNPs&GO were used to predict potentially harmful and disease-causing nsSNPs in CXCR4. Additionally, their impact on protein stability was examined by I-mutant 3.0, MUpro, Consurf, and Netsurf 2.0, combined with conservation and solvent accessibility analyses. Structural analysis with normal and mutant residues of the protein harboring these disease-associated functional SNPs was conducted using TM-align and SWIS MODEL, with visualization aided by PyMOL and the BIOVINA Discovery Studio Visualizer. The functional impact of wild-type and mutated CXCR4 variants was evaluated through molecular docking with its natural ligand CXCR4-modulator 1, using the PyRx tool. Non-coding SNPs in the 3' -UTR were investigated for their regulatory effects on miRNA binding sites using PolymiRTS. Five non-coding SNPs were identified in the 3'-UTR that can disrupt existing miRNA binding sites or create new ones. Non-coding SNPs in the 5' and 3'-UTRs, as well as in intronic regions, were also examined for their potential roles in gene expression regulation. Furthermore, RegulomeDB databases were employed to assess the regulatory potential of these non-coding SNPs based on chromatin state and protein binding regulation. In the mostly annotated variant (ENSP00000241393) of the CXCR4 gene, we found 23 highly deleterious and pathogenic nsSNPs and these were selected for in-depth analysis. Among the 23 nsSNPs, five (G55V, H79P, L80P, H113P, and P299L) displayed notable structural alternation, with elevated RMSD values and reduced TM (TM-score) values. A molecular docking study revealed the significant impact of the H113P variant on the protein-ligand binding affinity, supported by MD simulation over 100 nanoseconds, which highlighted substantial stability differences between wild-type and H113P mutated proteins during ligand binding. This comprehensive analysis shed light on the potential functional consequences of genetic variation in the CXCR genes, offering valuable insights into the implications of disease susceptibility and may pave the way for future therapeutic interventions.

Transcription repressor BACH2 redirects short-lived terminally differentiated effector into long-lived memory cells. We postulate that BACH2-mediated long-lived memory programs promote HIV-1 persistence in gut CD4+ T cells. We coupled single-cell DOGMA-seq and TREK-seq to capture chromatin accessibility, transcriptome, surface proteins, T cell receptor, HIV-1 DNA and HIV-1 RNA in 100,744 gut T cells from ten aviremic HIV-1+ individuals and five HIV-1- donors. BACH2 was the leading transcription factor that shaped gut tissue resident memory T cells (TRMs) into long-lived memory with restrained interferon-induced effector function. We found that HIV-1-infected cells were enriched in TRMs (80.8%). HIV-1-infected cells had increased BACH2 transcription factor accessibility, TRM (CD49a, CD69, CD103) and survival (IL7R) gene expression, and Th17 polarization (RORC, CCR6). In vitro gut CD4+ T cell infection revealed preferential infection and persistence of HIV-1 in CCR6+ TRMs. Overall, we found BACH2-driven TRM program promotes HIV-1 persistence and BACH2 as a new therapeutic target.

Introducing students to primary scientific literature is essential for establishing scientific literacy; however, students can feel overwhelmed by the amount of information within a research article. In our virology discussion courses, we address this issue by creating a student-centered and active learning environment. Students present and guide discussions on the rationales, background, methods, results, and conclusions from research literature in class. This approach has been applied in both undergraduate and graduate settings for students studying microbiology, with different expectations and criteria between the two groups. Student evaluations collected from three semesters were positive toward the teaching methods. Students praised the inclusion of "landmark discovery" articles, which examined paradigm-shifting concepts in virology. Undergraduates expressed their increased confidence in comprehending scientific literature and highlighted the impact of group work. In a critique of the course, students suggested more variety in the topics covered and inclusion of recent publications. Overall, our enhanced approach and methods improved student experiences with primary scientific literature and promoted student learning.

Protein homeostasis is maintained by the opposing action of ubiquitin ligase and deubiquitinase, two important components of the ubiquitin-proteasome pathway, and contributes to both normal physiological and pathophysiological processes. The current study aims to delineate the roles of ubiquitin-specific protease 15 (USP15), a member of the largest deubiquitinase family, in HIV-1 gene expression and replication.

We took advantage of highly selective and specific ubiquitin variants (UbV), which were recently designed and developed for USP15, and ascertained the inhibitory effects of USP15 on HIV-1 gene expression and production by transfection and Western blotting. We also used real-time RT-PCR, transcription factor profiling, subcellular fractionation, immunoprecipitation followed by Western blotting to determine the transcription factors involved and the underlying molecular mechanisms.

