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Current Opinion in HIV and AIDS Mar 2017Sensitive, scalable and affordable assays are critically needed for monitoring the success of interventions for preventing, treating and attempting to cure HIV... (Review)
Review
PURPOSE OF REVIEW
Sensitive, scalable and affordable assays are critically needed for monitoring the success of interventions for preventing, treating and attempting to cure HIV infection. This review evaluates current and emerging technologies that are applicable for both surveillance of HIV drug resistance (HIVDR) and characterization of HIV reservoirs that persist despite antiretroviral therapy and are obstacles to curing HIV infection.
RECENT FINDINGS
Next-generation sequencing (NGS) has the potential to be adapted into high-throughput, cost-efficient approaches for HIVDR surveillance and monitoring during continued scale-up of antiretroviral therapy and rollout of preexposure prophylaxis. Similarly, improvements in PCR and NGS are resulting in higher throughput single genome sequencing to detect intact proviruses and to characterize HIV integration sites and clonal expansions of infected cells.
SUMMARY
Current population genotyping methods for resistance monitoring are high cost and low throughput. NGS, combined with simpler sample collection and storage matrices (e.g. dried blood spots), has considerable potential to broaden global surveillance and patient monitoring for HIVDR. Recent adaptions of NGS to identify integration sites of HIV in the human genome and to characterize the integrated HIV proviruses are likely to facilitate investigations of the impact of experimental 'curative' interventions on HIV reservoirs.
Topics: Genotyping Techniques; HIV; HIV Infections; Humans; Microbial Sensitivity Tests; Proviruses
PubMed: 28059958
DOI: 10.1097/COH.0000000000000344 -
AIDS Research and Therapy Dec 2022The development of antiretroviral therapy (ART) has been effective in suppressing HIV replication. However, severe drug toxicities due to the therapy and its failure in... (Review)
Review
The development of antiretroviral therapy (ART) has been effective in suppressing HIV replication. However, severe drug toxicities due to the therapy and its failure in targeting the integrated proviral genome have led to the introduction of a new paradigm of gene-based therapies. With its effective inhibition and high precision, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) or CRISPR/Cas9 has emerged as an effective genome editing tool in the last decade. Mediated by guide RNAs (gRNAs), Cas9 endonuclease acts like genetic scissors that can modify specific target sites. With this concept, CRISPR/Cas9 has been used to target the integrated proviral HIV-1 genome both in in vitro as well as in vivo studies including non-human primates. The CRISPR has also been tested for targeting latent HIV-1 by modulating the proviral transcription with the help of a specialized Cas9 mutant. Overcoming the limitations of the current therapy, CRISPR has the potential to become the primary genome editing tool for eradicating HIV-1 infection. In this review, we summarize the recent advancements of CRISPR to target the proviral HIV-1 genome, the challenges and future prospects.
Topics: Humans; Animals; HIV-1; CRISPR-Cas Systems; HIV Infections; Virus Latency; HIV Seropositivity; Proviruses
PubMed: 36457057
DOI: 10.1186/s12981-022-00483-y -
Nature Feb 2023Human immunodeficiency virus 1 (HIV-1) reservoir cells persist lifelong despite antiretroviral treatment but may be vulnerable to host immune responses that could be...
Human immunodeficiency virus 1 (HIV-1) reservoir cells persist lifelong despite antiretroviral treatment but may be vulnerable to host immune responses that could be exploited in strategies to cure HIV-1. Here we used a single-cell, next-generation sequencing approach for the direct ex vivo phenotypic profiling of individual HIV-1-infected memory CD4 T cells from peripheral blood and lymph nodes of people living with HIV-1 and receiving antiretroviral treatment for approximately 10 years. We demonstrate that in peripheral blood, cells harbouring genome-intact proviruses and large clones of virally infected cells frequently express ensemble signatures of surface markers conferring increased resistance to immune-mediated killing by cytotoxic T and natural killer cells, paired with elevated levels of expression of immune checkpoint markers likely to limit proviral gene transcription; this phenotypic profile might reduce HIV-1 reservoir cell exposure to and killing by cellular host immune responses. Viral reservoir cells harbouring intact HIV-1 from lymph nodes exhibited a phenotypic signature primarily characterized by upregulation of surface markers promoting cell survival, including CD44, CD28, CD127 and the IL-21 receptor. Together, these results suggest compartmentalized phenotypic signatures of immune selection in HIV-1 reservoir cells, implying that only small subsets of infected cells with optimal adaptation to their anatomical immune microenvironment are able to survive during long-term antiretroviral treatment. The identification of phenotypic markers distinguishing viral reservoir cells may inform future approaches for strategies to cure and eradicate HIV-1.
