Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS.

The biological significance of chemical modifications to the ribonucleic acid (RNA) of human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding of the site-specific and context-dependent roles of these chemical modifications remains limited, primarily due to the absence of nucleotide-resolution mapping of modification sites. In this study, we present a method for achieving nucleotide-resolution mapping of chemical modification sites on HIV-1 RNA using liquid chromatography and tandem mass spectrometry (LC-MS/MS). LC-MS/MS, a powerful tool capable of directly analyzing native RNAs, has proven effective for mapping RNA modifications in small RNA molecules, including ribosomal RNA and transfer RNA. However, longer RNAs have posed challenges, such as the 9 Kb HIV-1 virion RNA, due to the complexity of and ambiguity in mass differences among RNase T1-cleaved RNA fragments in LC-MS/MS data. Here, we introduce a new target RNA enrichment method to isolate small local RNA fragments of HIV-1 RNA that potentially harbor site-specific N6-methyladenosine (m6A) modifications. In our initial trial, we used target-specific DNA probes only and encountered insufficient RNA fragmentation due to inefficient S1 digestion near the target site. Recognizing that inefficient S1 digestion by HIV-1 RNA is likely due to the formation of secondary structures in proximity to the target site, we designed multiple DNA probes annealing to various sites of HIV-1 RNA to better control the structures of RNA substrates for S1 digestion. The use of these non-target DNA probes significantly improved the isolation of more homogeneous target RNA fragments of approximately 50 bases in length. Oligonucleotide LC-MS/MS analysis of these isolated target RNA fragments successfully separated and detected both m6A-methylated and non-methylated oligomers at the two m6A-predicted sites. The principle of this new target enrichment strategy holds promise and should be broadly applicable to the analysis of any lengthy RNA that was previously deemed infeasible for investigation using oligonucleotide LC-MS/MS.

Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As.

Although the significance of chemical modifications on RNA is acknowledged, the evolutionary benefits and specific roles in human immunodeficiency virus (HIV-1) replication remain elusive. Most studies have provided only population-averaged values of modifications for fragmented RNAs at low resolution and have relied on indirect analyses of phenotypic effects by perturbing host effectors. Here we analysed chemical modifications on HIV-1 RNAs at the full-length, single RNA level and nucleotide resolution using direct RNA sequencing methods. Our data reveal an unexpectedly simple HIV-1 modification landscape, highlighting three predominant N6-methyladenosine (m6A) modifications near the 3' end. More densely installed in spliced viral messenger RNAs than in genomic RNAs, these m6As play a crucial role in maintaining normal levels of HIV-1 RNA splicing and translation. HIV-1 generates diverse RNA subspecies with distinct m6A ensembles, and maintaining multiple of these m6As on its RNAs provides additional stability and resilience to HIV-1 replication, suggesting an unexplored viral RNA-level evolutionary strategy.

CD3-immunotoxin mediated depletion of T cells in lymphoid tissues of rhesus macaques.

Selective T-cell depletion prior to cell or organ transplantation is considered a preconditioning regimen to induce tolerance and immunosuppression. An immunotoxin consisting of a recombinant anti-CD3 antibody conjugated with diphtheria toxin was used to eliminate T-cells. It showed significant T-cell depletion activity in the peripheral blood and lymph nodes in animal models used in previous studies. To date, a comprehensive evaluation of T-cell depletion and CD3 proliferation for all lymphoid tissues has not been conducted. Here, two rhesus macaques were administered A-dmDT390-SCFBdb (CD3-IT) intravenously at 25 µg/kg twice daily for four days. Samples were collected one day prior to and four days post administration. Flow cytometry and immunofluorescence staining were used to evaluate treatment efficiency accurately. Our preliminary results suggest that CD3-IT treatment may induce higher depletion of CD3 and CD4 T-cells in the lymph nodes and spleen, but is ineffective in the colon and thymus. The data showed a better elimination tendency of CD4 T-cells in the B-cell zone relative to the germinal center in the lymph nodes. Further, CD3-IT treatment may lead to a reduction in germinal center T follicular helper CD4 cells in the lymph nodes compared to healthy controls. The number of proliferating CD3 T-cell indicated that repopulation in different lymphoid tissues may occur four days post treatment. Our results provide insights into the differential efficacy of CD3-IT treatment and T-cell proliferation post treatment in different lymphoid tissues. Overall, CD3-IT treatment shows potential efficacy in depleting T-cells in the periphery, lymph nodes, and spleen, making it a viable preconditioning regimen for cell or organ transplantation. Our pilot study provides critical descriptive statistics and can contribute to the design of larger future studies.

