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Understanding the proviral role for TRAF6 interaction with the viral protease in flavivirus replication and pathogenesis

Award Number: R01AI153496
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Tumor necrosis factor receptor associated factor 6 (TRAF6) is central to host immune responses and is important for protective responses to pathogens. We report here a non-canonical role for TRAF6. The tick- borne flaviviruses (TBFVs) have evolved a unique mechanism to use TRAF6 to benefit infection. Understanding how the balance between TRAF6’s antiviral and proviral effects is tipped in favor of TBFV replication may reveal how flaviviruses overcome the cellular antiviral environment, to ultimately cause disease. We identified an interaction between TRAF6 and the TBFV protease, NS3pro. TRAF6 is an E3 ubiquitin ligase that is normally associated with antiviral functions in the context of virus infections. Surprisingly, TBFVs use TRAF6 to replicate in cells. Here, we show that TBFV NS3pro protein interaction with TRAF6 during infection supports TBFV replication. The proviral role of TRAF6 was not seen with mosquito-borne flaviviruses, consistent with the lack of a conserved TRAF6 binding motif. Disruption of the TRAF6-NS3pro interaction by site-directed mutagenesis resulted in a significant reduction of mature protease protein expression, and attenuation of in vitro virus replication. To investigate the TRAF6 proviral function, we assessed the ubiquitination status and found that NS3pro is ubiquitinated in the presence of TRAF6. The overall objective of this application is to determine how NS3-TRAF6 interaction affects protease structure and function(s), virus replication, and pathogenesis in a mouse model of TBFV infection. We hypothesize that NS3pro is activated by TRAF6 through direct interaction and ubiquitination, ultimately to promote productive virus replication. To test our hypothesis, we will use two specific aims. 1) To determine how TRAF6 benefits TBFV replication we will use cell biology and biochemical approaches to determine how NS3pro is modified by ubiquitination and how ubiquitination affects the structure and function of NS3pro. 2) To determine the proviral role of TRAF6 in primary cells (e.g., dendritic cells and neurons) and in pathogenesis in vivo we will use therapeutic and genetic methods to manipulate TRAF6 expression in primary cells and in a mouse model of TBFV pathogenesis. By understanding how TRAF6 benefits NS3pro and its role during infection, we will gain insight into the regulation of a critically important viral enzyme. Then by using a mouse model of TBFV infection we will determine the extent to which the NS3-TRAF6 interaction may be targeted for flavivirus drug development.


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 = $386,250 )
2024	2024	UNIVERSITY OF TOLEDO	3000 ARLINGTON AVE	TOLEDO	OH	43614	LUCAS	USA	Allergy and Infectious Diseases Research	000	5	4/18/2024	NON-COMPETING CONTINUATION	$386,250
														Subtotal = $386,250

Issue Date FY: 2023 ( Subtotal = $386,250 )
2023	2023	UNIVERSITY OF TOLEDO, THE	3000 ARLINGTON AVE	TOLEDO	OH	43614	LUCAS	USA	Allergy and Infectious Diseases Research	000	4	4/25/2023	NON-COMPETING CONTINUATION	$386,250
														Subtotal = $386,250

Issue Date FY: 2022 ( Subtotal = $386,250 )
2022	2022	UNIVERSITY OF TOLEDO, THE	3000 ARLINGTON AVE	TOLEDO	OH	43614	LUCAS	USA	Allergy and Infectious Diseases Research	000	3	4/18/2022	NON-COMPETING CONTINUATION	$386,250
														Subtotal = $386,250

Issue Date FY: 2021 ( Subtotal = $386,250 )
2021	2021	UNIVERSITY OF TOLEDO, THE	3000 ARLINGTON AVE	TOLEDO	OH	43614	LUCAS	USA	Allergy and Infectious Diseases Research	000	2	4/12/2021	NON-COMPETING CONTINUATION	$386,250
														Subtotal = $386,250

Issue Date FY: 2020 ( Subtotal = $385,833 )
2020	2020	UNIVERSITY OF TOLEDO, THE	3000 ARLINGTON AVE	TOLEDO	OH	43614	LUCAS	USA	Allergy and Infectious Diseases Research	000	1	5/13/2020	NEW	$385,833
														Subtotal = $385,833

Grand Total All Awards = $1,930,833

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Understanding the proviral role for TRAF6 interaction with the viral protease in flavivirus replication and pathogenesis

Award Number: R01AI153496

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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