Thursday, November 21, 2024
11/21/2024
Project Abstract: Retrovirus intasomes are integrase (IN) multimers bound to two viral DNA ends that are capable of concerted integration into a target DNA. Assembly mechanisms are unknown for IN multimers in different retrovirus intasomes, whose structures contain four IN subunits (prototype foamy virus, delta retroviruses), or 4 to 8 (alpha and beta retroviruses) or 4 to 12 and up to 16 subunits (lentiviruses). The 3-dimensional structure of different retrovirus intasomes has revealed remarkable architectural diversities besides conserved structural features and catalytic mechanisms. To understand the assembly mechanisms, we have determined 1) biophysically that the Rous sarcoma virus (RSV) tetrameric intasome is the precursor to the mature octameric intasome with four IN dimers; 2) intasome assembly in vitro is controlled by the C-terminal “tail” region comprising the last 17 residues of IN; and 3) the structure of RSV octameric intasome (containing four IN dimers and two viral DNA) and strand transfer complex (STC) with four IN dimers and a viral/target DNA substrate. We will determine the structure of RSV tetrameric intasome, a novel intermediate in RSV integration pathway by cryo-EM. We recently determined the structure of RSV octameric intasome by single particle cryo-EM at an overall resolution of 3.2 Å. The conserved intasome core (CIC) that contains the catalytic center for concerted integration had a resolution of 2.8 Å. The ensemble of conformations in the intasomes revealed significant dynamic flexibility of the two non-catalytic distal IN dimers, along with some movement of the two catalytic proximal dimers contained in the CIC, previously unrecognized in retrovirus intasomes. We hypothesize that there are ordered conformational transitions between key intermediates in the assembly pathway of RSV intasomes and the subsequent capture of the host target DNA. Our proposed studies will reveal how RSV IN first assembles the precursor tetrameric intasome en route to the mature octameric intasome by the following Aims. Aim 1. Determine the structure of the RSV intasomes. We will determine the structure of the RSV tetrameric intasome by cryo-EM. We will determine the first structure of intasome assembled with both U3 and U5 LTRs to provide novel insights into the assembly of intasomes most similar to biological complexes. Aim 2. Determine the functional significances of tetrameric and octameric intasomes. We will use structural and virological approaches to increase our knowledge of retrovirus integration, enhanced by near- atomic structure-based missense mutagenesis of RSV IN. We will focus on the transition of the tetrameric intasome to the octameric form to probe the independent functions of the distal and proximal IN dimers. Virological effects of single-point mutations in IN will be measured by their effect on reverse transcription and integration in virus infected cells and electron microscopy of virions to define phenotype under physiological conditions. These projects will expand our understanding of intasomes functional states and determine how higher order structures influence integration, and hence retroviral replication.
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