We first confirmed the specificity of USP15-mediated HIV-1 gene expression and virus production. We then showed that the inhibition of HIV-1 production by USP15 occurred at the transcription level, associated with an increased protein level of YY1, a known HIV-1 transcription repressor. Moreover, we demonstrated that USP15 regulated YY1 deubiquitination and stability. Lastly, we demonstrated that YY1 siRNA knockdown significantly diminished the inhibition of USP15 on HIV-1 gene expression and virus production.

These findings together demonstrate that stabilization of YY1 protein by USP15 deubiquitinating activity contributes to USP15-mediated inhibition of HIV-1 transcription and may help the development of USP15-specific UbV inhibitors as an anti-HIV strategy.

CCR5 is a class A GPCR and serves as one of the coreceptors facilitating HIV-1 entry into host cells. This receptor has vital roles in the immune system and is involved in the pathogenesis of different diseases. Various studies were conducted to understand its activation mechanism, including structural studies in which inactive and active states of the receptor were determined in complex with various binding partners. These determined structures provided opportunities to perform molecular dynamics (MD) simulations and to analyze conformational changes observed in the protein structures. The atomic-level dynamic studies allow us to explore the effects of ionizable residues on the receptor. Here, our aim was to investigate the conformational changes in CCR5 when it forms a complex with either the inhibitor maraviroc (MRV), an approved anti-HIV drug, or HIV-1 envelope protein GP120, and compare these changes to the receptor's apo form. In our simulations, we considered both ionized and protonated states of ionizable binding site residue GLU2837.39 in CCR5 as the protonation state of this residue was considered ambiguously in previous studies. Our molecular simulations results suggested that in fact, the change in the protonation state of GLU2837.39 caused interaction profiles to be different between CCR5 and its binding partners, GP120 or MRV. We observed that when the protonated state of GLU2837.39 was considered in complex with the envelope protein GP120, there were substantial structural changes in CCR5, indicating that it adopts a more active-like conformation. On the other hand, CCR5 in complex with MRV always adopted an inactive conformation regardless of the protonation state. Hence, the CCR5 coreceptor displays conformational heterogeneity not only depending on its binding partner but also influenced by the protonation state of the binding site binding site residue GLU2837.39. This outcome is also in accordance with some studies showing that GP120 binding could activate signaling pathways. This outcome could also have significant implications for discovering novel CCR5 inhibitors as anti-HIV drugs using in silico methods such as molecular docking, as it may be necessary to consider the protonated state of GLU2837.39.

Viral infections in humans often cause excessive inflammation. In some viral infections, inflammation can be serious and even fatal, while in other infections it can promote viral clearance. Viruses can escape from the host immune system via regulating inflammatory pathways, thus worsening the illness. MicroRNAs (miRNAs) are tiny non-coding RNA molecules expressed within diverse tissues as well as cells and are engaged in different normal pathological and physiological pathways. Emerging proof suggests that miRNAs can impact innate and adaptive immunity, inflammatory responses, cell invasion, and the progression of viral infections. We discuss some intriguing new findings in the current work, focusing on the impacts of different miRNAs on host inflammatory responses and virus-mediated inflammation. A better understanding of dysregulated miRNAs in viral infections could improve the identification, prevention, and treatment of several serious diseases.

HIV accelerates liver fibrosis attributable to multiple etiologies, including HCV, HBV, and steatotic liver disease. Evidence also suggests that HIV infection itself is associated with liver fibrogenesis. Recent studies have implicated Yes-associated protein 1 (YAP1) and the upstream lysophosphatidic acid (LPA)/PI3K/AKT pathway as critical regulators of hepatic fibrogenesis, and suggest a connection to HIV-related liver fibrosis. However, the relationship between YAP/PI3K/AKT pathway activation and HIV-related liver fibrosis remains uncertain.

qPCR, western blot, immunofluorescence, and ELISA (replicates n ≥3) were performed in an unbiased humanized mouse model (NRG-hu HSC mice, n = 6), the precision cut liver slice ex vivo model, and both traditional in vitro models as well as a 3D spheroid system.

YAP target gene mRNA and protein levels (ANKRD, CTGF, CYR61) were upregulated across all models exposed to HIV. Humanized mice infected with HIV had significant increases in the percentage of YAP-positive nuclei (2.2-fold) and the percentage area of Sirius Red collagen staining (3.3-fold) compared to control mice. Serum concentrations of LPA were increased 5.8-fold in people living with HIV compared to healthy controls. Modulation of LPAR1, PI3K, and AKT by either inhibitors or small-interfering RNAs abrogated the fibrotic effects of HIV exposure and downregulated YAP target genes within cultured liver cells.

The LPAR/PI3K/AKT axis is vital for the activation of YAP and hepatic fibrogenesis due to HIV infection. This novel mechanistic insight suggests new pharmacologic targets for treatment of liver fibrosis in people living with HIV.