Topics: Humans; Anti-Retroviral Agents; CD4-Positive T-Lymphocytes; HIV Infections; HIV-1; Proviruses; Viral Load; Virus Latency; Phenotype; Immunologic Memory; Lymph Nodes; Cell Survival; CD28 Antigens; Receptors, Interleukin-21
PubMed: 36599977
DOI: 10.1038/s41586-022-05538-8 -
Cell Jan 2022HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur...
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.
Topics: Aged; Base Sequence; Biological Evolution; Chromatin; Clone Cells; DNA, Viral; Epigenesis, Genetic; Female; HIV-1; Humans; Ionomycin; Male; Middle Aged; Phylogeny; Proviruses; RNA, Viral; Tetradecanoylphorbol Acetate; Transcription, Genetic; Virus Integration; Virus Latency
PubMed: 35026153
DOI: 10.1016/j.cell.2021.12.011 -
Viruses Apr 2022HTLV-1 uveitis (HU) is the third clinical entity to be designated as an HTLV-1-associated disease. Although HU is considered to be the second-most frequent... (Review)
Review
HTLV-1 uveitis (HU) is the third clinical entity to be designated as an HTLV-1-associated disease. Although HU is considered to be the second-most frequent HTLV-1-associated disease in Japan, information on HU is limited compared to that on adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy (HAM). Recent studies have addressed several long-standing uncertainties about HU. HTLV-1-related diseases are known to be caused mainly through vertical transmission (mother-to-child transmission), but emerging HTLV-1 infection by horizontal transmission (such as sexual transmission) has become a major problem in metropolitan areas, such as Tokyo, Japan. Investigation in Tokyo showed that horizontal transmission of HTLV-1 was responsible for HU with severe and persistent ocular inflammation. The development of ATL and HAM is known to be related to a high provirus load and hence involves a long latency period. On the other hand, factors contributing to the development of HU are poorly understood. Recent investigations revealed that severe HU occurs against a background of Graves' disease despite a low provirus load and short latency period. This review highlights the recent knowledge on HU and provides an update on the topic of HU in consideration of a recent nationwide survey.
Topics: Adult; Female; HTLV-I Infections; Human T-lymphotropic virus 1; Humans; Infectious Disease Transmission, Vertical; Paraparesis, Tropical Spastic; Proviruses; Uveitis
PubMed: 35458524
DOI: 10.3390/v14040794 -
Nature Feb 2019A stable latent reservoir for HIV-1 in resting CD4 T cells is the principal barrier to a cure. Curative strategies that target the reservoir are being tested and require...
A stable latent reservoir for HIV-1 in resting CD4 T cells is the principal barrier to a cure. Curative strategies that target the reservoir are being tested and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation may underestimate the reservoir size because one round of activation does not induce all proviruses. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection.
Topics: CD4-Positive T-Lymphocytes; Carrier State; Cell Line; DNA, Viral; Defective Viruses; HIV Infections; HIV-1; Humans; Lymphocyte Activation; Polymerase Chain Reaction; Proviruses; Virus Latency
PubMed: 30700913
DOI: 10.1038/s41586-019-0898-8 -
The Journal of Clinical Investigation Jun 2023Brain microglia (MG) may serve as a human immunodeficiency virus 1 (HIV) reservoir and ignite rebound viremia following cessation of antiretroviral therapy (ART), but...
