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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.

BackgroundAntiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown.MethodsWe undertook an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5'-leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets.ResultsClones carrying proviruses with 5'-leader defects can cause persistent NSV up to approximately 103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor (MSD) site and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced noninfectious virions containing viral RNA, but lacking envelope.ConclusionThese findings show that proviruses with 5'-leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5'-leader can help in understanding failure to completely suppress viremia.FundingOffice of the NIH Director and National Institute of Dental and Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases (NIAID), NIH, to the PAVE, BEAT-HIV, and DARE Martin Delaney collaboratories.

To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). The CRISPR/Cas9 system develops an adaptive immune resistance to foreign plasmids and viruses by creating site-specific DNA double-stranded breaks (DSBs). Here we adapt the CRISPR/Cas9 system to human cells for intracellular defense against foreign DNA and viruses. Using HIV-1 infection as a model, our results demonstrate that the CRISPR/Cas9 system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression and replication in human cells. We show that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas9 can be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge. These results unveil the potential of the CRISPR/Cas9 system as a new therapeutic strategy against viral infections. The CRISPR/Cas9 system can be used for genome editing. Here, Liao et al . show that the system can be adapted to inhibit HIV expression and replication, excise the integrated HIV genome and provide long-term protection against new infections in human cells, including pluripotent stem cells.

CRISPR-associated protein 9 (Cas9)-mediated genome editing provides a promising cure for HIV-1/AIDS; however, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an all-in-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9). The quadruplex sgRNAs/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem/progenitor cells from HIV-1 Tg26 transgenic mice. Intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 excised HIV-1 proviral DNA and significantly reduced viral RNA expression in several organs/tissues of Tg26 mice. In EcoHIV acutely infected mice, intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 reduced systemic EcoHIV infection, as determined by live bioluminescence imaging. Additionally, this quadruplex vector induced efficient proviral excision, as determined by PCR genotyping in the liver, lungs, brain, and spleen. Finally, in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection, successful proviral excision was detected by PCR genotyping in the spleen, lungs, heart, colon, and brain after a single intravenous injection of quadruplex sgRNAs/saCas9 AAV-DJ/8. In conclusion, in vivo excision of HIV-1 proviral DNA by sgRNAs/saCas9 in solid tissues/organs can be achieved via AAV delivery, a significant step toward human clinical trials. [Display omitted] Yin et al. use multiplex CRISPR/Cas9 genome editing technology to excise the HIV-1 provirus in a precise manner in three different HIV-1 animal models via in vivo AAV gene delivery. The feasibility of HIV excision in infected cells in vivo paves the way toward human clinical trials to cure HIV-1 infection.

Viruses are important ecological, biogeochemical, and evolutionary drivers in every environment. Upon infection, they often cause the lysis of the host cell. However, some viruses exhibit alternative life cycles, such as chronic infections without cell lysis. The nature and the impact of chronic infections in prokaryotic host organisms remains largely unknown. Here, we characterize a novel haloarchaeal virus, Haloferax volcanii pleomorphic virus 1 (HFPV-1), which is currently the only virus infecting the model haloarchaeon Haloferax volcanii DS2, and demonstrate that HFPV-1 and H. volcanii are a great model system to study virus–host interactions in archaea. HFPV-1 is a pleomorphic virus that causes a chronic infection with continuous release of virus particles, but host and virus coexist without cell lysis or the appearance of resistant cells. Despite an only minor impact of the infection on host growth, we uncovered an extensive remodeling of the transcriptional program of the host (up to 1,049 differentially expressed genes). These changes are highlighted by a down-regulation of two endogenous provirus regions in the host genome, and we show that HFPV-1 infection is strongly influenced by a cross-talk between HFPV-1 and one of the proviruses mediated by a superinfection-like exclusion mechanism. Furthermore, HFPV-1 has a surprisingly wide host range among haloarchaea, and purified virus DNA can cause an infection after transformation into the host, making HFPV-1 a candidate for being developed into a genetic tool for a range of so far inaccessible haloarchaea.

Non-suppressible HIV-1 viremia (NSV) is defined as persistent low-level viremia on antiretroviral therapy (ART) without evidence of ART non-adherence or significant drug resistance. Unraveling the mechanisms behind NSV would broaden our understanding of HIV-1 persistence. Here we analyzed plasma virus sequences in eight ART-treated individuals with NSV (88% male) and show that they are composed of large clones without evidence of viral evolution over time in those with longitudinal samples. We defined proviruses that match plasma HIV-1 RNA sequences as ‘producer proviruses’, and those that did not as ‘non-producer proviruses’. Non-suppressible viremia arose from expanded clones of producer proviruses that were significantly larger than the genome-intact proviral reservoir of ART-suppressed individuals. Integration sites of producer proviruses were enriched in proximity to the activating H3K36me3 epigenetic mark. CD4 + T cells from participants with NSV demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, participants with NSV showed significantly lower HIV-specific CD8 + T cell responses compared with untreated viremic controllers with similar viral loads. We identified potential critical host and viral mediators of NSV that may represent targets to disrupt HIV-1 persistence. Understanding the heterogeneity of HIV infection, such as in persons with non-suppressible HIV-1 viremia despite adherence to antiretroviral treatment, is crucial to better tailor therapeutic interventions to abrogate HIV-1 persistence.