There are currently no FDA-approved treatments for cirrhosis, while liver disease is the second leading cause of mortality among people living with HIV after AIDS. Increased lysophosphatidic acid concentrations and AKT activation after HIV infection found in recent work suggest that the Hippo pathway may be a key regulator of HIV-related fibrogenesis. By linking lysophosphatidic acid signaling, YAP activation, and HIV-related fibrogenesis, this mechanism presents a target for future research into therapeutic interventions for not only HIV but also other liver diseases, e.g. metabolic dysfunction- or alcohol-associated liver disease.

HIV-1 envelope glycoproteins (Envs) of most primary HIV-1 strains exist in closed conformation and infrequently sample open states, limiting access to internal epitopes. Thus, immunogen design aims to mimic the closed Env conformation as preferred target for eliciting broadly neutralizing antibodies (bnAbs). Here we identify incompletely closed Env conformations of 6 out of 13 transmitted/founder (T/F) strains that are sensitive to antibodies that recognize internal epitopes typically exposed on open Envs. A 3.6 Å cryo-electron microscopy structure of unliganded, incompletely closed T/F Envs (1059-SOSIP) reveals protomer motion that increased sampling of states with incompletely closed trimer apex. We reconstruct de novo the post-transmission evolutionary pathway of a second T/F. Evolved viruses exhibit increased Env resistance to cold, soluble CD4 and 19b, all of which correlate with closing of the adapted Env trimer. Lastly, we show that the ultra-broad N6 bnAb efficiently recognizes different Env conformations and exhibits improved antiviral breadth against VRC01-resistant Envs isolated during the first-in-humans antibody-mediated-prevention trial.

Interactions between human immunodeficiency virus type 1 (HIV-1) and the host factors or restriction factors of its target cells determine the cell's susceptibility to, and outcome of, infection. Factors intrinsic to the cell are involved at every step of the HIV-1 replication cycle, contributing to productive infection and replication, or severely attenuating the chances of success. Furthermore, factors unique to certain cell types contribute to the differences in infection between these cell types. Understanding the involvement of these factors in HIV-1 infection is a key requirement for the development of anti-HIV-1 therapies. As the list of factors grows, and the dynamic interactions between these factors and the virus are elucidated, comprehensive and up-to-date summaries that recount the knowledge gathered after decades of research are beneficial to the field, displaying what is known so that researchers can build off the groundwork of others to investigate what is unknown. Herein, we aim to provide a review focusing on protein host factors, both well-known and relatively new, that impact HIV-1 replication in a positive or negative manner at each stage of the replication cycle, highlighting factors unique to the various HIV-1 target cell types where appropriate.

The secretome is composed of cell surface membrane proteins and extracellular secreted proteins that are synthesized via secretory machinery, accounting for approximately one-third of human protein-encoding genes and playing central roles in cellular communication with the external environment. Secretome protein-protein interactions (SPPIs) mediate cell proliferation, apoptosis, and differentiation, as well as stimulus- or cell-specific responses that regulate a diverse range of biological processes. Aberrant SPPIs are associated with diseases including cancer, immune disorders, and illness caused by infectious pathogens. Identifying the receptor/ligand for a secretome protein or pathogen can be a challenging task, and many SPPIs remain obscure, with a large number of orphan receptors and ligands, as well as viruses with unknown host receptors, populating the SPPI network. In addition, proteins with known receptors/ligands may also interact with alternative uncharacterized partners and exert context-dependent effects. In the past few decades, multiple varied approaches have been developed to identify SPPIs, and these methods have broad applications in both basic and translational research. Here, we review and discuss the technologies for SPPI profiling and the application of these technologies in identifying novel targets for immunotherapy and anti-infectious agents.

The chemokine co-receptors CXCR4 and CCR5 mediate HIV entry and signal transduction necessary for viral infection. However, to date only the CCR5 antagonist maraviroc is approved for treating HIV-1 infection. Given that approximately 50% of late-stage HIV patients also develop CXCR4-tropic virus, clinical anti-HIV CXCR4 antagonists are needed. Here, we describe a novel allosteric CXCR4 antagonist TIQ-15 which inhibits CXCR4-tropic HIV-1 infection of primary and transformed CD4 T cells. TIQ-15 blocks HIV entry with an IC50 of 13 nM. TIQ-15 also inhibits SDF-1α/CXCR4-mediated cAMP production, cofilin activation, and chemotactic signaling. In addition, TIQ-15 induces CXCR4 receptor internalization without affecting the levels of the CD4 receptor, suggesting that TIQ-15 may act through a novel allosteric site on CXCR4 for blocking HIV entry. Furthermore, TIQ-15 did not inhibit VSV-G pseudotyped HIV-1 infection, demonstrating its specificity in blocking CXCR4-tropic virus entry, but not CXCR4-independent endocytosis or post-entry steps. When tested against a panel of clinical isolates, TIQ-15 showed potent inhibition against CXCR4-tropic and dual-tropic viruses, and moderate inhibition against CCR5-tropic isolates. This observation was followed by a co-dosing study with maraviroc, and TIQ-15 demonstrated synergistic activity. In summary, here we describe a novel HIV-1 entry inhibitor, TIQ-15, which potently inhibits CXCR4-tropic viruses while possessing low-level synergistic activities against CCR5-tropic viruses. TIQ-15 could potentially be co-dosed with the CCR5 inhibitor maraviroc to block viruses of mixed tropisms.

