# Project 3. Integration

> **NIH NIH U54** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2024 · $259,200

## Abstract

Project 3 – Integration
Retroviral replication requires integration of reverse-transcribed viral DNA into a host cell chromosome. This
process is catalyzed by integrase (IN) in the context of the stable nucleoprotein complex, containing a multimer
of IN assembled on viral DNA ends and termed the intasome. Characterization of in vitro-assembled intasomes
from many retroviral species elucidated conserved as well as genus-specific features. Lentiviral intasomes
harbor large IN homo-oligomers, containing as many as 16 subunits. However, the intasome represents only a
small part of the pre-integration complex (PIC) that assembles and mediates intracellular trafficking of the
intasome during virus infection. Furthermore, it is currently unclear how the HIV-1 PIC interfaces with its target,
chromatinized host cell genomic DNA. This project aims to characterize the structure and properties of the
HIV/lentiviral integration machinery using complementary top-down and bottom-up approaches. From the top-
down, we will leverage several novel affinity reagents to enrich for PICs from cellular extracts and then study
hemi-purified native HIV-1 PICs using advanced DNA footprinting and microscopy techniques. Experiments
using HIV-1 PICs will reveal the function of the IN-binding partner LEDGF/p75 and its cognate epigenetic
modification (H3K36me3) in integration in the context of chromatin. With the bottom-up approach, we will take
advantage of the intasome from maedi-visna virus (MVV, an ovine lentivirus), which, unlike HIV/SIV intasomes,
can be assembled in vitro as monodispersed preparations. This 0.6-MDa complex will allow us to characterize
the dynamics and the structure of the complex between the lentiviral intasome and chromatin at high temporal
and spatial resolutions. The proposed studies will shed light on the architecture of the lentiviral DNA integration
apparatus and how it interfaces with host factors and chromosomes, while avoiding suicidal autointegration.
Given that IN inhibitors are used worldwide to treat people living with HIV-1, our results will provide
unprecedented details of the biological machine that is the target of these highly successful medicines and could
reveal new ways to attack the PIC for therapeutic intervention.

## Key facts

- **NIH application ID:** 10835092
- **Project number:** 5U54AI170791-03
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Peter Cherepanov
- **Activity code:** U54 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $259,200
- **Award type:** 5
- **Project period:** 2022-07-01 → 2027-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10835092

## Citation

> US National Institutes of Health, RePORTER application 10835092, Project 3. Integration (5U54AI170791-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10835092. Licensed CC0.

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