Parallel analysis of transcription, integration, and sequence of single HIV-1 proviruses
2022; 185 (2): 266-+
HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur in some cells, challenging the concept of viral latency. Applying an assay for profiling the transcriptional activity and the chromosomal locations of individual proviruses, we describe a global genomic and epigenetic map of transcriptionally active and silent proviral species and evaluate their longitudinal evolution in persons receiving suppressive ART. Using genome-wide epigenetic reference data, we show that proviral transcriptional activity is associated with activating epigenetic chromatin features in linear proximity of integration sites and in their inter- and intrachromosomal contact regions. Transcriptionally active proviruses were actively selected against during prolonged ART; however, this pattern was violated by large clones of virally infected cells that may outcompete negative selection forces through elevated intrinsic proliferative activity. Our results suggest that transcriptionally active proviruses are dynamically evolving under selection pressure by host factors.
View details for DOI 10.1016/j.cell.2021.12.011
View details for Web of Science ID 000746172100008
View details for PubMedID 35026153
View details for PubMedCentralID PMC8809251
Signatures of immune selection in intact and defective proviruses distinguish HIV-1 elite controllers.
Science translational medicine
2021; 13 (624): eabl4097
Increasing evidence suggests that durable drug-free control of HIV-1 replication is enabled by effective cellular immune responses that may induce an attenuated viral reservoir configuration with a weaker ability to drive viral rebound. Here, we comprehensively tracked effects of antiviral immune responses on intact and defective proviral sequences from elite controllers (ECs), analyzing both classical escape mutations and HIV-1 chromosomal integration sites as biomarkers of antiviral immune selection pressure. We observed that, within ECs, defective proviruses were commonly located in permissive genic euchromatin positions, which represented an apparent contrast to autologous intact proviruses that were frequently located in heterochromatin regions; this suggests differential immune selection pressure on intact versus defective proviruses in ECs. In comparison to individuals receiving antiretroviral therapy, intact and defective proviruses from ECs showed reduced frequencies of escape mutations in cytotoxic T cell epitopes and antibody contact regions, possibly due to the small and poorly inducible reservoir that may be insufficient to drive effective viral escape in ECs. About 15% of ECs harbored nef deletions in intact proviruses, consistent with increased viral vulnerability to host immunity in the setting of nef dysfunction. Together, these results suggest a distinct signature of immune footprints in proviral sequences from ECs.
View details for DOI 10.1126/scitranslmed.abl4097
View details for PubMedID 34910552
Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19.
2020; 183 (1): 143-157.e13
Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.
View details for DOI 10.1016/j.cell.2020.08.025
View details for PubMedID 32877699
View details for PubMedCentralID PMC7437499
Distinct viral reservoirs in individuals with spontaneous control of HIV-1.
2020; 585 (7824): 261-267
Sustained, drug-free control of HIV-1 replication is naturally achieved in less than 0.5% of infected individuals (here termed 'elite controllers'), despite the presence of a replication-competent viral reservoir1. Inducing such an ability to spontaneously maintain undetectable plasma viraemia is a major objective of HIV-1 cure research, but the characteristics of proviral reservoirs in elite controllers remain to be determined. Here, using next-generation sequencing of near-full-length single HIV-1 genomes and corresponding chromosomal integration sites, we show that the proviral reservoirs of elite controllers frequently consist of oligoclonal to near-monoclonal clusters of intact proviral sequences. In contrast to individuals treated with long-term antiretroviral therapy, intact proviral sequences from elite controllers were integrated at highly distinct sites in the human genome and were preferentially located in centromeric satellite DNA or in Krüppel-associated box domain-containing zinc finger genes on chromosome 19, both of which are associated with heterochromatin features. Moreover, the integration sites of intact proviral sequences from elite controllers showed an increased distance to transcriptional start sites and accessible chromatin of the host genome and were enriched in repressive chromatin marks. These data suggest that a distinct configuration of the proviral reservoir represents a structural correlate of natural viral control, and that the quality, rather than the quantity, of viral reservoirs can be an important distinguishing feature for a functional cure of HIV-1 infection. Moreover, in one elite controller, we were unable to detect intact proviral sequences despite analysing more than 1.5 billion peripheral blood mononuclear cells, which raises the possibility that a sterilizing cure of HIV-1 infection, which has previously been observed only following allogeneic haematopoietic stem cell transplantation2,3, may be feasible in rare instances.
View details for DOI 10.1038/s41586-020-2651-8
View details for PubMedID 32848246
View details for PubMedCentralID PMC7837306
Early antiretroviral therapy in neonates with HIV-1 infection restricts viral reservoir size and induces a distinct innate immune profile.