A highly critical event in a virus's life cycle is successfully entering a given host. This process begins when a viral glycoprotein interacts with a target cell receptor, which provides the molecular basis for target virus-host cell interactions for novel drug discovery. Over the years, extensive research has been carried out in the field of virus-host cell interaction, generating a massive number of genetic and molecular data sources. These datasets are an asset for predicting virus-host interactions at the molecular level using machine learning (ML), a subset of artificial intelligence (AI). In this direction, ML tools are now being applied to recognize patterns in these massive datasets to predict critical interactions between virus and host cells at the protein-protein and protein-sugar levels, as well as to perform transcriptional and translational analysis. On the other end, deep learning (DL) algorithms-a subfield of ML-can extract high-level features from very large datasets to recognize the hidden patterns within genomic sequences and images to develop models for rapid drug discovery predictions that address pathogenic viruses displaying heightened affinity for receptor docking and enhanced cell entry. ML and DL are pivotal forces, driving innovation with their ability to perform analysis of enormous datasets in a highly efficient, cost-effective, accurate, and high-throughput manner. This review focuses on the complexity of virus-host cell interactions at the molecular level in light of the current advances of ML and AI in viral pathogenesis to improve new treatments and prevention strategies.

Atherosclerotic vascular disease disproportionately affects persons living with HIV (PLWH) compared to those without. The reasons for the excess risk include dysregulated immune response and inflammation related to HIV infection itself, comorbid conditions, and co-infections. Here, we review an updated understanding of immune and inflammatory pathways underlying atherosclerosis in PLWH, including effects of viral products, soluble mediators and chemokines, innate and adaptive immune cells, and important co-infections. We also present potential therapeutic targets which may reduce cardiovascular risk in PLWH.

CCR5-tropic viruses are preferentially transmitted during perinatal HIV-1 infection. CCR5 density on CD4 + T-cells likely impacts susceptibility to HIV-1 infection.

Fifty-two mother-infant dyads were enrolled. All mothers were living with HIV-1, 27 of the infants acquired HIV-1 in utero and 25 infants remained uninfected.

CCR5 density, together with frequencies of CD4 + and CD8 + T-cells expressing immune activation (CCR5, ICOS and HLA-DR) and immune checkpoint (TIGIT and PD-1) markers, were measured in whole blood from the dyads close to delivery.

Compared with mothers who did not transmit, mothers who transmitted HIV-1 had less exposure to ART during pregnancy ( P = 0.015) and higher plasma viral load close to delivery ( P = 0.0005). These mothers, additionally, had higher CCR5 density on CD4 + and CD8 + T-cells and higher frequencies of CCR5, ICOS and TIGIT-expressing CD8 + T-cells. Similarly, compared with infants without HIV-1, infants with HIV-1 had higher CCR5 density on CD4 + and CD8 + T-cells and higher frequencies of CCR5, TIGIT, and PD-1-expressing CD4 + and CD8 + T-cells as well as higher frequencies of HLA-DR-expressing CD8 + T-cells. CCR5 density on maternal CD4 + T-cells remained significantly associated with transmission after adjusting for maternal viral load and CD4 + T cell counts. Mother-infant dyads with shared high CCR5 density phenotypes had the highest risk of transmission/acquisition of infection compared with dyads with shared low-CCR5 density phenotypes.

This study provides strong evidence of a protective role for a combined mother-infant low CD4 + T-cell CCR5 density phenotype in in-utero transmission/acquisition of HIV-1.

