Cherepanov lab

Chromatin Structure and Mobile DNA Laboratory

 Retroviral DNA integration

Three-dimensional model of the retroviral intasome and chemical structures of some of the clinical HIV-1 integrase inhibitors studied in the lab

Retroviral replication requires stable insertion of the viral genetic material into cellular genome. The catalytic events associated with this process are carried out by the virus-derived enzyme integrase (IN).

Retroviral INs are structurally and mechanistically related to a diverse group of enzymes including bacterial and eukaryotic transposases, V(D)J recombinase RAG1/2, ribonuclease H, and the catalytic subunit of the RNA-induced silencing complex (RISC).

This superfamily is distinguished by the characteristic structural fold, organization of the active site and metal-dependent catalysis. As pathogens, viruses present an obvious societal burden and, being of the three essential retroviral enzymes, IN is an important target for anti-HIV/AIDS drug development. On the flipside, the unique ability of retroviruses to efficiently integrate their genetic material into host cell chromosomal DNA makes them ideal genome manipulation tools.

In principle, a single application of a retroviral vector may be sufficient to correct a genetic deficiency, make cells resistant to an infection or help the immune system to fight off cancer. Yet, uncontrolled integration of retroviral vectors poses an inherent risk of insertional mutagenesis and, consequently, serious side effects.

Over the years we determined a collection of crystal structures, which illustrated the mechanism of the retroviral integration process and revealed the mode of action of clinical HIV-1 IN inhibitors, such as Raltegravir. We also determined crystal structures of Transportin 3 (Tnpo3), the B-karyopherin involved in the early steps of HIV-1 replication in addition to its role in nuclear import of cellular Ser/Arg-rich splicing factors.

The brunt of our current efforts is currently directed towards understanding the rules of engagement between the retroviral integration machinery and the cellular environment. We hope that our research will yield the keys to developing safer retroviral vectors as well as novel concepts for anti-HIV/AIDS therapies. The two major projects aim to elucidate the structural and functional features of the interface between integrase and chromatin and the identification and the roles of host cell factors during integration.

Crystal structure of the intasome after engaging target (genomic) DNA in the strand transfer complex

Figure 1. (A) Crystal structure of the intasome after engaging target (genomic) DNA in the strand transfer complex. (B) IN active site before (top) and after binding Raltegravir (RAL). Active site residues comprising the active site DDE motif of IN are indicated. (C) Crystal structure of TNPO3 in complex with its cellular cargo ASF/SF2. This panel is adapted from Maertens et al., Proc Natl Acad Sci USA, 2014, 111:2728–33.