Science translational medicine
2019; 11 (520)
Neonatal HIV-1 infection is associated with rapidly progressive and frequently fatal immune deficiency if left untreated. Immediate institution of antiretroviral therapy (ART), ideally within hours after birth, may restrict irreversible damage to the developing neonatal immune system and possibly provide opportunities for facilitating drug-free viral control during subsequent treatment interruptions. However, the virological and immunological effects of ART initiation within hours after delivery have not been systematically investigated. We examined a unique cohort of neonates with HIV-1 infection from Botswana who started ART shortly after birth and were followed longitudinally for about 2 years in comparison to control infants started on treatment during the first year after birth. We demonstrate multiple clear benefits of rapid antiretroviral initiation, including an extremely small reservoir of intact proviral sequences, a reduction in abnormal T cell immune activation, a more polyfunctional HIV-1-specific T cell response, and an innate immune profile that displays distinct features of improved antiviral activity and is associated with intact proviral reservoir size. Together, these data offer rare insight into the evolutionary dynamics of viral reservoir establishment in neonates and provide strong empirical evidence supporting the immediate initiation of ART for neonates with HIV-1 infection.
View details for DOI 10.1126/scitranslmed.aax7350
View details for PubMedID 31776292
View details for PubMedCentralID PMC8397898
HIV-1 DNA sequence diversity and evolution during acute subtype C infection.
2019; 10 (1): 2737
Little is known about the genotypic make-up of HIV-1 DNA genomes during the earliest stages of HIV-1 infection. Here, we use near-full-length, single genome next-generation sequencing to longitudinally genotype and quantify subtype C HIV-1 DNA in four women identified during acute HIV-1 infection in Durban, South Africa, through twice-weekly screening of high-risk participants. In contrast to chronically HIV-1-infected patients, we found that at the earliest phases of infection in these four participants, the majority of viral DNA genomes are intact, lack APOBEC-3G/F-associated hypermutations, have limited genome truncations, and over one year show little indication of cytotoxic T cell-driven immune selections. Viral sequence divergence during acute infection is predominantly fueled by single-base substitutions and is limited by treatment initiation during the earliest stages of disease. Our observations provide rare longitudinal insights of HIV-1 DNA sequence profiles during the first year of infection to inform future HIV cure research.
View details for DOI 10.1038/s41467-019-10659-2
View details for PubMedID 31227699
View details for PubMedCentralID PMC6588551
Intact HIV-1 proviruses accumulate at distinct chromosomal positions during prolonged antiretroviral therapy.
The Journal of clinical investigation
2019; 129 (3): 988-998
Chromosomal integration of genome-intact HIV-1 sequences into the host genome creates a reservoir of virally infected cells that persists throughout life, necessitating indefinite antiretroviral suppression therapy. During effective antiviral treatment, the majority of these proviruses remain transcriptionally silent, but mechanisms responsible for viral latency are insufficiently clear. Here, we used matched integration site and proviral sequencing (MIP-Seq), an experimental approach involving multiple displacement amplification of individual proviral species, followed by near-full-length HIV-1 next-generation sequencing and corresponding chromosomal integration site analysis to selectively map the chromosomal positions of intact and defective proviruses in 3 HIV-1-infected individuals undergoing long-term antiretroviral therapy. Simultaneously, chromatin accessibility and gene expression in autologous CD4+ T cells were analyzed by assays for transposase-accessible chromatin using sequencing (ATAC-Seq) and RNA-Seq. We observed that in comparison to proviruses with defective sequences, intact HIV-1 proviruses were enriched for non-genic chromosomal positions and more frequently showed an opposite orientation relative to host genes. In addition, intact HIV-1 proviruses were preferentially integrated in either relative proximity to or increased distance from active transcriptional start sites and to accessible chromatin regions. These studies strongly suggest selection of intact proviruses with features of deeper viral latency during prolonged antiretroviral therapy, and may be informative for targeting the genome-intact viral reservoir.
View details for DOI 10.1172/JCI124291
View details for PubMedID 30688658
View details for PubMedCentralID PMC6391088
Effect of analytical treatment interruption and reinitiation of antiretroviral therapy on HIV reservoirs and immunologic parameters in infected individuals.