Background: The mechanisms of the formation of immunological competence against tuberculosis (TB), and especially those associated with HIV co-infection, remain poorly understood. However, there is an urgent need for risk recurrence predictive biomarkers, as well as for predictors of successful treatment outcomes. The goal of the study was to identify possible immunological markers of TB recurrence in individuals with HIV/TB co-infection. Methods: The plasma levels of IFN-γ, TNF-α, IL-10, and IL-1β (cytokines which play important roles in the immune activation and protection against Mycobacterium tuberculosis) were measured using ELISA EIA-BEST kits. The cytokine concentrations were determined using a standard curve obtained with the standards provided by the manufacturer of each kit. Results: A total of 211 individuals were enrolled in the study as follows: 62 patients with HIV/TB co-infection, 52 with HIV monoinfection, 52 with TB monoinfection, and 45 healthy donors. Out of the 62 patients with HIV/TB, 75.8% (47) of patients were newly diagnosed with HIV and TB, and 24.2% (15) displayed recurrent TB and were newly diagnosed with HIV. Decreased levels of IFN-γ, TNF-α, and IL-10 were observed in patients with HIV/TB when compared with HIV and TB patients. However, there was no difference in IFN-γ, TNF-α, or IL-10 secretion between both HIV/TB groups. At the same time, an almost 4-fold decrease in Il-1β levels was detected in the HIV/TB group with TB recurrence when compared with the HIV/TB group (p = 0.0001); a 2.8-fold decrease when compared with HIV patients (p = 0.001); and a 2.2-fold decrease with newly diagnosed TB patients (p = 0.001). Conclusions: Significantly decreased Il-1β levels in HIV/TB patients' cohort with secondary TB indicate that this cytokine can be a potential biomarker of TB recurrence.

Latency reversing agents (LRAs) such as protein kinase C (PKC) modulators can reduce rebound-competent HIV reservoirs in small animal models. Furthermore, administration of natural killer (NK) cells following LRA treatment improves this reservoir reduction. It is currently unknown why the combination of a PKC modulator and NK cells is so potent and whether exposure to PKC modulators may augment NK cell function in some way.

Primary human NK cells were treated with PKC modulators (bryostatin-1, prostratin, or the designed, synthetic bryostatin-1 analog SUW133), and evaluated by examining expression of activation markers by flow cytometry, analyzing transcriptomic profiles by RNA sequencing, measuring cytotoxicity by co-culturing with K562 cells, assessing cytokine production by Luminex assay, and examining the ability of cytokines and secreted factors to independently reverse HIV latency by co-culturing with Jurkat-Latency (J-Lat) cells.

PKC modulators increased expression of proteins involved in NK cell activation. Transcriptomic profiles from PKC-treated NK cells displayed signatures of cellular activation and enrichment of genes associated with the NFκB pathway. NK cell cytotoxicity was unaffected by prostratin but significantly decreased by bryostatin-1 and SUW133. Cytokines from PKC-stimulated NK cells did not induce latency reversal in J-Lat cell lines.

Although PKC modulators have some significant effects on NK cells, their contribution in "kick and kill" strategies is likely due to upregulating HIV expression in CD4+ T cells, not directly enhancing the effector functions of NK cells. This suggests that PKC modulators are primarily augmenting the "kick" rather than the "kill" arm of this HIV cure approach.

Induction of antibodies (Abs) against the conformational CD4-induced (CD4i) epitope is frequent in HIV-1 infection. However, the mechanism of development of anti-CD4i Abs is unclear. We used anti-idiotypic (aID) monoclonal Abs (mAbs) of anti-CD4i mAbs to isolate anti-CD4i mAbs from infected subjects and track the causative antigens. One anti-aID mAb sorted from infected subjects by aID mAbs had the characteristics of anti-CD4i Abs, including IGHV1-69 usage and ability to bind to HIV-1 Env enhanced by sCD4. Critical amino acid sequences for the binding of six anti-aID mAbs, with shared idiotope to anti-CD4i mAbs, were analysed by phage display. The identified amino acid sequences showed similarity to proteins from human microbiota and infectious agents. Peptides synthesized from Caudoviricetes sp and Vibrio vulnificus based on the identified sequences were reactive to most anti-aID and some anti-CD4i mAbs. These results suggest that anti-CD4i Abs may evolve from B cells primed by microorganisms.

Structural dynamics of human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein mediate cell entry and facilitate immune evasion. Single-molecule FRET using peptides for Env labeling revealed structural dynamics of Env, but peptide use risks potential effects on structural integrity/dynamics. While incorporating noncanonical amino acids (ncAAs) into Env by amber stop-codon suppression, followed by click chemistry, offers a minimally invasive approach, this has proved to be technically challenging for HIV-1. Here, we develope an intact amber-free HIV-1 system that overcomes hurdles of preexisting viral amber codons. We achieved dual-ncAA incorporation into Env on amber-free virions, enabling single-molecule Förster resonance energy transfer (smFRET) studies of click-labeled Env that validated the previous peptide-based labeling approaches by confirming the intrinsic propensity of Env to dynamically sample multiple conformational states. Amber-free click-labeled Env also enabled real-time tracking of single virion internalization and trafficking in cells. Our system thus permits in-virus bioorthogonal labeling of proteins, compatible with studies of virus entry, trafficking, and egress from cells.

