HIV integration in the human brain is linked to microglial activation and 3D genome remodeling.

To explore genome organization and function in the HIV-infected brain, we applied single-nuclei transcriptomics, cell-type-specific chromosomal conformation mapping, and viral integration site sequencing (IS-seq) to frontal cortex from individuals with encephalitis (HIVE) and without (HIV+). Derepressive changes in 3D genomic compartment structures in HIVE microglia were linked to the transcriptional activation of interferon (IFN) signaling and cell migratory pathways, while transcriptional downregulation and repressive compartmentalization of neuronal health and signaling genes occurred in both HIVE and HIV+ microglia. IS-seq recovered 1,221 brain integration sites showing distinct genomic patterns compared with peripheral lymphocytes, with enrichment for sequences newly mobilized into a permissive chromatin environment after infection. Viral transcription occurred in a subset of highly activated microglia comprising 0.33% of all nuclei in HIVE brain. Our findings point to disrupted microglia-neuronal interactions in HIV and link retroviral integration to remodeling of the microglial 3D genome during infection.

HIV-1 infection of genetically engineered iPSC-derived central nervous system-engrafted microglia in a humanized mouse model.

IMPORTANCE: Our mouse model is a powerful tool for investigating the genetic mechanisms governing central nervous system (CNS) human immunodeficiency virus type-1 (HIV-1) infection and latency in the CNS at a single-cell level. A major advantage of our model is that it uses induced pluripotent stem cell-derived microglia, which enables human genetics, including gene function and therapeutic gene manipulation, to be explored in vivo, which is more challenging to study with current hematopoietic stem cell-based models for neuroHIV. Our transgenic tracing of xenografted human cells will provide a quantitative medium to develop new molecular and epigenetic strategies for reducing the HIV-1 latent reservoir and to test the impact of therapeutic inflammation-targeting drug interventions on CNS HIV-1 latency.

Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice.

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

P2X1 Selective Antagonists Block HIV-1 Infection through Inhibition of Envelope Conformation-Dependent Fusion.

Purinergic receptors are well-established modulators of inflammatory processes, primarily through detection of extracellular nucleotides that are released by dying or infected cells. Emerging literature has demonstrated that inhibition of these inflammatory receptors can block HIV-1 productive infection and HIV-1-associated inflammation. The specificity of receptor type and mechanism of interaction has not yet been determined. Here, we characterize the inhibitory activity of P2X1 receptor antagonists, NF279 and NF449, in cell lines, primary cells, and a variety of HIV-1 envelope (Env) clades. NF279 and NF449 blocked productive infection at the level of viral membrane fusion, with a range of inhibitory activities against different HIV-1 Env isolates. A mutant virus carrying a truncation deletion of the C-terminal tail of HIV-1 Env glycoprotein 41 (gp41) showed reduced sensitivity to P2X1 antagonists, indicating that the sensitivity of inhibition by these molecules may be modulated by Env conformation. In contrast, a P2X7 antagonist, A438079, had a limited effect on productive infection and fusion. NF279 and NF449 interfered with the ability of the gp120 variable regions 1 and 2 (V1V2)-targeted broadly neutralizing antibody PG9 to block productive infection, suggesting that these drugs may antagonize HIV-1 Env at gp120 V1V2 to block viral membrane fusion. Our observations indicate that P2X1 antagonism can inhibit HIV-1 replication at the level of viral membrane fusion through interaction with Env. Future studies will probe the nature of these compounds in inhibiting HIV-1 fusion and the development of small molecules to block HIV-1 entry via this mechanism.IMPORTANCE While effective treatment can lower the severe morbidity and mortality associated with HIV-1 infection, patients infected with HIV-1 suffer from significantly higher rates of noncommunicable comorbidities associated with chronic inflammation. Emerging literature suggests a key role for P2X1 receptors in mediating this chronic inflammation, but the mechanism is still unknown. Here, we demonstrate that HIV-1 infection is reduced by P2X1 receptor antagonism. This inhibition is mediated by interference with HIV-1 Env and can impact a variety of viral clades. These observations highlight the importance of P2X1 antagonists as potential novel therapeutics that could serve to block a variety of different viral clades with additional benefits for their anti-inflammatory properties.