2018; 14 (1): e1006792
Therapeutic strategies aimed at achieving antiretroviral therapy (ART)-free HIV remission in infected individuals are under active investigation. Considering the vast majority of HIV-infected individuals experience plasma viral rebound upon cessation of therapy, clinical trials evaluating the efficacy of curative strategies would likely require inclusion of ART interruption. However, it is unclear what impact short-term analytical treatment interruption (ATI) and subsequent reinitiation of ART have on immunologic and virologic parameters of HIV-infected individuals. Here, we show a significant increase of HIV burden in the CD4+ T cells of infected individuals during ATI that was correlated with the level of plasma viral rebound. However, the size of the HIV reservoirs as well as immune parameters, including markers of exhaustion and activation, returned to pre-ATI levels 6-12 months after the study participants resumed ART. Of note, the proportions of near full-length, genome-intact and structurally defective HIV proviral DNA sequences were similar prior to ATI and following reinitiation of ART. In addition, there was no evidence of emergence of antiretroviral drug resistance mutations within intact HIV proviral DNA sequences following reinitiation of ART. These data demonstrate that short-term ATI does not necessarily lead to expansion of the persistent HIV reservoir nor irreparable damages to the immune system in the peripheral blood, warranting the inclusion of ATI in future clinical trials evaluating curative strategies.
View details for DOI 10.1371/journal.ppat.1006792
View details for PubMedID 29324842
View details for PubMedCentralID PMC5764487
Transcriptional Changes during Naturally Acquired Zika Virus Infection Render Dendritic Cells Highly Conducive to Viral Replication.
2017; 21 (12): 3471-3482
Although dendritic cells are among the human cell population best equipped for cell-intrinsic antiviral immune defense, they seem highly susceptible to infection with the Zika virus (ZIKV). Using highly purified myeloid dendritic cells isolated from individuals with naturally acquired acute infection, we here show that ZIKV induces profound perturbations of transcriptional signatures relative to healthy donors. Interestingly, we noted a remarkable downregulation of antiviral interferon-stimulated genes and innate immune sensors, suggesting that ZIKV can actively suppress interferon-dependent immune responses. In contrast, several host factors known to support ZIKV infection were strongly upregulated during natural ZIKV infection; these transcripts included AXL, the main entry receptor for ZIKV; SOCS3, a negative regulator of ISG expression; and IDO-1, a recognized inducer of regulatory T cell responses. Thus, during in vivo infection, ZIKV can transform the transcriptome of dendritic cells in favor of the virus to render these cells highly conducive to ZIKV infection.
View details for DOI 10.1016/j.celrep.2017.11.087
View details for PubMedID 29262327
View details for PubMedCentralID PMC5751936
Extensive virologic and immunologic characterization in an HIV-infected individual following allogeneic stem cell transplant and analytic cessation of antiretroviral therapy: A case study.
2017; 14 (11): e1002461
Notwithstanding 1 documented case of HIV-1 cure following allogeneic stem cell transplantation (allo-SCT), several subsequent cases of allo-SCT in HIV-1 positive individuals have failed to cure HIV-1 infection. The aim of our study was to describe changes in the HIV reservoir in a single chronically HIV-infected patient on suppressive antiretroviral therapy who underwent allo-SCT for treatment of acute lymphoblastic leukemia.We prospectively collected peripheral blood mononuclear cells (PBMCs) by leukapheresis from a 55-year-old man with chronic HIV infection before and after allo-SCT to measure the size of the HIV-1 reservoir and characterize viral phylogeny and phenotypic changes in immune cells. At day 784 post-transplant, when HIV-1 was undetectable by multiple measures-including PCR measurements of both total and integrated HIV-1 DNA, replication-competent virus measurement by large cell input quantitative viral outgrowth assay, and in situ hybridization of colon tissue-the patient consented to an analytic treatment interruption (ATI) with frequent clinical monitoring. He remained aviremic off antiretroviral therapy until ATI day 288, when a low-level virus rebound of 60 HIV-1 copies/ml occurred, which increased to 1,640 HIV-1 copies/ml 5 days later, prompting reinitiation of ART. Rebounding plasma HIV-1 sequences were phylogenetically distinct from proviral HIV-1 DNA detected in circulating PBMCs before transplantation. The main limitations of this study are the insensitivity of reservoir measurements, and the fact that it describes a single case.allo-SCT led to a significant reduction in the size of the HIV-1 reservoir and a >9-month-long ART-free remission from HIV-1 replication. Phylogenetic analyses suggest that the origin of rebound virus was distinct from the viruses identified pre-transplant in the PBMCs.
View details for DOI 10.1371/journal.pmed.1002461
View details for PubMedID 29182633
View details for PubMedCentralID PMC5705162
Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.
The Journal of clinical investigation
2017; 127 (7): 2689-2696
HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.
View details for DOI 10.1172/JCI93289
View details for PubMedID 28628034
View details for PubMedCentralID PMC5490740