TY - JOUR
T1 - Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation
AU - Medica, Samuel
AU - Crawford, Lindsey B.
AU - Denton, Michael
AU - Min, Chan Ki
AU - Jones, Taylor A.
AU - Alexander, Timothy
AU - Parkins, Christopher J.
AU - Diggins, Nicole L.
AU - Streblow, Gabriel J.
AU - Mayo, Adam T.
AU - Kreklywich, Craig N.
AU - Smith, Patricia
AU - Jeng, Sophia
AU - McWeeney, Shannon
AU - Hancock, Meaghan H.
AU - Yurochko, Andrew
AU - Cohen, Michael S.
AU - Caposio, Patrizia
AU - Streblow, Daniel N.
N1 - Publisher Copyright:
© 2023 Medica et al.
PY - 2023/10
Y1 - 2023/10
N2 - Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 –RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.
AB - Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 –RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.
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U2 - 10.1371/journal.ppat.1011682
DO - 10.1371/journal.ppat.1011682
M3 - Article
C2 - 37782657
AN - SCOPUS:85174212794
SN - 1553-7366
VL - 19
JO - PLoS pathogens
JF - PLoS pathogens
IS - 10 October
M1 - e1011682
ER -