In situ structural biology of HIV-1 transcription and latency in CD4⁺ t cells

The National Institute of Allergy and Infectious Diseases awarded the University of Oxford $170,980 on June 23, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to define the structural basis of HIV persistence through in situ structural biology of HIV-1 transcription and latency in CD4 T cells.

The project applies correlative light and electron microscopy (CLEM), cryo-electron tomography (cryo-ET), and cryo-focused ion beam milling to visualize individual HIV proviruses within intact nuclei in both CEM cell lines and primary CD4 T cells. The work addresses how chromatin organization, proximity to nuclear bodies, nucleosome positioning, and access to transcriptional machinery determine whether integrated proviruses remain latent or actively transcribe. Aim 1 establishes dual-reporter strategies in CEM cells to label proviral DNA and nascent RNA, enabling identification of active and silent proviruses and mapping of their nuclear niches by live-cell imaging and correlative cryo-ET. Aim 2 extends these methods to physiologically relevant primary CD4 T cells to classify distinct nuclear structural states and test whether latency states are structurally distinguishable and remodelable.

Work is performed in Great Britain, United Kingdom. The period of performance runs through May 31, 2028. This is a Project Grant assistance type.

Abstract

Human immunodeficiency virus type 1 (HIV-1) persists in long-lived CD4⁺ t cells despite effective antiretroviral therapy, where integrated proviruses remain silent or intermittently active. This latent reservoir is the major barrier to cure. Whether a provirus is latent or productive depends on its nuclear environment, including chromatin organization, proximity to nuclear bodies, positioning of nucleosomes, and access to transcriptional machinery. Current knowledge from the past 30 years of study is based largely on bulk assays that average across cells and require artificial enrichment of "latent" populations, masking the true heterogeneity of proviral states. Microscopy-based structural biology offers a unique solution. Correlative light and electron microscopy (clem) and cryo-electron tomography (cryo-ET) enable direct visualization of individual proviruses in intact nuclei, allowing transcriptionally active and silent proviruses to be examined side by side. This approach provides a population-based nanoscale view of latency and active transcription, revealing diverse structural states. The goal of this project is to define the structural basis of HIV persistence. We will apply cryo-clem, cryo-focused ION beam (cryo-FIB) milling, and cryo-ET to visualize the nuclear environments of HIV proviruses in both CEM cell lines and primary CD4⁺ t cells. We hypothesize that nanoscale chromatin and nuclear body architecture determines proviral competence and that distinct latency states are structurally distinguishable and remodelable. Aim 1 will establish dual-reporter strategies in CEM cells to label proviral DNA and nascent RNA, enabling identification of active and silent proviruses and mapping of their nuclear niches by live-cell imaging and correlative cryo-ET. Aim 2 will extend these methods to physiologically relevant primary CD4⁺ t cells, classifying distinct nuclear structural features associated with latency and transcription. This project will deliver the first in situ structural characterization of HIV proviruses in primary CD4⁺ t cells. The results will reveal nanoscale determinants of reservoir stability, clarify how nuclear architecture regulates proviral activity, and inform rational cure strategies. More broadly, the work will establish imaging approaches generally applicable to transcriptional regulation in both viral and host systems.