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Characterizing functionally relevant HIV-1 structures by correlative light and cryo-electron microcopy (CLEM)

Award Number: R21AI174879
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 01/18/2023
PERIOD OF PERFORMANCE END DATE: 12/31/2025

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Characterizing functionally relevant HIV-1 structures by correlative light and cryo-electron microcopy (CLEM) - Project Summary The HIV-1 capsid protects the viral genome from host innate sensors and mediates the nuclear import of pre- integration complexes. At some point during intra-cellular trafficking the capsid must disassemble to release the reverse transcribing vDNA for its integration into host-genes. The underlying mechanisms and the cellular location of capsid disassembly, which is commonly referred to as uncoating, remain unclear. Recent evidence points to the possibility to transport a few apparently intact capsids (dimensions ~30 x 60 nm) through a dilated (~60 nm) nuclear pore complex and into the nucleus1-3. The relevance of these few capsids to infection remains unclear, and their prior dynamics inside cells unknown. An unbiased and detailed structural analysis of relevant intra-cellular HIV-1 uncoating structures by cryo-EM remains highly challenging. Notably, in situ cryo-EM necessitates the use of cryo-focused ion beam (cryo-FIB) milling to generate a thin (100-300nm) sheet of vitreous ice referred to as a ‘lamella’ inside rapidly frozen cells. Procedures to precisely control the location in a cell where the lamella is prepared remains underdeveloped and a bottleneck for in situ structural investigation, especially of sparsely located HIV-1 specimen. The scientific premise of this proposal is to develop a robust 3D correlative light and cryo-EM (3D-CLEM) workflow to facilitate cryo-FIB ‘lamella’ preparation at precise intra-cellular sites containing HIV-1, and to characterize relevant capsid structures and correlate to their functional dynamics by live-cell imaging. This project will leverage, (1) fluorescence live-cell imaging of HIV-1 infection to locate relevant capsids that are respectively poised for nuclear entry and integration4, 5 and correlate their dynamics to capsid structures determined by 3D-CLEM, cryo-FIB lamella preparation and high-resolution cryo-electron tomography (cryo-ET); (2) develop automated methods to locate HIV-1 macrostructures on a lamella using low-resolution cryo-ET maps via a convoluted neural network (CNN)-trained software6 to un-biasedly pick capsid templates and 3D-HIV structures on a lamella, for high-resolution cryo-ET structural imaging and classification. The development and validation of the 3D-CLEM workflow and automated macrostructure localization procedure will improve throughput in cryo-EM imaging of HIV-1 structures inside cells, and improve our understanding of virus cell biology, which is of high public health significance.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = $227,999 )
2024	2024	FLORIDA STATE UNIVERSITY	874 TRADITIONS WAY	TALLAHASSEE	FL	32306	LEON	USA	Allergy and Infectious Diseases Research	000	2	12/6/2023	NON-COMPETING CONTINUATION	$205,199
2024	2024	FLORIDA STATE UNIVERSITY	874 TRADITIONS WAY	TALLAHASSEE	FL	32306	LEON	USA	Allergy and Infectious Diseases Research	001	2	5/17/2024	NON-COMPETING CONTINUATION	$22,800
														Subtotal = $227,999

Issue Date FY: 2023 ( Subtotal = $192,500 )
2023	2023	FLORIDA STATE UNIVERSITY	874 TRADITIONS WAY	TALLAHASSEE	FL	32306	LEON	USA	Allergy and Infectious Diseases Research	000	1	1/18/2023	NEW	$192,500
														Subtotal = $192,500

Grand Total All Awards = $420,499

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Characterizing functionally relevant HIV-1 structures by correlative light and cryo-electron microcopy (CLEM)

Award Number: R21AI174879

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 01/18/2023

PERIOD OF PERFORMANCE END DATE: 12/31/2025

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