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2–18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

Leukemogenic viruses like human T-lymphotropic virus and bovine leukemia virus (BLV) presumably persist in the host partly by latent integration of the provirus in a fraction of infected cells, leading to accumulative increase in the outgrowth of transformed cells. Furthermore, viral infection also correlates with a blockade of the apoptotic mechanisms concomitant with an apparent latency of the host cell. Conceptually, induction of viral or cellular gene expression could thus also be used as a therapeutic strategy against retroviral-associated leukemia. Here, we provide evidence that valproate, an inhibitor of deacetylases, activates BLV gene expression in transient transfection experiments and in short-term cultures of primary B-lymphocytes. In vivo, valproate injection into newly BLV-inoculated sheep did not abrogate primary infection. However, valproate treatment, in the absence of any other cytotoxic drug, was efficient for leukemia/lymphoma therapy in the sheep model leading to decreased lymphocyte numbers (respectively from 25.6, 35.7, and 46.5 x 10(3) cells per mm3 to 1.0, 10.6, and 24.3 x 10(3) cells per mm3 in three leukemic sheep) and tumor regression (from >700 cm3 to undetectable). The concept of a therapy that targets the expression of viral and cellular genes might be a promising treatment of adult T cell leukemia or tropical spastic paraparesis/human T-lymphotropic virus-associated myelopathy, diseases for which no satisfactory treatment exists so far.

Defective HIV-1 proviruses represent a population of viral genomes that are selected for by immune pressures, and clonally expanded to dominate the persistent HIV-1 proviral genome landscape. There are examples of RNA and protein expression from these compromised genomes which are generated by a variety of mechanisms. Despite the evidence that these proviruses are transcribed and translated, their role in HIV pathogenesis has not been fully explored. The potential for these genomes to participate in immune stimulation is particularly relevant considering the accumulation of cells harboring these defective proviruses over the course of antiretroviral therapy in people living with HIV. The expression of defective proviruses in different cells and tissues could drive innate sensing mechanisms and inflammation. They may also alter antiviral T cell responses and myeloid cell functions that directly contribute to HIV-1 associated chronic comorbidities. Understanding the impact of these defective proviruses needs to be considered as we advance cure strategies that focus on targeting the diverse population of HIV-1 proviral genomes. Graphical abstract

HIV establishes a persistent viral reservoir in the brain despite viral suppression in blood to undetectable levels on antiretroviral therapy (ART). The brain viral reservoir in virally suppressed HIV+ individuals is not well-characterized. In this study, intact, defective, and total HIV proviral genomes were measured in frontal lobe white matter from 28 virally suppressed individuals on ART using the intact proviral DNA assay (IPDA). HIV gag DNA/RNA levels were measured using single-copy assays and expression of 78 genes related to inflammation and white matter integrity was measured using the NanoString platform. Intact proviral DNA was detected in brain tissues of 18 of 28 (64%) individuals on suppressive ART. The median proviral genome copy numbers in brain tissue as measured by the IPDA were: intact, 10 (IQR 1–92); 3′ defective, 509 (225–858); 5′ defective, 519 (273–906); and total proviruses, 1063 (501–2074) copies/106 cells. Intact proviral genomes accounted for less than 10% (median 8.3%) of total proviral genomes in the brain, while 3′ and 5′ defective genomes accounted for 44% and 49%, respectively. There was no significant difference in median copy number of intact, defective, or total proviruses between groups stratified by neurocognitive impairment (NCI) vs. no NCI. In contrast, there was an increasing trend in intact proviruses in brains with vs. without neuroinflammatory pathology (56 vs. 5 copies/106 cells, p = 0.1), but no significant differences in defective or total proviruses. Genes related to inflammation, stress responses, and white matter integrity were differentially expressed in brain tissues with >5 vs. +5 intact proviruses/106 cells. These findings suggest that intact HIV proviral genomes persist in the brain at levels comparable to those reported in blood and lymphoid tissues and increase CNS inflammation/immune activation despite suppressive ART, indicating the importance of targeting the CNS reservoir to achieve HIV cure.