Changing the course of the human immunodeficiency virus type I (HIV-1) pandemic is a high public health priority with approximately 39 million people currently living with HIV-1 (PLWH) and about 1.5 million new infections annually worldwide. Broadly neutralizing antibodies (bnAbs) typically target highly conserved sites on the HIV-1 envelope glycoproteins (Envs), which mediate viral entry, and block the infection of diverse HIV-1 strains. But different mechanisms of HIV-1 resistance to bnAbs prevent robust application of bnAbs for therapeutic and preventive interventions.

Here we report the development of a new database that provides data and computational tools to aid the discovery of resistant features and may assist in analysis of HIV-1 resistance to bnAbs. Bioinformatic tools allow identification of specific patterns in Env sequences of resistant strains and development of strategies to elucidate the mechanisms of HIV-1 escape; comparison of resistant and sensitive HIV-1 strains for each bnAb; identification of resistance and sensitivity signatures associated with specific bnAbs or groups of bnAbs; and visualization of antibody pairs on cross-sensitivity plots. The database has been designed with a particular focus on user-friendly and interactive interface. Our database is a valuable resource for the scientific community and provides opportunities to investigate patterns of HIV-1 resistance and to develop new approaches aimed to overcome HIV-1 resistance to bnAbs.

HIResist is freely available at https://hiresist.ahc.umn.edu/.

Chemokines are important immune system proteins, many of which mediate inflammation due to their function to activate and cause chemotaxis of leukocytes. An important anti-inflammatory strategy is therefore to bind and inhibit chemokines, which leads to the need for biophysical studies of chemokines as they bind various possible partners. Because a successful anti-chemokine drug should bind at low concentrations, techniques such as fluorescence anisotropy that can provide nanomolar signal detection are required. To allow fluorescence experiments to be carried out on chemokines, a method is described for the production of fluorescently labeled chemokines. First, a fusion-tagged chemokine is produced in Escherichia coli, then efficient cleavage of the N-terminal fusion partner is carried out with lab-produced enterokinase, followed by covalent modification with a fluorophore, mediated by the lab-produced sortase enzyme. This overall process reduces the need for expensive commercial enzymatic reagents. Finally, we utilize the product, vMIP-fluor, in binding studies with the chemokine binding protein vCCI, which has great potential as an anti-inflammatory therapeutic, showing a binding constant for vCCI:vMIP-fluor of 0.37 ± 0.006 nM. We also show how a single modified chemokine homolog (vMIP-fluor) can be used in competition assays with other chemokines and we report a Kd for vCCI:CCL17 of 14 μM. This work demonstrates an efficient method of production and fluorescent labeling of chemokines for study across a broad range of concentrations.

The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein precursor (gp160) trimerizes, is modified by high-mannose glycans in the endoplasmic reticulum, and is transported via Golgi and non-Golgi secretory pathways to the infected cell surface. In the Golgi, gp160 is partially modified by complex carbohydrates and proteolytically cleaved to produce the mature functional Env trimer, which is preferentially incorporated into virions. Broadly neutralizing antibodies (bNAbs) generally recognize the cleaved Env trimer, whereas poorly neutralizing antibodies (pNAbs) bind the conformationally flexible gp160. We found that expression of bNAbs, pNAbs, or soluble/membrane forms of the receptor, CD4, in cells producing HIV-1 all decreased viral infectivity. Four patterns of co-expressed ligand:Env were observed: (i) ligands (CD4, soluble CD4-Ig, and some pNAbs) that specifically recognize the CD4-bound Env conformation resulted in uncleaved Envs lacking complex glycans that were not incorporated into virions; (ii) other pNAbs produced Envs with some complex carbohydrates and severe defects in cleavage, which were relieved by brefeldin A treatment; (iii) bNAbs that recognize gp160 as well as mature Envs resulted in Envs with some complex carbohydrates and moderate decreases in virion Env cleavage; and (iv) bNAbs that preferentially recognize mature Envs produced cleaved Envs with complex glycans in cells and on virions. The low infectivity observed upon co-expression of pNAbs or CD4 could be explained by disruption of Env trafficking, reducing the level of Env and/or increasing the fraction of uncleaved Env on virions. In addition to bNAb effects on virion Env cleavage, the secreted bNAbs neutralized the co-expressed viruses.IMPORTANCEThe Env trimers on the HIV-1 mediate virus entry into host cells. Env is synthesized in infected cells, modified by complex sugars, and cleaved to form a mature, functional Env, which is incorporated into virus particles. Env elicits antibodies in infected individuals, some of which can neutralize the virus. We found that antibodies co-expressed in the virus-producing cell can disrupt Env transit to the proper compartment for cleavage and sugar modification and, in some cases, block incorporation into viruses. These studies provide insights into the processes by which Env becomes functional in the virus-producing cell and may assist attempts to interfere with these events to inhibit HIV-1 infection.

