CROI 2025 Abstract eBook

Abstract eBook

Oral Abstracts

135

Direct Visualization of HIV-1 Nuclear Import and Its Interplay With the Nuclear Pore Zhen Hou 1 , Stanley Fronik 1 , Yao Shen 1 , Christopher Thompson 2 , Sarah Neumann 2 , Peijun Zhang 1 1 University of Oxford, Oxford, UK, 2 Thermo Fisher Scientific, San Francisco, CA, USA Background: The nuclear import of HIV-1 into the host nucleus marks a milestone of the viral infection, making it a potential drug target for effective prophylaxis and treatment. However, understanding this process has been challenged by its dynamic nature and the scarcity of HIV-1 cores in infected cells. Methods: To elucidate the mechanisms underlying the translocation of HIV-1 capsids into the nucleus and their interactions with nuclear pore complexes (NPCs), we developed a robust system that recapitulates the nuclear import of HIV-1 cores into intact nuclei using mechanically lysed or detergent permeabilized T cells with purified HIV-1 cores. We employed cryo-focused ion beam (cryo-FIB) milling and planar lift-out, guided by cryogenic correlative fluorescence light and electron microscopy (cryo-CLEM), to prepare thin lamellae of T-cell nuclei containing HIV-1 cores. These developments enabled the following correlative cryo-electron tomography (cryo-ET) analysis of HIV-1 core nuclear import in situ . Results: By utilizing these technologies, we achieved a productive capture of HIV-1 cores at different stages of nuclear import, with an unprecedented correlation efficiency of over 52%. Among the 510 HIV-1 cores identified in our tomograms, 87% of cores were specifically associated with nuclei. Statistical analyses of core morphology and size revealed, intriguingly, that tube-shaped and small HIV-1 cores were more advantageous in nuclear import compared to larger cone-shaped cores. Moreover, some large HIV-1 cores appeared to undergo deformation during the passage. Interestingly, NPCs, on the other end, were observed to dilate and occasionally crack, upon the entry of HIV-1 cores. We further observed the surface of capsid is coated with distinct densities, potentially corresponding to FG meshes, when passing through the NPCs. Conclusions: This work introduces a robust in situ system with a highly efficient correlative cryo-ET workflow and provides novel insights into the nuclear import of HIV-1, which not only advances HIV-1 research but also opens a new avenue for studies on nuclear import mechanisms. Visualizing the Cell Biology of HIV Latency and Reactivation Jonathan Karn, Frederick Kizito, Konstantin Leskov, Uri Mbonye, Muda Yang, Anna Agaponova, Kien Nguyen Case Western Reserve University, Cleveland, OH, USA Background: Studies using the Jurkat T-cell model have identified numerous factors crucial for initiating HIV-1 transcription. However, a significant limitation of transformed cells is their inability to replicate the transitions between active effector cells and quiescent memory T cells, which are the key drivers of HIV latency in vivo. Methods: We use refined primary cell models for HIV latency that accurately replicate the transitions between active effector cells and quiescent memory T cells. Our approach combines an HIV Cas-FISH immunofluorescence assay to trace changes in HIV DNA localization and immunofluorescence to identify key transcription factors. In parallel, the cells were analyzed by single-cell (sc) RNA-Seq, snRNA-Seq, AB-Seq, and ATAC-Seq, ensuring a comprehensive understanding of the process. Results: As activated effector cells transition quiescence, HIV proviral DNA moves from the nuclear Intermediate Euchromatic Compartment (IEC) to the perinucleolar compartment (PNC). Strikingly, the localization of proviruses to the PNC was blocked by integrase inhibitor raltegravir, which showed that this was due to spatial genome rearrangements. Dramatic changes in histone methylation, cellular metabolism, and transcriptome changes accompany the transition into quiescence. snRNA-Seq and scATAC-Seq show that the subset of genes that are known HIV integration sites become more inaccessible when cells enter quiescence. During the reactivation of latently infected cells through the T-cell receptor, nascent viral mRNA transcripts emanating from proviruses in the PNC were detected. There was the rapid accumulation of the viral trans-activator Tat and its proviral reactivation regulatory partners, P-TEFb and 7SK snRNA, and RNA processing factors in large interchromatin granule clusters, which assembled near the provirus, and a shift of the proviruses away from the nucleolus. For all latency reversing agens (LRAs), there is a strong correlation between the extent of P-TEFb biogenesis and their potency. HIV proviruses in latently infected memory T cells from patients also accumulated in the PNC and showed identical patterns of nuclear rearrangements after cellular reactivation.

