Structural and sequencing analysis of local target DNA recognition by MLV integrase.

S Aiyer,G T Montelione,A Chandar,M J Roth,N Malani,P Rossi,W M Schneider,F D Bushman

doi:10.1093/nar/gkv410

Abstract

Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD β1-β2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD β1-β2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD β1-β2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection.

Highlights

Retroviral integrase (IN) proteins mediate an indispensable step in the retrovirus replication, the irreversible integration of viral cDNA into the host genome
We report the comprehensive analysis and validation of the solution nuclear magnetic resonance (NMR) structure of the murine leukemia virus (MLV) IN C-terminus (IN C)-terminal domain (CTD), which is consistent with other retroviral CTD domains in adopting an SH3 fold
Homology modeling and structural analysis of the MLV IN catalytic core domain (CCD) was greatly assisted by the available prototype foamy virus (PFV) IN intasome coordinates [15], which we used as template

Summary

Introduction

Retroviral integrase (IN) proteins mediate an indispensable step in the retrovirus replication, the irreversible integration of viral cDNA into the host genome. The first step in integration involves removal of the terminal dinucleotide of the viral long terminal repeats (LTRs) produced by reverse transcription, exposing the 3 OH group of the invariant CA dinucleotide. The processed LTR DNA ends are inserted into the host genome through an energy-independent transesterification step known as the strand transfer reaction [1]. For murine leukemia virus (MLV), integration results in a target site duplication (TSD) of 4 bp, 3 to the invariant viral CA dinucleotide. MLV integration preferentially occurs at transcription start sites and CpG islands [2]. More recently it has been shown that strong enhancers are the predominant targets of MLV integration [3,4]

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Results

Conclusion