1 | Dynamic Stress Response to RNA Virus Infections

Using long-term live-cell imag­ing microscopy, we showed that chron­ic infec­tion with hepati­tis C virus in com­bi­na­tion with type I inter­fer­on (IFN) treat­ment induces a dynam­ic host cell stress response that can be visu­al­ized by recur­ring cycles of assem­bly and dis­as­sem­bly of SGs (Rug­gieri et al., 2012). HCV-induced SG-on and SG-off phas­es is reg­u­lat­ed at the lev­el of the eukary­ot­ic ini­ti­a­tion fac­tor 2 alpha (eIF2α) by the antag­o­nis­tic action of the two main switch­es, PKR, the stress kinase detect­ing viral dou­ble-strand­ed (ds) RNA, and GADD34, the stress-induced reg­u­la­to­ry sub­unit of Pro­tein Phos­phatase 1 (Fig­ure 1) . We recent­ly elu­ci­dat­ed the com­plex reg­u­la­tion of this response with help of quan­ti­ta­tive math­e­mat­i­cal mod­el­ling and dis­cov­ered that this dynam­ic stress response is reg­u­lat­ed by a sto­chas­tic process with mem­o­ry. Thus, the moment at which cells form SGs is neg­a­tive­ly cor­re­lat­ed with the dura­tion of the pre­ced­ing SG phase. This approach also evi­denced the impor­tance of inher­ent cell-to-cell vari­a­tions in mod­u­lat­ing SG dynam­ics, specif­i­cal­ly in the lev­els of PKR and GADD34 (Klein, Kallen­berg­er et al., 2022). Build­ing on these results, we now ques­tion how the estab­lish­ment of the antivi­ral response mod­u­lates SG for­ma­tion and cell adap­ta­tion to stress dur­ing acute viral infection.

2 | Regulation of GADD34, the Stress-induced Regulatory Subunit of Phosphatase PP1

A key pre­dic­tion of our math­e­mat­i­cal mod­el of the inte­grat­ed stress response was the key role of GADD34, both at the pro­tein and at the mRNA lev­el, in the adap­ta­tion to repeat­ed stress. We recent­ly iden­ti­fied an AU-rich ele­ment (ARE), a reg­u­la­to­ry ele­ment known to desta­bi­lize mRNAs, in the 3′ untrans­lat­ed region (UTR) of PPP1R15A mRNA, encod­ing GADD34 (Fig­ure 2). Under nor­mal con­di­tions, we found that PPP1R15A ARE is rec­og­nized by pro­teins of the TTP fam­i­ly that pro­mote rapid mRNA decay. Upon expo­sure to dif­fer­ent types of stress, as TTP pro­teins are inac­ti­vat­ed, PPP1R15A mRNA is tran­sient­ly sta­bi­lized, allow­ing for rapid GADD34 trans­la­tion and pro­mot­ing entry into the stress adap­ta­tion phase. With this work, we uncov­ered the role of PPP1R15A mRNA turnover in shap­ing the dynam­ics of stress adap­ta­tion and cell sen­si­tiv­i­ty to repet­i­tive stress expo­sure and iden­ti­fied GADD34 as the mol­e­c­u­lar mem­o­ry of the acti­vat­ed inte­grat­ed stress response (Magg, Manet­to et al., 2024). By study­ing the post-tran­scrip­tion­al reg­u­la­tion of PPP1R15A mRNA, we have real­ized that basal lev­els of GADD34 are crit­i­cal and reg­u­lat­ed by addi­tion­al mech­a­nisms, which we are cur­rent­ly exploring.

3 | Assembly of Stress Granule like Condensates in Droplets

Bot­tom up approach­es mak­ing use of puri­fied SG pro­teins such as G3BP1, the main SG nucle­at­ing pro­tein, have revealed excit­ing fea­tures of how these bio­mol­e­c­u­lar con­den­sates dynam­i­cal­ly form by the phase sep­a­ra­tion. How­ev­er, in cells, SG con­den­sa­tion also results from mul­ti­ple inter­ac­tions with RNAs and RNA-bind­ing pro­teins, which great­ly com­plex­i­fy this mod­el. To tack­le this chal­lenge, we opt­ed for the com­bined use of sim­ple microflu­idics and flu­o­res­cence microscopy, to estab­lish a droplet-based method that bridges the gap between in vit­ro sys­tems and liv­ing cells, pro­vid­ing a plat­form to study stress-induced phase sep­a­ra­tion under phys­i­o­log­i­cal conditions.

4 | Translational Control by Flaviviruses

Fla­vivirus­es such as DENV, ZIKV and WNV have a pos­i­tive sense sin­gle-strand­ed RNA genome with a type I cap at their 5’ end and a non-polyadeny­lat­ed 3′ untrans­lat­ed region. Viral genomes there­fore com­pete with host mRNAs for their trans­la­tion. We have shown that fla­vivirus infec­tion induces a severe repres­sion of the host cell trans­la­tion in human cells, which is uncou­pled from the cel­lu­lar stress response (Roth et al., 2017). Despite this repres­sion, trans­la­tion of viral genomes is main­tained while host trans­la­tion is repressed. This work sug­gest­ed an uncon­ven­tion­al and virus-spe­cial­ized trans­la­tion ini­ti­a­tion mech­a­nism that we are cur­rent­ly investigating.

