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PubMed2026

Suppression of HIV-1 replication in CEM-A cell cultures by trans-splicing group I introns targeting PAS/PBS sequences and conditionally expressing ΔN-Bax.

Anti-HIV group I introns containing antisense guide sequences directed against the HIV-1 primer activation signal and primer-binding site (PAS/PBS) were designed and evaluated. Because PAS/PBS sequences are present in the viral RNA species examined, these RNAs can serve as trans-splicing substrates. The introns were active against both artificial target RNAs and viral RNA generated during infection. Cleavage and degradation of targeted viral RNA may have contributed to suppression, whereas inclusion of a 3' exon encoding the proapoptotic protein ΔN-Bax was associated with increased programmed cell death and may have augmented suppression of viral replication. In cultured CEM-A cells, transgene expression of these introns markedly suppressed HIV-1 replication, with p24 levels falling below the assay detection limit in selected clones. RESULTS: RT-PCR and sequence analysis detected splice products containing the expected PAS/PBS junctions. In the dual-luciferase assay, intron expression reduced normalized Gaussia luciferase signal by approximately 70% relative to the negative control. Qualitative Annexin V imaging and caspase-3 assays were consistent with infection-dependent apoptosis after ΔN-Bax splice-product formation. Transient expression of each intron in HEK293T cells followed by infection with VSV-G-pseudotyped HIV-1NL4-3 at an MOI of 2 reduced p24 levels by approximately 50% at 4 days post-infection. Construct 128L produced the strongest RT-PCR band under the tested conditions and was selected for subsequent experiments. A canonical splice product and a low-abundance noncanonical splice product were detected; both involved the intended HIV-derived target RNA, although transcriptome-wide off-target splicing was not assessed. Heterogeneous transformed HEK293T populations showed an approximately 2-log10 reduction in p24. In selected clonal HEK293T and CEM-A lines, p24 was below the assay detection limit at the measured endpoints, including up to 90 days after infection in some CEM-A clones. CONCLUSIONS: PAS/PBS-targeting group I introns suppressed HIV-1-associated p24 production in the tested cell-culture models. Linking the introns to a ΔN-Bax 3' exon was associated with infection-dependent apoptosis and may further limit viral replication and spread. The use of highly conserved, functionally constrained target sequences may reduce the likelihood of escape, but viral evolution and transcriptome-wide off-target effects were not assessed. This conditional death-upon-infection strategy warrants further evaluation in primary-cell and in vivo models.

Humans↗

PubMed2025

Aberrant DNA methylation of genes regulating CD4+ T cell HIV-1 reservoir in women with HIV.

BACKGROUND: The HIV-1 reservoir in CD4+ T cells (HRCD4) pose a major challenge to curing HIV, with many of its mechanisms still unclear. HIV-1 DNA integration and immune responses may alter the host's epigenetic landscape, potentially silencing HIV-1 replication. METHODS: This study used bisulphite capture DNA methylation sequencing in CD4+ T cells from the blood of 427 virally suppressed women with HIV to identify differentially methylated sites and regions associated with HRCD4. RESULTS: The average total HRCD4 size was 1409 copies per million cells, with most proviruses defective and only a small proportion intact. The study identified 245 differentially methylated CpG sites and 85 regions linked to HRCD4 size, with 52% of significant sites in intronic regions. Genes associated with HRCD4 were involved in viral replication, HIV-1 latency and cell growth and apoptosis. HRCD4 size was inversely related to DNA methylation of interferon signalling genes and positively associated with methylation at known HIV-1 integration sites. HRCD4-associated genes were enriched on the pathways related to immune defence, transcription repression and host-virus interactions. CONCLUSIONS: These findings suggest that HIV-1 reservoir is linked to aberrant DNA methylation in CD4+ T cells, offering new insights into epigenetic mechanisms of HIV-1 latency and potential molecular targets for eradication strategies. KEY POINTS: Study involved 427 women with HIV. Identified 245 aberrant DNA methylation sites and 85 methylation regions in CD4+ T cells linked to the HIV-1 reservoir. Highlighted genes are involved in viral replication, immune defence, and host genome integration. Findings suggest potential molecular targets for eradication strategies.

Humans↗

PubMed2025

Kinetic Investigation of Resistance to Islatravir Conferred by Mutations in HIV-1 Reverse Transcriptase.