The type-I interferon (IFN) response constitutes the major innate immune pathway against viruses in mammals. Despite its critical importance for antiviral defence, this pathway is inactive during early embryonic development. There seems to be an incompatibility between the IFN response and pluripotency, the ability of embryonic cells to develop into any cell type of an adult organism. Instead, pluripotent cells employ alternative ways to defend against viruses that are typically associated with safeguard mechanisms against transposable elements. The absence of an inducible IFN response in pluripotent cells and the constitutive activation of the alternative antiviral pathways have led to the hypothesis that embryonic cells are highly resistant to viruses. However, some findings challenge this interpretation. We have performed a meta-analysis that suggests that the susceptibility of pluripotent cells to viruses is directly correlated with the presence of receptors or co-receptors for viral adhesion and entry. These results challenge the current view of pluripotent cells as intrinsically resistant to infections and raise the fundamental question of why these cells have sacrificed the major antiviral defence pathway if this renders them susceptible to viruses.

Chemokines are cytokines whose primary role is cellular activation and stimulation of leukocyte migration. They perform their various functions by interacting with G protein-coupled cell surface receptors (GPCRs) and are involved in the regulation of many biological processes such as apoptosis, proliferation, angiogenesis, hematopoiesis or organogenesis. They contribute to the maintenance of the homeostasis of lymphocytes and coordinate the function of the immune system. However, chemokines and their receptors are sometimes hijacked by some pathogens to infect the host organism. For a given chemokine receptor, there is a wide structural, organizational and conformational diversity. In this review, we describe the evidence for structural variety reported for the chemokine receptor CCR5, how this variability can be exploited by HIV-1 to infect its target cells and what therapeutic solutions are currently being developed to overcome this problem.

Trogocytosis is the intercellular transfer of membrane and membrane-associated proteins between cells. Trogocytosis is an underappreciated phenomenon that has historically routinely been dismissed as an artefact. With a greater understanding of the process and the implications it has on biological systems, trogocytosis has the potential to become a paradigm changer. The presence on a cell of molecules they don't endogenously express can alter the biological activity of the cell and could also lead to the acquisition of new functions. To better appreciate this phenomenon, it is important to understand how these intercellular membrane exchanges influence the function and activity of the donor and the recipient cells. In this chapter, we will examine how the molecules acquired by trogocytosis influence the biology of a variety of systems including mammalian fertilization, treatment of hemolytic disease of the newborn, viral and parasitic infections, cancer immunotherapy, and immune modulation.

Viruses require host cells to replicate and proliferate, which indicates that viruses hijack the cellular machinery. Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4-positive T cells, and efficiently uses cellular proteins to replicate. Cells already have proteins that inhibit the replication of the foreign HIV-1, but their function is suppressed by viral proteins. Intriguingly, HIV-1 infection also changes the cellular metabolism to aerobic glycolysis. This phenomenon has been interpreted as a cellular response to maintain homeostasis during viral infection, yet HIV-1 efficiently replicates even in this environment. In this review, we discuss the regulatory role of glycolytic enzymes in viral replication and the impact of aerobic glycolysis on viral infection by introducing various host proteins involved in viral replication. Furthermore, we would like to propose a "glyceraldehyde-3-phosphate dehydrogenase-induced shock (G-shock) and kill strategy" that maximizes the antiviral effect of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to eliminate latently HIV-1-infected cells.

The class I HLA genotype has been widely recognized as a factor influencing HIV disease progression in treatment-naïve subjects. However, little is known regarding its role in HIV disease course and how it influences the size of the viral reservoir once anti-retroviral therapy (ART) is started. Here, leveraging on cutting-edge bioinformatic tools, we explored the relationship between HLA class I and the HIV reservoir in a cohort of 90 people living with HIV (PLWH) undergoing ART and who achieved viral suppression. Analysis of HLA allele distribution among patients with high and low HIV reservoir allowed us to document a predominant role of HLA-B and -C genes in regulating the size of HIV reservoir. We then focused on the analysis of HIV antigen (Ag) repertoire, by investigating immunogenetic parameters such as the degree of homozygosity, HLA evolutionary distance and Ag load. In particular, we used two different bioinformatic algorithms, NetMHCpan and MixMHCpred, to predict HLA presentation of immunogenic HIV-derived peptides and identified HLA-B*57:01 and HLA-B*58:01 among the highest ranking HLAs in terms of total load, suggesting that their previously reported protective role against HIV disease progression might be linked to a more effective viral recognition and presentation to Cytotoxic T lymphocytes (CTLs). Further, we speculated that some peptide-HLA complexes, including those produced by the interaction between HLA-B*27 and the HIV Gag protein, might be particularly relevant for the efficient regulation of HIV replication and containment of the HIV reservoir. Last, we provide evidence of a possible synergistic effect between the CCR5 ∆32 mutation and Ag load in controlling HIV reservoir.

