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Analysis of target DNA binding sites through NMR structural analysis of the MLV IN CTD and homology modeling of the CCD domains.
Genomic integration sites of MLV in HEK293mCAT cells
Target-site selection by retroviral integrase (IN) proteins has profound effects on viral pathogenesis. We describe the solution NMR 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).  The MLV IN CTD adopts an SH3 domain fold followed by a long unstructured tail. We have obtained a concordant structural model of the MLV IN CCD using SWISS-MODEL, MMM-tree and I-TASSER servers. Using the PFV IN target capture complex X-ray structure and structure-based sequence alignment, residues within the CCD ?2 helical region and the CTD ?1-?2 loop predicted to bind target DNA in the context of the active MLV intasome were identified. The role of these residues in vivo was analyzed by point mutations and motif interchanges.  Chimeric viruses with substitutions at the CCD ?2 helical region and the CTD ?1-?2 loop result in viable virus and point mutants within those interchanges function similar to predicted PFV IN residue functions. Next-generation sequencing and analysis of local integration target sites indicate the CCD ?2 helical region, in particular P187, interacts with the sequence outside the target site duplication (TSD), whereas the CTD ?1-?2 loop bind to residues within TSD.