Brain microglia (MG) may serve as a human immunodeficiency virus 1 (HIV) reservoir and ignite rebound viremia following cessation of antiretroviral therapy (ART), but they have yet to be proven to harbor replication-competent HIV. Here, we isolated brain myeloid cells (BrMCs) from nonhuman primates and rapid autopsy of people with HIV (PWH) on ART and sought evidence of persistent viral infection. BrMCs predominantly displayed microglial markers, in which up to 99.9% of the BrMCs were TMEM119+ MG. Total and integrated SIV or HIV DNA was detectable in the MG, with low levels of cell-associated viral RNA. Provirus in MG was highly sensitive to epigenetic inhibition. Outgrowth virus from parietal cortex MG in an individual with HIV productively infected both MG and PBMCs. This inducible, replication-competent virus and virus from basal ganglia proviral DNA were closely related but highly divergent from variants in peripheral compartments. Phenotyping studies characterized brain-derived virus as macrophage tropic based on the ability of the virus to infect cells expressing low levels of CD4. The lack of genetic diversity in virus from the brain suggests that this macrophage-tropic lineage quickly colonized brain regions. These data demonstrate that MG harbor replication-competent HIV and serve as a persistent reservoir in the brain.
Topics: Animals; Humans; Microglia; HIV-1; Brain; Macrophages; Proviruses; HIV Infections
PubMed: 37317962
DOI: 10.1172/JCI167417 -
Proceedings of the National Academy of... May 2023Treatment of HIV-1-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was...
Treatment of HIV-1-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4 T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.
Topics: Mice; Animals; Humans; Anti-Retroviral Agents; Gene Editing; HIV Infections; HIV Seropositivity; Proviruses; Receptors, CCR5
PubMed: 37126704
DOI: 10.1073/pnas.2217887120 -
The Journal of Clinical Investigation Jul 2023Human endogenous retroviruses (HERVs) are ancestral viral relics that constitute nearly 8% of the human genome. Although normally silenced, the most recently integrated...
Human endogenous retroviruses (HERVs) are ancestral viral relics that constitute nearly 8% of the human genome. Although normally silenced, the most recently integrated provirus HERV-K (HML-2) can be reactivated in certain cancers. Here, we report pathological expression of HML-2 in malignant gliomas in both cerebrospinal fluid and tumor tissue that was associated with a cancer stem cell phenotype and poor outcomes. Using single-cell RNA-Seq, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells (NPC-like) that drive cellular plasticity. Using CRISPR interference, we demonstrate that HML-2 critically maintained glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models. Additionally, we demonstrate that HML-2 critically regulated embryonic stem cell programs in NPC-derived astroglia and altered their 3D cellular morphology by activating the nuclear transcription factor OCT4, which binds to an HML-2-specific long-terminal repeat (LTR5Hs). Moreover, we discovered that some glioblastoma cells formed immature retroviral virions, and inhibiting HML-2 expression with antiretroviral drugs reduced reverse transcriptase activity in the extracellular compartment, tumor viability, and pluripotency. Our results suggest that HML-2 fundamentally contributes to the glioblastoma stem cell niche. Because persistence of glioblastoma stem cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target.
Topics: Humans; Animals; Mice; Endogenous Retroviruses; Glioblastoma; Stem Cell Niche; Proviruses
PubMed: 37395282
DOI: 10.1172/JCI167929 -
Microbiology Spectrum Feb 2015Over 40% of mammalian genomes comprise the products of reverse transcription. Among such retrotransposed sequences are those characterized by the presence of long... (Review)
Review
Over 40% of mammalian genomes comprise the products of reverse transcription. Among such retrotransposed sequences are those characterized by the presence of long terminal repeats (LTRs), including the endogenous retroviruses (ERVs), which are inherited genetic elements closely resembling the proviruses formed following exogenous retrovirus infection. Sequences derived from ERVs make up at least 8 to 10% of the human and mouse genomes and range from ancient sequences that predate mammalian divergence to elements that are currently still active. In this chapter we describe the discovery, classification and origins of ERVs in mammals and consider cellular mechanisms that have evolved to control their expression. We also discuss the negative effects of ERVs as agents of genetic disease and cancer and review examples of ERV protein domestication to serve host functions, as in placental development. Finally, we address growing evidence that the gene regulatory potential of ERV LTRs has been exploited multiple times during evolution to regulate genes and gene networks. Thus, although recently endogenized retroviral elements are often pathogenic, those that survive the forces of negative selection become neutral components of the host genome or can be harnessed to serve beneficial roles.
Topics: Animals; Endogenous Retroviruses; Gene Expression Regulation, Viral; Genetic Diseases, Inborn; Host-Pathogen Interactions; Humans; Mammals; Neoplasms; Proviruses
PubMed: 26104559
DOI: 10.1128/microbiolspec.MDNA3-0009-2014