# The HUSH complex in HIV-1 latency

> **NIH NIH R37** · UNIV OF MASSACHUSETTS MED SCH WORCESTER · 2024 · $837,467

## Abstract

PROJECT SUMMARY (See instructions):
Antiretroviral therapy (ART) suppresses HIV-1 viremia but does not cure HIV-1 infection. Inability to cure is attributed 
to HIV-1 proviruses in long-lived memory CD4+ T cells, most of which are transcriptionally silent and resistant to 
extirpation. Better understanding of the factors that regulate provirus transcription could offer new strategies for 
effective cure of HIV-1 infection. This grant proposal stems from our observation that vpx/vpr from across primate 
immunodeficiency virus phylogeny increase provirus transcription by degrading the Human Silencing Hub (HUSH) 
complex. The immediate goals are to better understand host and viral determinants of HUSH-mediated transcriptional 
silencing of the provirus. Aim 1 will be to use unbiased, genome-wide, HUSH loss-of-function screens to identify host 
factors required in trans for HUSH-mediated silencing. We will also identify cellular proteins near HUSH-silenced 
proviruses using a dCas9–APEX2 fusion protein (C-BERST) that is targeted to proviruses with sgRNAs. Functional 
relevance of hits will be confirmed and mechanistic hypotheses about how these factors contribute to provirus 
silencing will be tested. We will assess the contribution of our hits to HIV-1 latency by quantitating cell-associated HIV1 RNA after candidate gene disruption in stimulated CD4+ T cells from people living with HIV-1 on ART. Aim 2 will be 
to characterize cis-acting requirements for detection and silencing by HUSH. Promoters and transcripts from HUSH 
targeted proviruses and transgenes will be engineered to finely map and characterize the determinants of HUSH 
sensitivity. HIV-1 sequences cloned from people living with HIV-1 will be tested to determine if HUSH exerts selective 
pressure on the virus during establishment of HIV-1 latency in vivo. If HIV-1 infection is to be treated by perturbing 
HUSH, the consequences of these interventions on CD4+ T cell function and development need to be better 
understood. In Aim 3, the effect of HUSH on the chromatin landscape in 1° CD4+ T cells will be assessed using RNASeq, ATAC-Seq, and CUT&Tag for multiple chromatin features. HUSH-sensitive genes identified here, which include 
endogenous retroviruses, will be used iteratively as reporters for screens in Aim 1. HUSH components will be knocked 
out in human CD34+ hematopoietic stem cells (HSCs) and these cells will be used to reconstitute a model immune 
system in which the effect of HUSH knockout on development and chromatin landscape of CD4+ T cells will be 
assessed. Finally, the effect of HUSH knockout on HIV-1 replication and establishment of latency will be assessed 
within this experimental model of HIV-1 infection.

## Key facts

- **NIH application ID:** 10886200
- **Project number:** 4R37AI147868-06
- **Recipient organization:** UNIV OF MASSACHUSETTS MED SCH WORCESTER
- **Principal Investigator:** JEREMY LUBAN
- **Activity code:** R37 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $837,467
- **Award type:** 4C
- **Project period:** 2019-07-01 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10886200

## Citation

> US National Institutes of Health, RePORTER application 10886200, The HUSH complex in HIV-1 latency (4R37AI147868-06). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10886200. Licensed CC0.

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