P2X Antagonists Inhibit HIV-1 Productive Infection and Inflammatory Cytokines Interleukin-10 (IL-10) and IL-1ß in a Human Tonsil Explant Model.

HIV-1 causes a persistent infection of the immune system that is associated with chronic comorbidities. The mechanisms that underlie this inflammation are poorly understood. Emerging literature has implicated proinflammatory purinergic receptors and downstream signaling mediators in HIV-1 infection. This study probed whether inhibitors of purinergic receptors would reduce HIV-1 infection and HIV-1-stimulated inflammation. An ex vivo human tonsil histoculture infection model was developed to support HIV-1 productive infection and stimulated the inflammatory cytokine interleukin-1 beta (IL-1ß) and the immunosuppressive cytokine interleukin-10 (IL-10). This study tests whether inhibitors of purinergic receptors would reduce HIV-1 infection and HIV-1-stimulated inflammation. The purinergic P2X1 receptor antagonist NF449, the purinergic P2X7 receptor antagonist A438079, and azidothymidine (AZT) were tested in HIV-1-infected human tonsil explants to compare levels of inhibition of HIV-1 infection and HIV-stimulated inflammatory cytokine production. All drugs limited HIV-1 productive infection, but P2X-selective antagonists (NF449 and A438079) significantly lowered HIV-stimulated IL-10 and IL-1ß. We further observed that P2X1- and P2X7-selective antagonists can act differentially as inhibitors of both HIV-1 infection and HIV-1-stimulated inflammation. Our findings highlight the differential effects of HIV-1 on inflammation in peripheral blood compared to those in lymphoid tissue. For the first time, we demonstrate that P2X-selective antagonists act differentially as inhibitors of both HIV-1 infection and HIV-1-stimulated inflammation. Drugs that block these pathways can have independent inhibitory activities against HIV-1 infection and HIV-induced inflammation.IMPORTANCE Patients who are chronically infected with HIV-1 experience sequelae related to chronic inflammation. The mechanisms of this inflammation have not been elucidated. Here, we describe a class of drugs that target the P2X proinflammatory signaling receptors in a human tonsil explant model. This model highlights differences in HIV-1 stimulation of lymphoid tissue inflammation and peripheral blood. These drugs serve to block both HIV-1 infection and production of IL-10 and IL-1ß in lymphoid tissue, suggesting a novel approach to HIV-1 therapeutics in which both HIV-1 replication and inflammatory signaling are simultaneously targeted.

Sequential trafficking of Env and Gag to HIV-1 T cell virological synapses revealed by live imaging.

BACKGROUND: HIV infection is enhanced by cell adhesions that form between infected and uninfected T cells called virological synapses (VS). VS are initiated by an interaction between Env and CD4 on cell surfaces and result in the recruitment of virus assembly to the site of cell-cell contact. However, the recruitment of Env to the VS and its relationship to Gag recruitment is not well defined. RESULTS: To study the trafficking of HIV-1 Env through the VS, we constructed a molecular clone of HIV carrying a green fluorescent protein-Env fusion protein called, HIV Env-isfGFP-∆V1V2. The Env-isfGFP-∆V1V2 fusion protein does not produce virus particles on its own, but can be rescued by cotransfection with full-length HIV constructs and produce virus particles that package the fluorescent Env. These rescued fluorescent Env can participate in VS formation and can be used to directly image CD4-dependent Env transfer across VS from donor to target cells. The movements of fluorescently tagged Gag and Env to the VS and transfer into target cells can be also tracked through live imaging. Time lapse live imaging reveals evidence of limited Env accumulation at the site of cell-cell contact shortly after cell adhesion, followed by Gag re-distribution to contact area. Both Gag and Env can be recruited to form button-like spots characteristic of VS. CONCLUSIONS: Env and Gag are recruited to the VS in a coordinated temporal sequence and subsequently transfer together across the synapse into the target cell. Env accumulations, when observed, are earlier than Gag re-distribution to the contact area during formation of VS.

Reduced Potency and Incomplete Neutralization of Broadly Neutralizing Antibodies against Cell-to-Cell Transmission of HIV-1 with Transmitted Founder Envs.

