# Single-cell capture of on-ART SIV transcription reveals TGF-β–mediated metabolic control of viral latency

## TL;DR

This study shows that blocking TGF-β with a drug called galunisertib can reverse SIV latency in monkeys by changing the metabolism of infected T cells.

## Contribution

The study introduces a novel SIV Transcripts Capture Assay (SCAP) to detect rare virus-expressing cells and identifies TGF-β as a key regulator of viral latency through metabolic suppression.

## Key findings

- Galunisertib caused metabolic reprogramming in CD4+ T cells, activating glycolysis and fatty acid metabolism.
- SIV-expressing cells were metabolically quiescent before treatment and activated after galunisertib.
- The study found increased inflammatory macrophages and fewer tissue-resident memory T cells after treatment.

## Abstract

We previously demonstrated that blocking TGF-β with galunisertib, a safe, orally available small drug, reactivated latent SIV in vivo by shifting T cells toward a transitional effector phenotype. Here, we investigated the mechanisms underlying this effect using single-cell RNA sequencing, metabolic profiling, and high-dimensional spectral flow cytometry of samples from SIV-infected, antiretroviral therapy–treated (ART-treated) macaques before and after galunisertib. To characterize virus-transcribing, infected cells during ART, we developed a novel, sensitive SIV Transcripts Capture Assay (SCAP) that detected 127 SIV-expressing cells within lymph node single-cell transcriptome libraries. Galunisertib drove broad metabolic reprogramming in CD4+ T cells, with transcriptional upregulation of inflammatory and mitochondrial biosynthesis pathways, confirmed by Seahorse profiling. Metabolomics revealed increased energy metabolites and amino acids and enhanced metabolic flux without proliferation. SIV transcript–positive cells before galunisertib were metabolically quiescent compared with cells without detectable viral transcripts. After galunisertib, virus-expressing cells showed a dramatic metabolic activation, with upregulation of glycolysis, fatty acid metabolism, and TNF-α signaling. High-dimensional flow cytometry demonstrated effects beyond CD4+ T cells, including fewer tissue-resident memory T cells, but more inflammatory macrophages. In conclusion, SCAP represents a specific tool for characterizing rare SIV-infected cells transcribing virus during ART, and it reveals TGF-β as a key mediator of viral latency in vivo through metabolic suppression.

Characterization of cells producing virus in tissues in macaques with SIV on therapy points to metabolic reprogramming as a way to reverses SIV latency.