Islatravir (EFdA) is a novel nucleoside reverse transcriptase translocation inhibitor (NRTTI) that potently blocks HIV-1 replication in vivo. Its unique structural features in contrast to nucleoside reverse transcriptase inhibitors (NRTIs), particularly the 4'-ethynyl and 3'-hydroxy groups, contribute to its high clinical potency. Once intracellularly activated to EFdA 5'-triphosphate (EFdA-TP), it competes with dATP for incorporation by HIV-1 reverse transcriptase (RT) during HIV-1 genomic replication. The 4'-ethynyl group of incorporated EFdA-MP interacts with a hydrophobic pocket of HIV-1 RT, hindering DNA translocation and terminating DNA synthesis. The M184V mutation, commonly associated with resistance to NRTIs such as lamivudine and emtricitabine, and the M184V/A114S mutations, both located within the hydrophobic pocket, were shown to reduce Islatravir susceptibility in cell-based viral resistance selection assays. To elucidate the mechanisms by which these mutations affect Islatravir inhibition, we employed pre-steady-state kinetics to investigate their impact on EFdA-TP incorporation by HIV-1 RT using both DNA and RNA templates. We found that M184V had a modest effect on EFdA-TP incorporation efficiency, increasing it 2-fold with the DNA template and decreasing it 3-fold with the RNA template. In contrast, M184V/A114S significantly inhibited EFdA-TP incorporation, reducing its incorporation efficiency 5.4-fold with the DNA template and 181-fold with the RNA template. These reductions were primarily attributable to corresponding decreases in EFdA-TP incorporation rate constants of 18-fold and 105-fold, respectively. These results suggest that, unlike FDA-approved NRTIs, the clinical efficacy of Islatravir, may not be substantially compromised by the M184V mutation alone but will be significantly reduced by the M184V/A114S mutations.

HIV Reverse Transcriptase↗

PubMed2026

PTBP1 at the host-virus interface: mechanistic roles in viral RNA translation, replication, and immune modulation.

Viruses require the involvement of host RNA binding proteins for completion of important steps of their life cycle. Polypyrimidine tract binding protein 1 (PTBP1) is an RNA-binding protein found ubiquitously which performs important regulatory functions like alternative splicing, RNA stability, RNA localization, and translation by virtue of its four RRMs and shuttling between nucleus and cytoplasm. There is increasing evidence showing that many viruses make use of such regulatory roles of PTBP1 to facilitate their gene expression and replication. This review describes the existing mechanistic knowledge about the PTBP1 functions during viral infection, paying attention to the role of PTBP1 in viral RNA translation, viral RNA genome replication, and regulation of host antiviral response. Special attention is paid to the regulation by PTBP1 of IRES-dependent translation of enteroviruses and hepatitis C virus, as well as to the PTBP1 contribution to RNA stabilization, long-distance RNA interactions, and genome cyclization of flaviviruses such as dengue virus and Japanese encephalitis virus. Recent data on the PTBP1 function in coronavirus RNA metabolism are discussed as well. Furthermore, the role of PTBP1 in being both proviral and antiviral is reviewed in terms of innate immunity signalling pathways, stress granule biology, and virus-host interaction. Finally, we will explore the possibility of PTBP1 being used as a host-directed antiviral drug target despite the hurdles in doing so considering its multifunctionality as an essential cellular RNA-binding protein.

Polypyrimidine Tract-Binding Protein↗

PubMed2025

A New Type of Nonsuppressible Viremia Produced by HIV-Infected Macrophage.

BACKGROUND: HIV-1 RNA typically declines rapidly after initiation of antiretroviral therapy (ART); often reaching undetectable levels within a few weeks and remaining undetectable by standard assays. However, some patients on ART have persistent nonsuppressible viremia (NSV) that does not respond to treatment optimization or intensification. NSV can emerge at the time of ART initiation (primary NSV) or after being ART-suppressed (secondary NSV). Here, we examine mechanisms producing primary NSV in four people on ART. METHODS: Blood samples were collected from four participants who, despite being adherent to ART, required approximately a year or more to become virologically suppressed. Viral RNA and proviral DNA genomes were sequenced to examine HIV-1 drug resistance, genome intactness and genetic diversity. The ability of HIV-1 Envs to facilitate efficient entry into cells expressing low levels of CD4 (a proxy for macrophage tropism) was assessed. RESULTS: Before ART, the blood contained HIV-1 RNA genomes that were adapted to replication in CD4+ T cells and rapidly decayed after ART initiation. During ART, the blood contained HIV-1 genomes that were drug sensitive, genetically diverse, macrophage-tropic, not evolving and often had defects in vpr. CONCLUSIONS: Our results suggest that in individuals with primary NSV, ART stopped virus replication, but large pools of long-lived, HIV-infected macrophage continued to produce virus. This is mechanistically distinct from secondary NSV produced by CD4+ T cell clones. In addition, defects in vpr independently accumulation in macrophage-tropic lineages found in three participants, suggesting that vpr may impact survival of, or virus production from, HIV-infected macrophage.

