The free energy landscape of retroviral integration

Willem Vanderlinden,Pauline J Kolbeck,Jan Lipfert,Tine Brouns,Lukas F Milles,Philipp U Walker,Philipp C Nickels,Wolfgang Ott,Zeger Debyser

doi:10.1038/s41467-019-12649-w

Abstract

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo.

Highlights

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle
We further show how auxiliary DNAbinding interfaces enable dynamic target site selection and undergo conformational changes on strand transfer, suggesting a mechanism for engaging cellular machines involved in strand transfer complex (STC) resolution and repair
Samples prepared by incubating pBR322 plasmids with 10 nM wild-type (WT) cleaved intermediate (CI) in reaction buffer and direct deposition for atomic force microscopy (AFM) imaging resulted in ~25% intasome-bound plasmids overall (Fig. 1d, e)

Summary

Introduction

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Spuma-, beta-/gamma-, and lentiviral intasomes are composed of integrase tetramers, octamers, or hexadecamers, respectively[8–13] Despite this architectural diversity, a genus-independent intasome core is conserved throughout the available structures. A recent single-molecule study found a highly inefficient target capture and/or strand transfer, resulting in a short-lived STC16 These results challenge the hypothesis that efficient integration is achieved through an increasing stability of consecutive nucleoprotein intermediates[2,17–19]. It remains unclear whether and how retroviruses have evolved a mechanism for a high strand-transfer yield and what processes follow strand-transfer completion. We further show how auxiliary DNAbinding interfaces enable dynamic target site selection and undergo conformational changes on strand transfer, suggesting a mechanism for engaging cellular machines involved in STC resolution and repair

Methods

Results

Conclusion