[R21] Combining CRISPR-Cas9 base editing and proteomics to determine how filovirus VP24 proteins modulate ubiquitination and viral replication
Ente: National Institute of Allergy and Infectious Diseases
Scadenza: 2028-05-31
Importo max: 462.000 EUR
Paese: US
Descrizione
Summary
The filovirus family includes deadly, emerging/re-emerging, zoonotic viruses such as Marburg virus (MARV) and
Ebola virus (EBOV). A recently described filovirus is Měnglà virus (MLAV) which was identified in a Rousettus
bat in China. VP24 proteins are unique to the filoviruses, engage key cellular signaling pathways and play critical
roles in viral infectivity and transmission. The VP24s from different filovirus lineages target different host
pathways. EBOV VP24 binds importin alpha (IMPα) proteins that mediate STAT1 nuclear import, thereby
blocking cellular responses to interferons (IFNs). MARV VP24 interacts with Keap1, a master regulator of
antioxidant responses that recruits Cullin 3-Roc1 E3 ubiquitin (Ub) ligase to direct the degradation of transcription
factor Nrf2. MARV VP24-Keap1 interaction frees Nrf2 from degradation, triggering a similar cytoprotective state.
We found that MLAV VP24 binds neither IMPα nor Keap1. We hypothesized that MLAV VP24 instead targets
distinct cellular signaling molecules. To address this, we performed comparative affinity purification-mass
spectroscopy (AP-MS) proteomics in Rousetttus cells, examining the EBOV, MARV and MLAV VP24s. As
expected, this identified IMPα and Keap1 as EBOV and MARV VP24 interactors, respectively. Consistent with
our hypothesis, we identified the host deubiqutinating enzymes USP15, USP11 and USP4, which are paralogs,
as unique MLAV VP24 interactors. Interestingly, USP15 regulates type I IFN responses and Nrf2-dependent
antioxidant responses, among many other pathways. Because MARV and MLAV VP24s engage regulators of
ubiquitination, we propose to define the Ub-regulated pathways targeted by each VP24, identify any overlap in
the pathways affected and measure how targeting of ubiquitination affects MARV and MLAV infection. To this
end, we will first perform mutational mapping to test the hypothesis that MLAV VP24 targets a region conserved
between USP15, USP11 and USP4 and to identify loss of binding mutants. We will use inducible lentiviral vectors
to deliver wildtype and loss of binding (to Keap1 or USPs) mutants for MARV and MLAV VP24 into Rousettus
and human cells, determine whether these affect deubiquitination of known USP15, USP11 and USP4 targets
and evaluate the impact of each VP24 on global ubiquitination in untreated, IFNα-treated and polyI:C transfected
cells. We will then use CRISPR-Cas9 base editing to generate enzymatically inactive endogenous USP15,
USP11, and USP4 and to disrupt Keap1 interaction with Nrf2. In parallel, we will use CRISPR-Cas9 knockout of
the same host factors. We will use these cells in combination with proteomics profiling of Ub to define the global
effects of these enzymes on host pathways. Finally, the base edited and knockout cells will be used, in
combination with MARV and MLAV replication cycle modeling assays that enable study of filovirus replication at
Biosafety Level 2, to determine the effect of these host enzymes on MARV and MLAV rep
Istituzione: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
PI: Christopher F Basler, Jeffrey R Johnson
Progetto: 1R21AI199908-01
Settori: National Institute of Allergy and Infectious Diseases
Vai al bando originale
Registrati gratis su Bandolo per trovare bandi compatibili con la tua azienda.