Elucidation of TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes

Yang, Emily; Huang, Serina; Jami‐Alahmadi, Yasaman; McInerney, Gerald M.; Wohlschlegel, James A.; Li, Melody M. H.

doi:10.1371/journal.ppat.1010743

Cited by 18 publications

(19 citation statements)

References 91 publications

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“…3XFLAG-ZAPS and -ZAPL were amplified from the pcDNA3.1-3XFLAG plasmids using primers to add ClaI and NotI restriction sites for ligation into the ePB vector (gift from Dr. Ali Brivanlou, Rockefeller University) (31). Full-length TRIM25 (gift from Dr. Jae U. Jung at Cleveland Clinic Lerner Research Institute) (49) was cloned into pcDNA3.1-myc as previously described (50). ZAP positive selection mutants were generated by the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA) or synthesized as a gene block (Twist Bioscience, South San Francisco, CA) with ClaI and NotI restriction sites and ligated into the ePB vector.…”

Section: Methodsmentioning

confidence: 99%

“…1µg/mL puromycin was added 48 hours post-transfection to select for ZAP KO 293T cells that have incorporated the ePB transposon. Our ZAPS WT and ZAPL WT cell lines were made by selecting single cell clones that follow two criteria: 1) robustly express ZAP following 24 hours of 1µg/mL doxycycline treatment, and 2) recapitulate differential alphaviral sensitivities (S3 Fig) similar to previously generated bulk cell lines with inducible ZAP expression (7,50). The mutant ZAP cell lines in this study were bulk cells that survived after puromycin selection.…”

Section: Methodsmentioning

confidence: 99%

“…SINV (Toto1101) (51), SINV expressing luciferase (Toto1101/Luc and Toto1101/Luc:ts6) (35), SINV expressing enhanced green fluorescent protein (EGFP) (TE/5’2J/GFP) (52), RRV expressing EGFP (gift from Dr. Mark Heise, University of North Carolina) (53), ONNV expressing EGFP (gift from Dr. Steve Higgs, Kansas State University) (54), CHIKV vaccine strain 181/clone 25 (gift from Scott Weaver, The University of Texas Medical Branch at Galveston) (55) expressing EGFP, and VEEV vaccine strain TC-83 expressing EGFP (gift from Dr. Ilya Frolov, University of Alabama at Birmingham) have been previously described (8,50). All alphaviral stocks were generated and titered in BHK-21 cells (35).…”

Section: Methodsmentioning

confidence: 99%

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Positive selection analyses identify a single WWE domain residue that shapes ZAP into a super restriction factor

Huang,

Girdner,

Nguyen

et al. 2023

Preprint

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The host interferon pathway upregulates intrinsic restriction factors in response to viral infection. Many of them block a diverse range of viruses, suggesting that their antiviral functions might have been shaped by multiple viral families during evolution. Virus-host conflicts have led to the rapid adaptation of viral and host proteins at their interaction hotspots. Hence, we can use evolutionary genetic analyses to elucidate antiviral mechanisms and domain functions of restriction factors. Zinc finger antiviral protein (ZAP) is a restriction factor against RNA viruses such as alphaviruses, in addition to other RNA, retro-, and DNA viruses, yet its precise antiviral mechanism is not fully characterized. Previously, an analysis of 13 primate ZAP identified 3 positively selected residues in the poly(ADP-ribose) polymerase-like domain. However, selective pressure from ancient alphaviruses and others likely drove ZAP adaptation in a wider representation of mammals. We performed positive selection analyses in 261 mammalian ZAP using more robust methods with complementary strengths and identified 7 positively selected sites in all domains of the protein. We generated ZAP inducible cell lines in which the positively selected residues of ZAP are mutated and tested their effects on alphavirus replication and known ZAP activities. Interestingly, the mutant in the second WWE domain of ZAP (N658A) is dramatically better than wild-type ZAP at blocking replication of Sindbis virus and other ZAP-sensitive alphaviruses due to enhanced viral translation inhibition. The N658A mutant inhabits the space surrounding the previously reported poly(ADP-ribose) (PAR) binding pocket, but surprisingly has reduced binding to PAR. In summary, the second WWE domain is critical for engineering a super restrictor ZAP and fluctuations in PAR binding modulate ZAP antiviral activity. Our study has the potential to unravel the role of ADP-ribosylation in the host innate immune defense and viral evolutionary strategies that antagonize this post-translational modification.Author summaryHost proteins and viral proteins that encounter one another are locked in a perpetual genetic arms race. In this evolutionary race, a mutation that confers a survival advantage will become more frequent in the population. By looking at the sequences of genes that are known to have antiviral roles in mammals, we can identify the exact sites where a host and viral protein have interacted and gain insight into how an antiviral protein works. Here, we identified these sites in zinc finger antiviral protein (ZAP), a host protein that blocks many different viruses. We found that changing one of the sites from the original amino acid to another dramatically improves ZAP’s antiviral activity against Sindbis virus, an alphavirus, due to improved inhibition of viral translation. Our mutation is also better at inhibiting other members in theAlphavirusgenus. We observed that our mutant ZAP has reduced ability to bind poly(ADP-ribose), a post-translational modification that is targeted by alphaviruses for productive infection. Our findings help us better understand how viruses have shaped the evolution of broad-spectrum host antiviral proteins, with great implications for the engineering of super restriction factors.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Positive selection analyses identify a single WWE domain residue that shapes ZAP into a super restriction factor