Journal Article↗

PubMed2025

Expression of intron-containing HIV-1 RNA induces NLRP1 inflammasome activation in myeloid cells.

Despite the success of antiretroviral therapy in suppressing plasma viremia in people living with human immunodeficiency virus type-1 (HIV-1), persistent viral RNA expression in tissue reservoirs is observed and can contribute to HIV-1-induced immunopathology and comorbidities. Infection of long-lived innate immune cells, such as tissue-resident macrophages and microglia may contribute to persistent viral RNA production and chronic inflammation. We recently reported that de novo cytoplasmic expression of HIV-1 intron-containing RNA (icRNA) in macrophages and microglia leads to MDA5 and MAVS-dependent innate immune sensing and induction of type I IFN responses, demonstrating that HIV icRNA is a pathogen-associated molecular pattern (PAMP). In this report, we show that cytoplasmic expression of HIV-1 icRNA also induces NLRP1 inflammasome activation and IL-1β secretion in macrophages and microglia in an RLR- and endosomal TLR-independent manner. Infection of both macrophages and microglia with either replication-competent or single-cycle HIV-1 induced IL-1β secretion, which was attenuated when cytoplasmic expression of viral icRNA was prevented. While IL-1β secretion was blocked by treatment with caspase-1 inhibitors or knockdown of NLRP1 or caspase-1 expression in HIV-infected macrophages, overexpression of NLRP1 significantly enhanced IL-1β secretion in an HIV-icRNA-dependent manner. Immunoprecipitation analysis revealed interaction of HIV-1 icRNA, but not multiply-spliced HIV-1 RNA, with NLRP1, suggesting that HIV-1 icRNA sensing by NLRP1 is sufficient to trigger inflammasome activation. Together, these findings reveal a pathway of NLRP1 inflammasome activation induced by de novo expressed HIV icRNA in HIV-infected myeloid cells.

HIV-1↗

PubMed2025

Genetic Evolution Between HIV-1 Groups M and O: HIV-1/MO Recombinant Forms.

HIV exhibits significant genetic diversity, with genetic recombination being a major evolutionary process. The co-circulation of HIV-1/M and HIV-1/O variants has led to the description of 20 HIV-1/M+O dual infections since 1998. Despite the genetic divergence between these variants, HIV-1/M+O dual infections have resulted in the emergence of HIV-1/MO intergroup recombinant forms, with 20 unique HIV-1/MO recombinant forms (URF_MO) currently described, raising the question of a possible benefit of the recombination and the modalities of their emergence. This review summarized the current knowledge of HIV-1/MO recombinant forms, including their virological and genetic characteristics, phylogenetic analysis, genome profiles, and breakpoints number and location. This study also identified the potential impacts of HIV-1/MO recombination on diagnosis, monitoring, and treatment, as well as the replicative capacity of such recombinants. This review highlighted the greater diversity and complexity of HIV-1/MO recombinants than originally thought, offering new research perspectives on their emergence and virological properties.

HIV-1↗

PubMed2025

Reverse transcription progression and genome length regulate HIV-1 core elasticity and disassembly.

