Thursday, April 3, 2025
4/3/2025
Correlative cryoET of the HIV-1 integration targeting in native T-lymphocytes - Title: Correlative cryoET of the HIV-1 integration targeting in native T-lymphocytes Project Summary Retroviruses, including human immunodeficiency virus type 1 (HIV-1), integrate their genetic material into the host cell genome during their replication cycle. In the case of HIV-1, upon entering a host cell, viral RNA is reverse transcribed into a double-stranded viral DNA (vDNA) by the viral reverse transcriptase. This vDNA, along with other viral and cellular proteins, forms a preintegration complex (PIC), which translocates into the host cell nucleus to the gene-dense transcriptionally active open chromatin regions where the viral DNA is inserted into the host cell genome by viral integrase. Integration is an essential step for establishing a persistent infection in the host. The integrated viral DNA becomes part of the host cell's genome as proviral DNA and its positioning within the chromatin architecture plays a pivotal role in HIV-1 pathogenesis. While extensive data from genetic, biochemical and imaging studies point to integration targeting at gene-dense and transcriptionally active chromatin, the chromatin architecture at these sites, however, has remained elusive. This is largely due to the difficulties in obtaining high-resolution in situ images of native chromatin in intact cells using cryoET, further compounded by the challenges to locate the integration targeting sites through correlative approaches. Building on our recent exciting results on resolving individual nucleosome and chromatin fiber structures within intact native T- lymphocytes using cutting-edge cryo-focused-ion-beam technology (cryoFIB) and cryoET, we aim to address these challenges by developing novel correlative technologies for integration targeting and determine their architectures using advanced in situ structural biology methodologies. Specifically, we will create capsid and integrase dual-labelled virions, develop Quantum Dot labelling of vDNA, along with fluorescence labelling of host factors in T-lymphocytes for the correlative approaches to determine the molecular architecture of integration targeting sites. We will further investigate structural alterations resulting from changes in the integration targeting by disrupting crucial interactions between HIV-1 and host factors. The proposed research will not only fill a critical knowledge gap in our understanding of HIV-1 integration targeting but also push the boundaries of correlative and in-situ cryoET to achieve resolutions previously considered unattainable, facilitating progress in studies beyond those proposed here.
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