Huang,

Girdner,

Nguyen

et al. 2023

Preprint

Self Cite

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“…The infectious clone plasmids of enhanced GFP (EGFP)-expressing or unlabeled CHIKV (vaccine strain 181/clone 25), EGFP-expressing or unlabeled ONNV (strain SG650), EGFP-expressing SINV (TE/5'2J/GFP), and EGFP-expressing RRV (strain T48) have been previously reported [67][68][69][70][71] . Propagations and titrations of virus stocks were generated in BHK-21 cells as previously described 72,73 . To infect THP-1 derived macrophages or primary monocyte derived macrophages, viruses were diluted in DPBS supplemented with 1% human AB serum (Omega Scientific) and 1% P/S, and added to cells at a multiplicity of infection (MOI) of 5 plaque-forming units (PFU)/cell.…”

Section: Methodsmentioning

confidence: 99%

Chikungunya virus glycoproteins transform macrophages into productive viral dissemination vessels

Yao

Ramachandran

et al. 2023

Preprint

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Despite their role as innate sentinels, macrophages are cellular reservoirs for chikungunya virus (CHIKV), a highly pathogenic arthropod-borne alphavirus that has caused unprecedented epidemics worldwide. Here, we took interdisciplinary approaches to elucidate the CHIKV determinants that subvert macrophages into virion dissemination vessels. Through comparative infection using chimeric alphaviruses and evolutionary selection analyses, we discovered for the first time that CHIKV glycoproteins E2 and E1 coordinate efficient virion production in macrophages with the domains involved under positive selection. We performed proteomics on CHIKV-infected macrophages to identify cellular proteins interacting with the precursor and/or mature forms of viral glycoproteins. We uncovered two E1-binding proteins, signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 (eIF3k), with novel inhibitory activities against CHIKV production. These results highlight how CHIKV E2 and E1 have been evolutionarily selected for viral dissemination likely through counteracting host restriction factors, making them attractive targets for therapeutic intervention.

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“…Dysregulated protein abundance and quality control is central to nearly all human diseases, spanning cardiovascular disease 1,2 , cancer [3][4][5] , neurodegeneration [6][7][8] , and viral infections 9,10 . The latter is particularly noteworthy given both the recent COVID-19 pandemic and the preponderance of mechanisms by which host proteostasis processes are co-opted by pro-viral factors [11][12][13][14][15] . Consequently, the development of pharmacological agents that modulate protein homeostasis is a burgeoning area for both functional biology and drug development.…”

Section: Introductionmentioning

confidence: 99%

Pervasive aggregation and depletion of host and viral proteins in response to cysteine-reactive electrophilic compounds

Julio,

Shikwana,

Truong

et al. 2023

Preprint

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Protein homeostasis is tightly regulated, with damaged or misfolded proteins quickly eliminated by the proteasome and autophagosome pathways. By co-opting these processes, targeted protein degradation technologies enable pharmacological manipulation of protein abundance. Recently, cysteine-reactive molecules have been added to the degrader toolbox, which offer the benefit of unlocking the therapeutic potential of ‘undruggable’ protein targets. The proteome-wide impact of these molecules remains to be fully understood and given the general reactivity of many classes of cysteine-reactive electrophiles, on- and off-target effects are likely. Using chemical proteomics, we identified a cysteine-reactive small molecule degrader of the SARS-CoV-2 non- structural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host chaperones together with activation of global cell stress response pathways. We find that cysteine-reactive electrophiles increase global protein ubiquitylation, trigger proteasome activation, and result in widespread aggregation and depletion of host proteins, including components of the nuclear pore complex. Formation of stress granules was also found to be a remarkably ubiquitous cellular response to nearly all cysteine-reactive compounds and degraders. Collectively, our study sheds light on complexities of covalent target protein degradation and highlights untapped opportunities in manipulating and characterizing proteostasis processes via deciphering the cysteine-centric regulation of stress response pathways.

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Elucidation of TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes

Cited by 18 publications

References 91 publications

Positive selection analyses identify a single WWE domain residue that shapes ZAP into a super restriction factor

Positive selection analyses identify a single WWE domain residue that shapes ZAP into a super restriction factor

Chikungunya virus glycoproteins transform macrophages into productive viral dissemination vessels

Pervasive aggregation and depletion of host and viral proteins in response to cysteine-reactive electrophilic compounds

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