Broadly neutralizing antibodies (bNAbs) have been isolated from HIV-1 patients and can potently block infection of a wide spectrum of HIV-1 subtypes. These antibodies define common epitopes shared by many viral isolates. While bNAbs potently antagonize infection with cell-free virus, inhibition of HIV-1 transmission from infected to uninfected CD4+ T cells through virological synapses (VS) has been found to require greater amounts of antibody. In this study, we examined two well-studied molecular clones and two transmitted/founder (T/F) clones for their sensitivities to a panel of bNAbs in cell-free and cell-to-cell infection assays. We observed resistance of cell-to-cell transmission to antibody neutralization that was reflected not only by reductions of antibody potency but also by decreases in maximum neutralization capacity relative to the levels seen with cell-free infections. BNAbs targeting different epitopes exhibited incomplete neutralization against cell-associated virus with T/F Envs, which was not observed with the cell-free form of the same virus. We further identified the membrane-proximal internal tyrosine-based sorting motif as a determinant that can affect the incomplete neutralization of these T/F clones in cell-to-cell infection. These findings indicate that the signal that affects surface expression and/or internalization of Env from the plasma membrane can modulate the presentation of neutralizing epitopes on infected cells. These results highlight that a fraction of virus can escape from high concentrations of antibody through cell-to-cell infection while remaining sensitive to neutralization in cell-free infection. The ability to fully inhibit cell-to-cell transmission may represent an important consideration in the development of antibodies for treatment or prophylaxis.IMPORTANCE In recent years, isolation of new-generation HIV-1 bNAbs has invigorated HIV vaccine research. These bNAbs display remarkable potency and breadth of coverage against cell-free virus; however, they exhibit a diminished ability to block HIV-1 cell-to-cell transmission. The mechanism(s) by which HIV-1 resists neutralization when transmitting through VS remains uncertain. We examined a panel of bNAbs for their ability to neutralize HIV-1 T/F viruses in cell-to-cell infection assays. We found that some antibodies exhibit not only reduced potency but also decreased maximum neutralization capacity or in vitro efficacy against cell-to-cell infection of HIV-1 with T/F Envs compared to cell-free infection of the same virus. We further identified the membrane-proximal internal tyrosine-based sorting motif YXXL as a determinant that can affect the incomplete neutralization phenotype of these T/F clones. When the maximum neutralization capacity falls short of 100%, this can have a major impact on the ability of antibodies to halt viral replication.

Visualization of HIV T Cell Virological Synapses and Virus-Containing Compartments by Three-Dimensional Correlative Light and Electron Microscopy.

Virological synapses (VS) are adhesive structures that form between infected and uninfected cells to enhance the spread of HIV-1. During T cell VS formation, viral proteins are actively recruited to the site of cell-cell contact where the viral material is efficiently translocated to target cells into heterogeneous, protease-resistant, antibody-inaccessible compartments. Using correlative light and electron microscopy (CLEM), we define the membrane topography of the virus-containing compartments (VCC) where HIV is found following VS-mediated transfer. Focused ion beam scanning electron microscopy (FIB-SEM) and serial sectioning transmission electron microscopy (SS-TEM) were used to better resolve the fluorescent Gag-containing structures within the VCC. We found that small punctate fluorescent signals correlated with single viral particles in enclosed vesicular compartments or surface-localized virus particles and that large fluorescent signals correlated with membranous Gag-containing structures with unknown pathological function. CLEM imaging revealed distinct pools of newly deposited viral proteins within endocytic and nonendocytic compartments in VS target T cells. IMPORTANCE: This study directly correlates individual virus-associated objects observed in light microscopy with ultrastructural features seen by electron microscopy in the HIV-1 virological synapse. This approach elucidates which infection-associated ultrastructural features represent bona fide HIV protein complexes. We define the morphology of some HIV cell-to-cell transfer intermediates as true endocytic compartments and resolve unique synapse-associated viral structures created by transfer across virological synapses.

Mechanisms of enhanced HIV spread through T-cell virological synapses.