We identify both canonical and novel human leukocyte antigen (HLA)-HIV associations, providing a first step toward improved understanding of HIV immune control among the understudied Honduras Mestizo population. Our results are relevant to understanding the protective or detrimental effects of HLA subtypes in Latin America because their unique HLA diversity poses challenges for designing vaccines against HIV and interpreting results from such vaccine trials. Likewise, the description of the HLA profile in an understudied population that shows a unique HLA immunogenetic background is not only relevant for HIV immunology but also relevant in population genetics, molecular anthropology, susceptibility to other infections, autoimmune diseases, and allograft transplantation.

HIV-1 envelope glycoproteins (Envs) mediate viral entry and are the sole target of neutralizing antibodies. Envs of most primary HIV-1 strains exist in a closed conformation and occasionally sample more open states. Thus, current knowledge guides immunogen design to mimic the closed Env conformation as the preferred target for eliciting broadly neutralizing antibodies (bnAbs) to block HIV-1 entry. Here we show that Env-preferred conformations of 6 out of 13 (46%) transmitted/founder (T/F) strains tested are incompletely closed. As a result, entry of these T/Fs into target cells is sensitive to antibodies that recognize internal epitopes exposed on open Env conformations. A cryo-electron microscopy structure of unliganded, incompletely closed T/F Envs (1059-SOSIP) at 3.6 Å resolution exhibits an asymmetric configuration of Env protomers with increased sampling of states with incompletely closed trimer apex. Double electron-electron resonance spectroscopy provided further evidence for enriched occupancy of more open Env conformations. Consistent with conformational flexibility, 1059 Envs were associated with resistance to most bnAbs that exhibit reduced potency against functional Env intermediates. To follow the fate of incompletely closed Env in patients, we reconstructed de novo the post-transmission evolutionary pathway of a second T/F Env (CH040), which is sensitive to the V3-targeting antibody 19b and highly resistant to most bnAbs. Evolved viruses exhibited increased resistance to cold, soluble CD4 and 19b, all of which correlate with closing of the adapted Env trimer. Lastly, we show a correlation between efficient neutralization of multiple Env conformations and increased antiviral breadth of CD4-binding site (CD4bs) bnAbs. In particular, N6 bnAb, which uniquely recognizes different Env conformations, efficiently neutralizes 50% of the HIV-1 strains that were resistant to VRC01 and transmitted during the first-in-humans antibody-mediated prevention trial (HVTN 704). VRC01-resistant Envs are incompletely closed based on their sensitivity to cold and on partial sensitivity to antibodies targeting internal, typically occluded, epitopes. Most VRC01-resistant Envs retain the VRC01 epitope according to VRC01 binding to their gp120 subunit at concentrations that have no significant effect on virus entry, and they exhibit cross resistance to other CD4bs bnAbs that poorly recognize functional Env intermediates. Our findings refine current knowledge of Env conformational states and provide guidance for developing new strategies for bnAb immunotherapy and Env-based immunogen design.

CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

HIV-1 generates remarkable intra- and inter-host viral diversity during infection. In response to dynamic selective pressures of the host environment, HIV-1 will evolve distinct phenotypes - biological features that provide fitness advantages. The transmitted form of HIV-1 has been shown to require a high density of CD4 on the target cell surface (as found on CD4+ T cells) and typically uses CCR5 as a co-receptor during entry. This phenotype is referred to as R5 T cell-tropic (or R5 T-tropic); however, HIV-1 can switch to a secondary co-receptor, CXCR4, resulting in a X4 T cell-tropic phenotype. Macrophage-tropic (or M-tropic) HIV-1 can evolve to efficiently enter cells expressing low densities of CD4 on their surface (such as macrophages/microglia). So far only CCR5-using M-tropic viruses have been found. M-tropic HIV-1 is most frequently found within the central nervous system, and infection of the CNS has been associated with neurological impairment. It has been shown that interferon resistance phenotypes have a selective advantage during transmission, but the underlying mechanism of this is still unclear. During untreated infection, HIV-1 evolves under selective pressure from both the humoral/antibody response and CD8+ T cell killing. Sufficiently potent antiviral therapy will suppress viral replication, but if the antiviral drugs are not sufficiently potent to stop replication then the replicating virus will evolve drug resistance. HIV-1 phenotypes are highly relevant to treatment efforts, clinical outcomes, vaccine studies, and cure strategies. Therefore, it is critical to understand the dynamics of the host environment that drive these phenotypes and how they affect HIV-1 pathogenesis. This review will provide a comprehensive discussion of HIV-1 entry, transmission, and drug resistance phenotypes. Finally, we will assess the methods used in previous and current research to characterize these phenotypes.

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.