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In situ structures of three components essential to human cytomegalovirus pathogenesis: genome-packaging machinery, capsid-associated tegument and prefusion glycoprotein complexes

Award Number: R01DE028583
ORGANIZATION: NATIONAL INSTITUTE OF DENTAL AND CRANIOFACIAL RESEARCH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Though latently infecting most of us asymptomatically, human cytomegalovirus (HCMV) is a leading viral cause of birth defects and can be life-threatening to immune-compromised individuals. As a member of the ß- herpesvirus subfamily of the Herpesviridae and the most structurally and genetically complex herpesvirus (e.g., its genome is twice that of chickenpox-causing varicella zoster virus, an a-herpesvirus), HCMV is one of the largest of all viruses and presents a major challenge to structure determination. Architecturally similar to other herpesviruses, HCMV is composed of a glycoprotein-containing envelope, a tegument layer, and a bacteriophage-like icosahedral capsid enclosing a genome of a single dsDNA molecule. Distinctive from members of the a- and ¿-herpesvirus subfamilies are two processes central to HCMV infection: 1) its large genome needs to be packaged through a portal complex and then stabilized by a unique tegument protein pp150; 2) the process of cell fusion by gB involves a unique pentameric glycoprotein complex gH/gL/UL128/UL130/UL131. These processes thus can be targeted for structure-guided design for novel vaccines and anti-virals against HCMV infections. By cryo electron microscopy (cryoEM), the PI’s group obtained the first three-dimensional structure of HCMV capsid at 18Å resolution in 1999, which was followed by progressive improvement in resolution, culminating at the recent 3.9Å resolution structure reported in Science. Our pilot studies resolved the portal complex and pp150-capsid interactions absent from a- and ¿-herpesvirus subfamilies. Furthermore, we have demonstrated atomic resolution structure determination for membrane protein complexes and—in collaboration with Merck—obtained preliminary cryoEM data for HCMV pentameric glycoprotein complexes. We hypothesize that our state-of-the-art technologies in electron-counting cryoEM, symmetry relaxation and local refinement methods, and HCMV BAC technologies together would now allow us to determine in situ structures of genome packaging/ejection portal complex, pp150 and glycoprotein complexes, and when combined with structure-guided mutagenesis, to identify essential hot-spot residues critical to the interactions among these proteins. Harnessing technology breakthroughs in cryoEM and structure-guided mutagenesis, the proposed research aims to: (1) obtain in situ structure of the portal of DNA genome packaging and ejection machinery at near-atomic resolution and identify residues critical to capsid assembly and stabilization; (2) determine the in situ structure of pp150 at about 2Å resolution and identify the chemical bonds between capsid and capsid-interacting pp150 residues, particularly the cys tetrad conserved among primate cytomegaloviruses; (3) obtain atomic structures of purified pentameric glycoprotein complex in complex with three neutralizing monoclonal antibodies, as well as their in situ pre-fusion glycoprotein structures on viral envelope by cryo electron tomography for comparison. The expected results should inform efforts in designing inhibitors and vaccines against HCMV infections.


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: 2023 ( Subtotal = $471,864 )
2023	2023	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	000	5	3/23/2023	NON-COMPETING CONTINUATION	$471,864
														Subtotal = $471,864

Issue Date FY: 2022 ( Subtotal = $468,353 )
2022	2022	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	000	4	3/24/2022	NON-COMPETING CONTINUATION	$316,028
2022	2022	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	003	4	4/25/2022	NON-COMPETING CONTINUATION	$151,117
2022	2022	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	005	4	9/20/2022	SUPPLEMENT FOR EXPANSION	$0
2022	2022	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	004	4	4/26/2022	NON-COMPETING CONTINUATION	-$119,513
2022	2022	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	001	4	3/28/2022	SUPPLEMENT FOR EXPANSION	$108,649
2022	2022	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	002	4	4/22/2022	SUPPLEMENT FOR EXPANSION	$12,072
														Subtotal = $468,353

Issue Date FY: 2021 ( Subtotal = $471,864 )
2021	2021	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90095	LOS ANGELES	USA	Oral Diseases and Disorders Research	000	3	4/19/2021	NON-COMPETING CONTINUATION	$471,864
														Subtotal = $471,864

Issue Date FY: 2020 ( Subtotal = $471,864 )
2020	2020	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90095	LOS ANGELES	USA	Oral Diseases and Disorders Research	001	2	4/17/2020	SUPPLEMENT FOR EXPANSION	$120,721
2020	2020	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90095	LOS ANGELES	USA	Oral Diseases and Disorders Research	000	2	3/31/2020	NON-COMPETING CONTINUATION	$351,143
														Subtotal = $471,864

Issue Date FY: 2019 ( Subtotal = $364,443 )
2019	2019	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90095	LOS ANGELES	USA	Oral Diseases and Disorders Research	000	1	2/15/2019	NEW	$364,443
														Subtotal = $364,443

Grand Total All Awards = $2,248,388

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In situ structures of three components essential to human cytomegalovirus pathogenesis: genome-packaging machinery, capsid-associated tegument and prefusion glycoprotein complexes

Award Number: R01DE028583

ORGANIZATION: NATIONAL INSTITUTE OF DENTAL AND CRANIOFACIAL RESEARCH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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