An elaborate network of cell-cell interactions in the immune system is essential for vertebrates to mount adaptive immune responses against invading pathogens. For lymphotropic viruses such as the human immunodeficiency virus type 1 (HIV-1), these immune cell interactions can also promote the spread of the virus within the host. The main target of HIV-1 infection is the CD4(+) helper T lymphocyte, a cell type that is responsible for coordinating immune responses and modulating effector responses to foreign antigens. As part of their normal immune surveillance duties, these cells migrate actively within lymphoid tissues and can travel from inductive sites to effector sites in search of their cognate antigen. For CD4(+) T cells, there is an ongoing search for a unique peptide antigen presented in the context of class II MHC that can activate a proliferative or tolerogenic response. This iterative and continual probing and interrogation of other cells determine the outcome of immune responses. Recent studies in vitro have revealed that the viral infection program induces cell-cell interactions called virological synapses between infected and uninfected CD4(+) T cells. These long-lived, virally induced adhesive contacts greatly enhance the rate of productive infection and may be central to the spread of the virus in vivo. Here, we review aspects of this efficient mode of cell-to-cell infection and the implications for our understanding of HIV-1 pathogenesis.

The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility.

BACKGROUND: The p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy. RESULTS: Remarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV. CONCLUSIONS: Our results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor.

HIV-1 Cell-Free and Cell-to-Cell Infections Are Differentially Regulated by Distinct Determinants in the Env gp41 Cytoplasmic Tail.

UNLABELLED: The HIV-1 envelope (Env) glycoprotein mediates viral entry during both cell-free and cell-to-cell infection of CD4(+) T cells. The highly conserved long cytoplasmic tail (CT) of Env is required in a cell type-dependent manner for optimal infectivity of cell-free virus. To probe the role of the CT in cell-to-cell infection, we tested a panel of mutations in the CT region that maintain or attenuate cell-free infection to investigate whether the functions of the CT are conserved during cell-free and cell-to-cell infection. The mutations tested included truncations of structural motifs in the gp41 CT and two point mutations in lentiviral lytic peptide 3 (LLP-3) previously described as disrupting the infectivity of cell-free virus. We found that small truncations of 28 to 43 amino acids (aa) or two LLP-3 point mutations, YW_SL and LL_RQ, severely impaired single-round cell-free infectivity 10-fold or more relative to wild-type full-length CT. These mutants showed a modest 2-fold reduction in cell-to-cell infection assays. Conversely, large truncations of 93 to 124 aa severely impaired cell-to-cell infectivity 20-fold or more while resulting in a 50% increase in infectivity of cell-free viral particles when produced in 293T cells. Intermediate truncations of 46 to 90 aa showed profound impairment of both modes of infection. Our results show that the abilities of Env to support cell-free and cell-to-cell infection are genetically distinct. These differences are cell type dependent for large-CT-truncation mutants. Additionally, point mutants in LLP-3 can maintain multiround propagation from cell-to-cell in primary CD4(+) T cells. IMPORTANCE: The functions of HIV Env gp41 CT remain poorly understood despite being widely studied in the context of cell-free infection. We have identified domains of the gp41 CT responsible for striking selective deficiencies in either cell-free or cell-to-cell infectivity. These differences may reflect a different intrinsic regulatory influence of the CT on cell-associated versus particle-associated Env or differential interaction with host or viral proteins. Our findings provide novel insight into the key regulatory potential of the gp41 CT in cell-free and cell-to-cell HIV-1 infection, particularly for short-truncation mutants of ≤43 amino acids or mutants with point mutations in the LLP-3 helical domain of the CT, which are able to propagate via cell-to-cell infection in the absence of infectious cell-free virus production. These mutants may also serve as tools to further define the contributions of cell-free and cell-to-cell infection in vitro and in vivo.

P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection.

UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) infection is chronic and presently still incurable. Antiretroviral drugs effectively suppress replication; however, persistent activation of inflammatory pathways remains a key cause of morbidity. Recent studies proposed that purinergic signaling is required for HIV-1 infection. Purinergic receptors are distributed throughout a wide variety of tissue types and detect extracellular ATP as a danger signal released from dying cells. We have explored how these pathways are involved in the transmission of HIV-1 from cell to cell through virological synapses. Infection of CD4+ T lymphocytes with HIV-1 in the presence of an inhibitor of P2X receptors effectively inhibited HIV-1 infection through both cell-free and cell-to-cell contact in a dose-dependent manner. Inhibition of direct cell-to-cell infection did not affect the formation of virological synapses or the subsequent cell-to-cell transfer of HIV-1. During both cell-free and cell-to-cell CD4+ T lymphocyte infection, purinergic antagonists blocked infection at the level of viral membrane fusion. During cell-to-cell transmission, we observed CXCR4 colocalization with the newly internalized virus particles within target lymphocytes and found that the purinergic antagonists did not impair the recruitment of the coreceptor CXCR4 to the site of Gag internalization in the target cell. In a screen of a library of purinergic antagonists, we found that the most potent inhibitors of HIV-1 fusion were those that target P2X receptors, while P2Y-selective receptor antagonists or adenosine receptor antagonists were ineffective. Our results suggest that P2X receptors may provide a therapeutic target and that purinergic antagonists may have potent activity against viral infection of CD4+ T lymphocytes by both cell-free and cell-to-cell transmission. IMPORTANCE: This study identifies purinergic antagonists to be potent inhibitors of HIV-1 cell-free and cell-to-cell-mediated infection and provides a stepwise determination of when these compounds inhibit HIV-1 infection. These data provide a rationale for the development of novel antiretroviral therapies that have a dual role in both direct antiviral activity and the reduction of HIV-associated inflammation. Purinergic antagonists are shown here to have equivalent efficacy in inhibiting HIV infection via cell-free and cell-to-cell infection, and it is shown that purinergic receptors could provide an attractive therapeutic anti-HIV target that might avoid resistance by targeting a host signaling pathway that potently regulates HIV infection. The high-throughput screen of HIV-1 fusion inhibitors further defines P2X-selective compounds among the purinergic compounds as being the most potent HIV entry inhibitors. Clinical studies on these drugs for other inflammatory indications suggest that they are safe, and thus, if developed for use as anti-HIV agents, they could reduce both HIV replication and HIV-related inflammation.

HIV-1 Vpu antagonism of tetherin inhibits antibody-dependent cellular cytotoxic responses by natural killer cells.

UNLABELLED: The type I interferon-inducible factor tetherin retains virus particles on the surfaces of cells infected with vpu-deficient human immunodeficiency virus type 1 (HIV-1). While this mechanism inhibits cell-free viral spread, the immunological implications of tethered virus have not been investigated. We found that surface tetherin expression increased the antibody opsonization of vpu-deficient HIV-infected cells. The absence of Vpu also stimulated NK cell-activating FcγRIIIa signaling and enhanced NK cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). The deletion of vpu in HIV-1-infected primary CD4(+) T cells enhanced the levels of antibody binding and Fc receptor signaling mediated by HIV-positive-patient-derived antibodies. The magnitudes of antibody binding and Fc signaling were both highly correlated to the levels of tetherin on the surfaces of infected primary CD4 T cells. The affinity of antibody binding to FcγRIIIa was also found to be critical in mediating efficient Fc activation. These studies implicate Vpu antagonism of tetherin as an ADCC evasion mechanism that prevents antibody-mediated clearance of virally infected cells. IMPORTANCE: The ability of the HIV-1 accessory factor to antagonize tetherin has been considered to primarily function by limiting the spread of virus by preventing the release of cell-free virus. This study supports the hypothesis that a major function of Vpu is to decrease the recognition of infected cells by anti-HIV antibodies at the cell surface, thereby reducing recognition by antibody-dependent clearance by natural killer cells.

HIV-1 interacts with human endogenous retrovirus K (HML-2) envelopes derived from human primary lymphocytes.