The structural and mechanical properties of the HIV-1 core are critical for successful infection, balancing stability for early replication and controlled disassembly for genome release. Recent studies have highlighted the role of core elasticity in nuclear entry, yet the molecular determinants regulating this property remain poorly understood. Here, atomic force microscopy (AFM) was used to investigate the relationship between reverse transcription progression, genome length, core elasticity, and disassembly. The results demonstrate that reverse transcription induces a gradual loss of elasticity, rendering the core increasingly brittle as DNA synthesis progresses. Cores containing shorter genomes remained highly elastic, whereas those with longer genomes exhibited increased brittleness, structural damage, and a higher degree of disassembly, after 4 hours of reverse transcription. Additionally, cores from an RNase H-deficient HIV-1 mutant retained high elasticity. These findings provide insight into the interplay between genome synthesis, core integrity, and nuclear entry, supporting a model in which reverse transcription-generated mechanical stress facilitates uncoating. Furthermore, early-stage reverse transcription preserved core elasticity, suggesting a temporal window for successful nuclear import before structural destabilization compromises infectivity.

HIV-1↗

PubMed2025

DHX15 inhibits mouse APOBEC3 deamination.

APOBEC3 family proteins are critical host factors that counteract and prevent the replication of retroviruses and other viruses through cytidine deamination. Human APOBEC3 proteins inactivate HIV-1 through the introduction of lethal mutations to viral genomes. In contrast, mouse APOBEC3 does not induce DNA hypermutation of murine retroviruses, although it retains functional cytidine deaminase activity. Why mouse APOBEC3 does not effectively deaminate murine retroviruses is still unknown. In this study, we found that the dead box helicase DHX15 interacts with mouse APOBEC3 and inhibits its deamination activity. DHX15 was packaged into murine leukemia virus (MLV) virions independent of its binding with APOBEC3. Moreover, DHX15 knockdown inhibited MLV replication and resulted in more G-to-A mutations in proviral DNA. Finally, DHX15 knockdown induced DNA damage in murine cells, suggesting that it plays a role in preserving genome integrity in cells expressing mouse APOBEC3 protein.

Animals↗

PubMed2024

The HIV-1 Transcriptional Program: From Initiation to Elongation Control.

A large body of work in the last four decades has revealed the key pillars of HIV-1 transcription control at the initiation and elongation steps. Here, I provide a recount of this collective knowledge starting with the genomic elements (DNA and nascent TAR RNA stem-loop) and transcription factors (cellular and the viral transactivator Tat), and later transitioning to the assembly and regulation of transcription initiation and elongation complexes, and the role of chromatin structure. Compelling evidence support a core HIV-1 transcriptional program regulated by the sequential and concerted action of cellular transcription factors and Tat to promote initiation and sustain elongation, highlighting the efficiency of a small virus to take over its host to produce the high levels of transcription required for viral replication. I summarize new advances including the use of CRISPR-Cas9, genetic tools for acute factor depletion, and imaging to study transcriptional dynamics, bursting and the progression through the multiple phases of the transcriptional cycle. Finally, I describe current challenges to future major advances and discuss areas that deserve more attention to both bolster our basic knowledge of the core HIV-1 transcriptional program and open up new therapeutic opportunities.

HIV-1↗

PubMed2026

Programmed ribosomal frameshifting triggers translational stress to promote viral replication.

Programmed ribosomal frameshifting (PRF) is a conserved viral strategy for expressing polyproteins from compact genomes. Although PRF is traditionally viewed as a structural mechanism, here we show that it functions as a regulatory signal that rewires host translation in favor of viral replication. A minimal SARS-CoV-2 PRF element is sufficient to activate the GCN2 arm of the integrated stress response (ISR) independently of the canonical ISR sensor ZAKα. This activation serves as a temporal switch during early infection to shut off host translation and is required for viral propagation in cells and human airway organoids. Proteomic and genetic screens identify DRG1 and IGF2BP3 as key mediators of PRF-induced GCN2 activation. We further show that this PRF-GCN2 axis is conserved in human immunodeficiency virus (HIV)-1 and West Nile virus, highlighting its broad relevance across RNA viruses. These findings reveal a sophisticated mechanism of viral translational control, highlighting PRF as a stress-inducing module that enhances viral replication.

RNA virus↗

PubMed2024

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

Hepatitis B virus↗

PubMed2026

In vivo genome editing of central nervous system SIV reservoirs in ART-suppressed rhesus macaques.