5 | Role of RNA Modifications in the Flavivirus Life Cycle

Chem­i­cal mod­i­fi­ca­tions of RNA affect all stages of the RNA life cycle, includ­ing splic­ing, sta­bil­i­ty and trans­la­tion. RNA mod­i­fi­ca­tions are also essen­tial for the host immune sys­tem to dis­tin­guish between self and for­eign RNA. Increas­ing evi­dence sug­gests that viral RNA genomes, whose struc­ture close­ly mim­ics that of cel­lu­lar mRNAs, are also high­ly mod­i­fied. How­ev­er, results on their detec­tion and map­ping are part­ly con­tro­ver­sial. We have decid­ed to take on this prob­lem by devel­op­ing new purifi­ca­tion approach­es and we aim to explore the epi­tran­scrip­tomics of viral genome RNA over the course of fla­vivirus infec­tion. This will enable us to assess its impact on the virus life cycle and host recognition.

Link to com­plete pub­li­ca­tion list: ORCID-iD: 0000–0001-9981–3308

• Magg V*, Manet­to A*, Kopp K, Wu C, Lind­ner D, Naghizadeh M, Schott J, Eke L, Welsch J, Kallen­berg­er SM, Haucke V, Lock­er N, Stoeck­lin G†, Rug­gieri A†. Turnover of PPP1R15A mRNA encod­ing GADD34 con­trols respon­sive­ness and adap­ta­tion to cel­lu­lar stress. 2024. Cell Reports 43(4):114069.
• Klein P*, Kallen­berg­er SM*, Roth H, Roth K, Ly-Har­tig TBN, Magg V, Aleš J, Tale­mi SR , Qiang Y, Wolf S, Olek­siuk O, Kurilov R, Di Ven­tu­ra B, Barten­schlager R, Eils R, Rohr K, Ham­precht FA, Höfer T, Fack­ler OT, Stoeck­lin G and Rug­gieri A. Tem­po­ral con­trol of the inte­grat­ed stress response by a sto­chas­tic mol­e­c­u­lar switch. 2022. Sci­ence Advances 8(12):eabk2022.
• Tale­mi SR, Barten­schlager M, Schmid B, Rug­gieri A†, Barten­schlager R†, Höfer T†. Dengue virus is sen­si­tive to inhi­bi­tion pri­or to pro­duc­tive repli­ca­tion. 2021. Cell Reports 37(2):109801.
• Rug­gieri A, Helm M, Cha­tel-Chaix L. An epi­ge­net­ic “extreme makeover”: the methy­la­tion of fla­vivi­ral RNA (and beyond). 2020. RNA Biol 18:1–13.
• Eier­mann N, Haneke K, Sun Z, Stoeck­lin G, Rug­gieri A. Dance with the dev­il: Stress gran­ules and sig­nal­ing in antivi­ral respons­es. 2020. Virus­es 12(9), 984.
• Haneke K, Schott J, Lind­ner D, Hol­lensen AK, Damgaard CK, Mongis C, Knop M, Palm W, Rug­gieri A, Stoeck­lin G. CDK1 cou­ples pro­lif­er­a­tion with pro­tein syn­the­sis. 2020. J Cell Biol. 219(3): e201906147.
• Bro­card M, Iade­va­ia V, Klein P, Hall B, Lewis G, Lu J, Burke J, Will­cocks M, Park­er R, Good­fel­low IG, Rug­gieri A, Lock­er N. Norovirus infec­tion results in eIF2α-inde­pen­dent host trans­la­tion shut-off and remod­els the G3BP1 inter­ac­tome evad­ing stress gran­ule for­ma­tion. 2020. PLoS Pathog. 16(1):e1008250.
• Rug­gieri, A. and Stoeck­lin, G. (2019). A Sig­nal to Con­dense. Nat Chem Biol. 15(1):5–6.
• Roth, H., Magg, V., Uch, F., Mutz, P., Klein, P., Haneke, K., Lohmann, V., Barten­schlager, R., Fack­ler, O.T., Lock­er, N., Stoeck­lin, G., Rug­gieri, A. (2017). Fla­vivirus infec­tion uncou­ples trans­la­tion sup­pres­sion from cel­lu­lar stress respons­es. mBio 8(1):e02150-16.
• Rug­gieri, A., Daz­ert, E., Metz, P., Hof­mann, S., Bergeest, J.-P., Mazur, J., Bankhead, P., Hiet, M.-S., Kallis, S., Alvisi, G., Samuel, C.E., Lohmann, V., Kader­ali, L., Rohr, K., Frese, M., Stoeck­lin, G., Barten­schlager, R. (2012). Dynam­ic oscil­la­tion of trans­la­tion and stress gran­ule for­ma­tion mark the cel­lu­lar response to virus infec­tion. Cell Host Microbe 12(1): 71–85.