UNLABELLED: Human endogenous retroviruses (HERVs) are viruses that have colonized the germ line and spread through vertical passage. Only the more recently acquired HERVs, such as the HERV-K (HML-2) group, maintain coding open reading frames. Expression of HERV-Ks has been linked to different pathological conditions, including HIV infection, but our knowledge on which specific HERV-Ks are expressed in primary lymphocytes currently is very limited. To identify the most expressed HERV-Ks in an unbiased manner, we analyzed their expression patterns in peripheral blood lymphocytes using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing. We observe that three HERV-Ks (KII, K102, and K18) constitute over 90% of the total HERV-K expression in primary human lymphocytes of five different donors. We also show experimentally that two of these HERV-K env sequences (K18 and K102) retain their ability to produce full-length and posttranslationally processed envelope proteins in cell culture. We show that HERV-K18 Env can be incorporated into HIV-1 but not simian immunodeficiency virus (SIV) particles. Moreover, HERV-K18 Env incorporation into HIV-1 virions is dependent on HIV-1 matrix. Taken together, we generated high-resolution HERV-K expression profiles specific for activated human lymphocytes. We found that one of the most abundantly expressed HERV-K envelopes not only makes a full-length protein but also specifically interacts with HIV-1. Our findings raise the possibility that these endogenous retroviral Env proteins could directly influence HIV-1 replication. IMPORTANCE: Here, we report the HERV-K expression profile of primary lymphocytes from 5 different healthy donors. We used a novel deep-sequencing technology (PacBio SMRT) that produces the long reads necessary to discriminate the complexity of HERV-K expression. We find that primary lymphocytes express up to 32 different HERV-K envelopes, and that at least two of the most expressed Env proteins retain their ability to make a protein. Importantly, one of them, the envelope glycoprotein of HERV-K18, is incorporated into HIV-1 in an HIV matrix-specific fashion. The ramifications of such interactions are discussed, as the possibility of HIV-1 target tissue broadening and immune evasion are considered.

Postintegration HIV-1 infection of cervical epithelial cells mediates contact-dependent productive infection of T cells.

The female genital epithelium plays a protective role against invading pathogens; however, sexual transmission of human immunodeficiency virus type 1 (HIV-1) still occurs in healthy women. To model virus-cell interactions in this barrier during sexual transmission, we studied the uptake and infection of ectocervical and endocervical cell lines with cell-free fluorescent protein-expressing recombinant HIV-1 carrying primary transmitted/founder envelope genes. We observed that a subset of both the ectocervical and endocervical epithelial cells become productively infected with cell-free HIV-1 in a CD4-independent manner. In addition, the ability of the semen-derived enhancer of virus infection (SEVI) to enhance virus-epithelial cell interactions was studied. This infection is increased approximately 2-5 fold when inoculation occurs in the presence of SEVI fibrils. Once infected, the epithelial cells are capable of transmitting the virus to target CD4 T cells in coculture in a contact-dependent manner that uses conventional CD4- and coreceptor-dependent entry. The infection of target CD4 T cells only occurs when de novo HIV-1 is produced within the epithelial cells. These findings suggest that a subset of cervical epithelial cells may be actively involved in establishing a systemic HIV infection and should be a target when designing prevention strategies to protect against HIV-1 sexual transmission.

HIV-1 interaction with an O-glycan-specific bacterial lectin enhances virus infectivity and resistance to neutralization by antibodies.

Bacteria dysbiosis has been associated with an increased risk of HIV-1 transmission and acquisition. The prevalent idea is that bacteria dysbiosis compromises mucosal integrity and promotes inflammatory conditions to cause recruitment and activation of immune cells that harbor or are targeted by HIV-1. However, it is also possible that HIV-1 directly binds bacteria or bacterial products to impact virus infectivity and transmissibility. This study evaluated HIV-1 interactions with bacteria through glycan-binding lectins. The Streptococcal Siglec-like lectin SLBR-N, which is part of the fimbriae shrouding the bacteria surface and recognizes α2,3 sialyated O-linked glycans, was noted for its ability to enhance HIV-1 infectivity in the context of cell-free infection and cell-to-cell transfer. Enhancing effects were recapitulated with O-glycan-binding plant lectins, signifying the importance of O-glycans. Conversely, N-glycan-binding bacterial lectins FimH and Msl had no effect. SLBR-N was demonstrated to capture and transfer infectious HIV-1 virions, bind to O-glycans on HIV-1 Env, and increase HIV-1 resistance to broadly neutralizing antibodies targeting different regions of Env. Hence, this study highlights the potential contribution of O-glycans in promoting HIV-1 infection through the exploitation of O-glycan-binding lectins from commensal bacteria at the mucosa.

HIV-1 infection is associated with depletion of germinal center B cells and a decrease in IgA + plasma cells in the GI tract.