Latent human immunodeficiency virus type 1 (HIV-1) reservoirs in the central nervous system (CNS) may sustain viral persistence and neuroinflammation contributing to HIV-associated neurocognitive disorders (HAND) despite suppressive ART. AAV9-delivered CRISPR has successfully edited SIV proviral DNA in peripheral tissues with acceptable safety profiles, but the extent of in vivo genome editing in the brain remains unclear. Using SIV-infected rhesus macaques, we mapped intact proviral DNA across CNS regions and tested systemic AAV9-CRISPR-Cas9 targeting conserved sites within Ψ packaging signal and Gag region. Ten adult rhesus macaques were infected with genetically barcoded SIVmac239, suppressed with ART, then randomized to receive intravenous AAV9-SaCas9 with dual gRNAs (Ψ + Gag) or a Cas9-only control. At necropsy after viral rebound, SIV genomes were detected in multiple brain regions as well as lymphoid tissues, confirming the CNS as a persistent reservoir during ART. Barcode analysis revealed region-specific patterns consistent with compartmentalized CNS persistence. In CRISPR-treated animals, proviral editing was measurable across anatomically distinct CNS sites. These findings demonstrate that intact and potentially replication-competent virus persists in the primate brain under ART and that systemic AAV9-CRISPR can reach and edit proviral DNA in this sanctuary, supporting genome editing as a strategy toward durable remission of CNS reservoirs.

ART↗

PubMed2025

HIV-1 inhibits IFITM3 expression to promote the infection of megakaryocytes.

Despite an undetectable plasma viral load as a result of antiretroviral therapy, HIV-1-infected individuals with poor immune reconstitution harbor infectious HIV-1 within their platelets. Megakaryocytes, as platelet precursors, are the likely cellular origin of these HIV-1-containing platelets. To investigate the mechanisms that allow megakaryocytes to support HIV-1 infection, we established in vitro models of viral infection using hematopoietic stem cell-derived megakaryocytes and the megakaryocytic MEG-01 cell line. We observed HIV-1 DNA provirus integration into the megakaryocyte cell genome, self-limiting virus production, and HIV-1 protein and RNA compartmentalization, which are hallmarks of HIV-1 infection in myeloid cells. In addition, following HIV-1 infection of megakaryocyte precursors, the expression of interferon-induced transmembrane protein 3 (IFITM3), an antiviral factor constitutively expressed in megakaryocytes, was inhibited in terminally differentiated HIV-1-infected megakaryocytes. IFITM3 knockdown in MEG-01 cells prior to infection led to enhanced HIV-1 infection, indicating that IFITM3 acts as an HIV-1 restriction factor in megakaryocytes. Together, these findings indicate that megakaryocyte precursors are susceptible to HIV-1 infection, leading to terminally differentiated megakaryocytes harboring virus in a process regulated by IFITM3. Megakaryocytes may thus constitute a neglected HIV-1 reservoir that warrants further study in order to develop improved antiretroviral therapies and to facilitate HIV-1 eradication.

Humans↗

PubMed2026

The brain as an HIV reservoir: Recent findings using autopsy tissues from people with HIV.

HIV persistence within anatomical reservoirs remains the primary barrier to achieving an HIV cure. While antiretroviral therapy effectively suppresses plasma viremia, it does not eliminate integrated proviral genomes that persist in long-lived cellular compartments. The central nervous system (CNS) is a clinically important HIV reservoir, characterized by immune privilege and the persistence of tissue-resident infection despite effective antiretroviral therapy (ART). Evidence from postmortem studies reveals that HIV DNA, RNA, and even intact replication-competent proviruses remain detectable in brain tissue from virally suppressed people with HIV. Evidence derived primarily from in situ approaches and viable-cell studies supports myeloid-lineage reservoirs, particularly microglia and CNS-associated macrophages, as key cellular sources of persistence, while the extent and biological relevance of astrocyte infection remains debated. These reservoirs exhibit transcriptional activity and are associated with chronic neuroinflammation, which may contribute to HIV-associated neurocognitive disorders, despite systemic viral suppression. Here, we synthesize recent findings from autopsy brain studies, including work enabled by major biorepositories, such as the National NeuroHIV Tissue Consortium and rapid-autopsy programs, including the Last Gift, both of which are essential for studying HIV reservoirs in the CNS. We summarize methodologies for detecting and characterizing HIV in brain tissue, highlight heterogeneous patterns of regional distribution and compartmentalization, and review emerging links between CNS persistence and neuroinflammation. We conclude with priorities for harmonized tissue processing, multi-modal single-cell and spatial profiling, and coordinated cross-cohort analyses to clarify the contribution of CNS reservoirs to neuroHIV pathogenesis and systemic rebound.

Humans↗