CD3e-immunotoxin spares CD62Llo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3ehi T cells.

CD3-epsilon(CD3e) immunotoxins (IT), a promising precision reagent for various clinical conditions requiring effective depletion of T cells, often shows limited treatment efficacy for largely unknown reasons. Tissue-resident T cells that persist in peripheral tissues have been shown to play pivotal roles in local and systemic immunity, as well as transplant rejection, autoimmunity and cancers. The impact of CD3e-IT treatment on these local cells, however, remains poorly understood. Here, using a new murine testing model, we demonstrate a substantial enrichment of tissue-resident Foxp3+ Tregs following CD3e-IT treatment. Differential surface expression of CD3e among T-cell subsets appears to be a main driver of Treg enrichment in CD3e-IT treatment. The surviving Tregs in CD3e-IT-treated mice were mostly the CD3edimCD62Llo effector phenotype, but the levels of this phenotype markedly varied among different lymphoid and nonlymphoid organs. We also found notable variations in surface CD3e levels among tissue-resident T cells of different organs, and these variations drive CD3e-IT to uniquely reshape T-cell compositions in local organs. The functions of organs and anatomic locations (lymph nodes) also affected the efficacy of CD3e-IT. The multi-organ pharmacodynamics of CD3e-IT and potential treatment resistance mechanisms identified in this study may generate new opportunities to further improve this promising treatment.

Comparison of CD3e Antibody and CD3e-sZAP Immunotoxin Treatment in Mice Identifies sZAP as the Main Driver of Vascular Leakage.

Anti-CD3-epsilon (CD3e) monoclonal antibodies (mAbs) and CD3e immunotoxins (ITs) are promising targeted therapy options for various T-cell disorders. Despite significant advances in mAb and IT engineering, vascular leakage syndrome (VLS) remains a major dose-limiting toxicity for ITs and has been poorly characterized for recent "engineered" mAbs. This study undertakes a direct comparison of non-mitogenic CD3e-mAb (145-2C11 with Fc-silentTM murine IgG1: S-CD3e-mAb) and a new murine-version CD3e-IT (saporin-streptavidin (sZAP) conjugated with S-CD3e-mAb: S-CD3e-IT) and identifies their distinct toxicity profiles in mice. As expected, the two agents showed different modes of action on T cells, with S-CD3e-mAb inducing nearly complete modulation of CD3e on the cell surface, while S-CD3e-IT depleted the cells. S-CD3e-IT significantly increased the infiltration of polymorphonuclear leukocytes (PMNs) into the tissue parenchyma of the spleen and lungs, a sign of increased vascular permeability. By contrast, S-CD3e-mAbs-treated mice showed no notable signs of vascular leakage. Treatment with control ITs (sZAP conjugated with Fc-silent isotype antibodies) induced significant vascular leakage without causing T-cell deaths. These results demonstrate that the toxin portion of S-CD3e-IT, not the CD3e-binding portion (S-CD3e-mAb), is the main driver of vascular leakage, thus clarifying the molecular target for improving safety profiles in CD3e-IT therapy.

The clonal repopulation of HSPC gene modified with anti-HIV-1 RNAi is not affected by preexisting HIV-1 infection.

Despite advances in hematopoietic stem/progenitor cell (HSPC) transplant for HIV-1-infected patients, the impact of a preexisting HIV-1 infection on the engraftment and clonal repopulation of HSPCs remains poorly understood. We have developed a long terminal repeat indexing-mediated integration site sequencing (LTRi-Seq) method that provides a multiplexed clonal quantitation of both anti-HIV-1 RNAi (RNA interference) gene-modified and control vector-modified cell populations, together with HIV-1-infected cells-all within the same animal. In our HIV-1-preinfected humanized mice, both therapeutic and control HSPCs repopulated efficiently without abnormalities. Although the HIV-1-mediated selection of anti-HIV-1 RNAi-modified clones was evident in HIV-1-infected mice, the organ-to-organ and intra-organ clonal distributions in infected mice were indistinguishable from those in uninfected mice. HIV-1-infected cells showed clonal patterns distinct from those of HSPCs. Our data demonstrate that, despite the substantial impact of HIV-1 infection on CD4+ T cells, HSPC repopulation remains polyclonal, thus supporting the use of HSPC transplant for anti-HIV treatment.