Conclusions: In contrast to transformed cells where proviruses are found primarily at the nuclear periphery, in primary memory T cells, P-TEFb highly restricts HIV transcription, and the nuclear architecture undergoes rearrangements that shape the transcriptional silencing and reactivation of proviral HIV.

Oral Abstracts

137

Single-Cell Spatial Profiling Identified Intact HIV+ Cells in Lymph Nodes Amare Eshetu 1 , Nadejda Beliakova-Bethell 2 , Antoine Chaillon 3 , Davey Smith 3 , Ya-Chi Ho 1 , Sara Gianella Weibel 3 1 Yale University, New Haven, CT, USA, 2 Veterans Affairs San Diego Healthcare System, San Diego, CA, USA, 3 University of California San Diego, La Jolla, CA, USA Background: How HIV reservoir is established in lymphoid tissues remain elusive. Methods: We obtained frozen lymph nodes from 2 viremic donors and 2 virally suppressed donors from the Last Gift Cohort. Lymph nodes from 2 uninfected donors served as negative controls. We performed single-cell spatial transcriptomic profiling (475 immune genes) of 11 tissue sections using 10x Genomics Xenium, which uses cell segmentation to identify bona fide single cells. To distinguish intact vs non-intact HIV RNA, we designed 5 sets of HIV probes targeting packaging signal, Rev-response element (RRE), unspliced HIV (gag-pol), splice junctions, and all HIV. A threshold of >2 HIV RNA reads/probe defines HIV RNA+ cells. Detection of both packaging signal and RRE defines cells having intact HIV. An adjusted P<0.05 with a log2 fold change >2 is considered significant. Results: We found that nonspecific RNA detection was a major caveat of this platform. We identified 791,108 cells (162,326 from viremic, 129,754 from virally suppressed, and 499,028 from uninfected donors). In viremic samples, we identified 1,967 HIV+ cells (1.21%, 12,117.6/million), including 17 cells having intact HIV RNA (104.7/million). Comparing non-intact HIV+ cells vs HIV– cells in viremic samples showed no differentially expressed genes, indicating the robustness of intact HIV detection. Of note, HIV– cells in viremic (vs virally suppressed) samples already showed upregulated type I IFN response ( IFI44L , MX1 , IFIT1 , IFIT2 , IFIT3 , ISG15 , APOBEC3G ), Th1 ( EOMES , IFNG , TNF ), cytotoxic ( NKG7 , CD8B , GZMH , GZMK , GZMB ), and exhaustion ( LAG3 , TIGIT ) genes, indicating immune responses to HIV in the tissues in uninfected cells. In viremic sample, HIV+ cells having intact HIV (vs uninfected cells) had higher levels of type I IFN response ( ISG20 , IRF8 ), cytotoxic ( PRF1 , GZMK , GZMH , NKG7 ), memory ( SELL , CCR7 , TCF7 ), activation ( TNFRSF9 , ICOS , CD40 , CD74 ), exhaustion ( TOX , PDCD1 ), Th1 ( TBX21 ), Th17 ( CCR6 ), homing ( CD69 , ITGB7 ), and HIV coreceptor ( CCR5 , CXCR4 ) genes. Of note, we found upregulation of BACH2 and BCL2A1 in intact HIV+ cells which may promote long-term survival. In contrast, in virally suppressed (vs uninfected donor) samples, IFITM3 and IL1B were the only upregulated type I IFN response genes, indicating minimal but detectable viral sensor expression. Conclusions: We identified immune profiles of cells having intact HIV in lymph nodes and the lymph node microenvironment during viremia and viral suppression.

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CROI 2025