Gastrointestinal (GI) B cells and plasma cells (PCs), critical to mucosal homeostasis, play an important role in the host response to HIV-1 infection. Here, high resolution mapping of human B cells and PCs from colon and ileum during both viremic and suppressed HIV-1 infection identified a significant reduction in germinal center (GC) B cells and Follicular Dendritic Cells (FDCs) during HIV-1 viremia. Further, IgA + PCs, the major cellular output of intestinal GCs were significantly reduced during viremic HIV-1 infection. PC-associated transcriptional perturbations, including type I interferon signaling persisted in antiretroviral therapy (ART) treated individuals, suggesting ongoing disruption of the intestinal immune milieu during ART. GI humoral immune perturbations associated with changes in intestinal microbiome composition and systemic inflammation. Herein, we highlight a key immune defect in the GI mucosa due to HIV-1 viremia, with major implications. One Sentence Summary: Major perturbations in intestinal GC dynamics in viremic HIV-1 infection relate to reduced IgA + plasma cells, systemic inflammation and microbiota changes.

Neutralization resistance of virological synapse-mediated HIV-1 Infection is regulated by the gp41 cytoplasmic tail.

Human immunodeficiency virus type 1 (HIV-1) infection can spread efficiently from infected to uninfected T cells through adhesive contacts called virological synapses (VSs). In this process, cell-surface envelope glycoprotein (Env) initiates adhesion and viral transfer into an uninfected recipient cell. Previous studies have found some HIV-1-neutralizing patient sera to be less effective at blocking VS-mediated infection than infection with cell-free virus. Here we employ sensitive flow cytometry-based infection assays to measure the inhibitory potency of HIV-1-neutralizing monoclonal antibodies (MAb) and HIV-1-neutralizing patient sera against cell-free and VS-mediated infection. To various degrees, anti-Env MAbs exhibited significantly higher 50% inhibitory concentration (IC(50)s) against VS-mediated infection than cell-free infection. Notably, the MAb 17b, which binds a CD4-induced (CD4i) epitope on gp120, displayed a 72-fold reduced efficacy against VS-mediated inocula compared to cell-free inocula. A mutant with truncation mutation in the gp41 cytoplasmic tail (CT) which is unable to modulate Env fusogenicity in response to virus particle maturation but which can still engage in cell-to-cell infection was tested for the ability to resist neutralizing antibodies. The ΔCT mutation increased cell surface staining by neutralizing antibodies, significantly enhanced neutralization of VS-mediated infection, and had reduced or no effect on cell-free infection, depending upon the antibody. Our results suggest that the gp41 CT regulates the exposure of key neutralizing epitopes during cell-to-cell infection and plays an important role in immune evasion. Vaccine strategies should consider immunogens that reflect Env conformations exposed on the infected cell surface to enhance protection against VS-mediated HIV-1 spread.

Viral Persistence in the Gut-Associated Lymphoid Tissue and Barriers to HIV Cure.

More than 40 years after the first reported cases of what then became known as acquired immunodeficiency syndrome (AIDS), tremendous progress has been achieved in transforming the disease from almost universally fatal to a chronic manageable condition. Nonetheless, the efforts to find a preventative vaccine or a cure for the underlying infection with Human Immunodeficiency Virus (HIV) remain largely unsuccessful. Many challenges intrinsic to the virus characteristics and host response need to be overcome for either goal to be achieved. This article will review the obstacles to an effective HIV cure, specifically the steps involved in the generation of HIV latency, focusing on the role of the gut-associated lymphoid tissue, which has received less attention compared with the peripheral blood, despite being the largest repository of lymphoid tissue in the human body, and a large site for HIV persistence.

HIV-1 infection of genetically engineered iPSC-derived central nervous system-engrafted microglia in a humanized mouse model.

The central nervous system (CNS) is a major human immunodeficiency virus type 1 reservoir. Microglia are the primary target cell of HIV-1 infection in the CNS. Current models have not allowed the precise molecular pathways of acute and chronic CNS microglial infection to be tested with in vivo genetic methods. Here, we describe a novel humanized mouse model utilizing human-induced pluripotent stem cell-derived microglia to xenograft into murine hosts. These mice are additionally engrafted with human peripheral blood mononuclear cells that served as a medium to establish a peripheral infection that then spread to the CNS microglia xenograft, modeling a trans-blood-brain barrier route of acute CNS HIV-1 infection with human target cells. The approach is compatible with iPSC genetic engineering, including inserting targeted transgenic reporter cassettes to track the xenografted human cells, enabling the testing of novel treatment and viral tracking strategies in a comparatively simple and cost-effective way vivo model for neuroHIV.