Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration

Abstract

Integrase is the key enzyme that mediates integration of retroviral DNA into cellular DNA which is essential for viral replication. Inhibitors of HIV-1 that target integrase recognize the nucleoprotein complexes formed by integrase and viral DNA substrate (intasomes) rather than the free enzyme. Atomic resolution structures of HIV-1 intasomes are therefore required to understand the mechanisms of inhibition and drug resistance. To date, prototype foamy virus (PFV) is the only retrovirus for which such structures have been determined. We show that PFV strand transfer complexes (STC) can be assembled on product DNA without going through the normal forward reaction pathway. The finding that a retroviral STC can be assembled in this way may provide a powerful tool to alleviate the obstacles that impede structural studies of nucleoprotein intermediates in HIV-1 DNA integration.

Allosteric Inhibition of Human Immunodeficiency Virus Integrase

“…In support of this, the HIV-1 IN(CCD-CTD) crystal structure (Protein Data Bank entry 1EX4 (53)) reveals a tetramer packing interface that is mediated solely by CTD-CTD interactions and bears no resemblance to the tetramers observed in the prototype foamy virus integrase in complex with DNA (62)(63)(64)(65)(66). Thus, we hypothesize that the open polymer formed in the presence of allosteric inhibitors proceeds through polymerization of the CTD as in the 1EX4 crystal lattice.…”

Retroviral DNA Integration

“…Snapshots of such symmetrized PFV intasomes, visualized in the states prior to and after 3′-processing, as well as in complex with target host DNA prior to and after strand transfer, are now available. 156,157,190 …”

Retroviral intasomes arising

“…Determined using X-ray crystallography, this structure was transformative in that it provided the initial glimpse of a functional IN multimer and also elucidated the mechanism of action of the clinical HIV IN strand transfer inhibitors [4]. Additional structures, which visualized the remaining PFV complexes along the integration pathway, were similarly reported over the ensuing 2 years (Figure 1) [2,5,6]. Today, the PFV intasome can be considered a minimalist assembly with the lowest order IN-to-viral DNA stoichiometry among retroviral intasomes characterized thus far [7].…”

“…While, in principle, one could envisage a functional intasome made of only two catalytic IN subunits, the smallest complex observed to-date is that of PFV, a bare-bones tetrameric CIC. Interestingly, the NED, NTD, and CTD of the outer IN subunits, which are disordered in all PFV intasome crystal structures [2,4–6], are dispensable for catalytic function in vitro [35]. However, the outer IN subunits were implicated in interactions with nucleosomal DNA during integration into chromatinized targets [36].…”