Dr. Alessia Rug­gieri, Ph.D.

Alessia.Ruggieri@med.uni-heidelberg.de

Phone: ++49-(0)6221–56-7761

Fax: ++49-(0)6221–56-4570

Translational control by RNA viruses

Projects

Through­out the infec­tion, virus­es elic­it mul­ti­ple host cell respons­es includ­ing innate immune and stress respons­es. Viral dou­ble-strand­ed (ds) RNA repli­ca­tion inter­me­di­ates trig­ger the acti­va­tion of the stress sen­tinel Pro­tein kinase R (PKR) which medi­ates the phos­pho­ry­la­tion of eIF2α, a crit­i­cal fac­tor for trans­la­tion ini­ti­a­tion. As an almost imme­di­ate result, polysomes dis­as­sem­ble, pro­tein syn­the­sis is sup­pressed. Stalled mRNAs con­den­sate with RNA-bind­ing pro­teins and form mem­brane-less stress gran­ules (SGs) which for­ma­tion is dynam­ic, reversible and dri­ven by cytoso­lic phase sep­a­ra­tion. To estab­lish pro­duc­tive infec­tions, virus­es have evolved mech­a­nisms to over­come trans­la­tion­al atten­u­a­tion that results from stress response induc­tion. My lab­o­ra­to­ry is try­ing to under­stand how RNA virus­es con­trol the host trans­la­tion machin­ery and the cel­lu­lar stress response to ensure their prog­e­ny pro­duc­tion. We explore the strate­gies evolved by dif­fer­ent mem­bers of the Fla­viviri­dae fam­i­ly such as hepati­tis C virus (HCV), dengue virus (DENV), Zika virus (ZIKV) and West Nile virus (WNV) to antag­o­nize or inverse­ly to uti­lize the host stress response path­way.

Reasearch Inter­ests:

  • Host Stress Response to RNA Virus­es

  • Stress Gran­ules

  • Trans­la­tion­al Repres­sion by Virus­es

  • Innate Sens­ing of Retro­virus­es

1 | Dynamic Stress Response to Hepatitis C Virus Infection

Using long-term live-cell imag­ing microscopy, we showed that HCV infec­tion in com­bi­na­tion with type I inter­fer­on (IFN) treat­ment induces a dynam­ic and oscil­lat­ing host cell stress response that can be visu­al­ized by cycles of assem­bly and dis­as­sem­bly of SGs (Rug­gieri et al., 2012). We recent­ly addressed the com­plex reg­u­la­tion of this dynam­ic process with help of quan­ti­ta­tive math­e­mat­i­cal mod­el­ling and are cur­rent­ly inves­ti­gat­ing the bio­log­i­cal func­tion of SGs in HCV chron­ic infec­tion. Our pre­vi­ous work was per­formed using two-dimen­sion­al cul­tures of hepatoma-derived Huh7 cells that are pro­lif­er­a­tive and have immune-com­pe­tence and lost their main hepat­ic func­tions. We are cur­rent­ly set­ting up three-dimen­sion­al hepa­to­cyte-like cul­tures which is com­pat­i­ble with long-term live cell imag­ing and light-sheet microscopy to inves­ti­gate HCV-induced stress response under more phys­i­o­log­i­cal con­di­tions.

Fig­ure 1. Math­e­mat­i­cal mod­el­ing of HCV-induced SG oscil­la­tions. A. Descrip­tion of the stress sig­nal­ing path­way acti­vat­ed by HCV infec­tion. PKR is acti­vat­ed by bind­ing to viral dsR­NA, dimer­izes and autophos­pho­ry­lates. Active PKR phos­pho­ry­lates its direct sub­strate eIF2α lead­ing to a bulk trans­la­tion ini­ti­a­tion shut­off, polysome dis­as­sem­bly and assem­bly of SGs. To pro­mote sur­vival, cells GADD34 is tran­scrip­tion­al­ly and trans­la­tion­al­ly upreg­u­lat­ed and in com­plex with the Pro­tein Phos­phatase 1 dephos­pho­ry­lates eIF2α. Trans­la­tion ini­ti­a­tion is reac­ti­vat­ed and SGs dis­as­sem­ble. As long as viral dsR­NA is present in the cell, these cycles of active and stalled trans­la­tion will occur. B. Math­e­mat­i­cal mod­el of SG oscil­la­tions.

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

HCV-induced oscil­lat­ing SG for­ma­tion is reg­u­lat­ed at the lev­el of the eukary­ot­ic ini­ti­a­tion fac­tor 2 alpha (eIF2α) by the antag­o­nis­tic action of two main switch­es, PKR and GADD34, the stress-induced reg­u­la­to­ry sub­unit of Pro­tein Phos­phatase 1. The math­e­mat­i­cal mod­el of oscil­lat­ing SGs iden­ti­fied GADD34 as an impor­tant and dynam­ic node of reg­u­la­tion, both at the pro­tein and at mRNA lev­els. We exper­i­men­tal­ly val­i­dat­ed that under nor­mal con­di­tions GADD34 is a short-lived pro­tein whose mRNA exhibits a rapid turnover and are cur­rent­ly inves­ti­gat­ing the sig­nal­ing path­ways respon­si­ble for GADD34 reg­u­la­tion upon stress induc­tion.

 

Fig­ure 2. The inte­grat­ed stress response.

3 | Translational Control by Flaviviruses

Fla­vivirus­es such as DENV, ZIKV and WNV have a (+) 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 recent­ly 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). Impor­tant­ly, 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.

Fig­ure 3. Polysome pro­files of Fla­vivirus-infect­ed Huh7 cells. This tech­nique allows the iden­ti­fi­ca­tion of trans­la­tion­al changes in host cells sub­mit­ted to dif­fer­ent envi­ron­men­tal stress­es includ­ing virus infec­tion by sep­a­rat­ing heav­ier ribo­some-asso­ci­at­ed mRNAs (active­ly trans­lat­ing mRNAs, polyso­mal ribo­somes) from the light sub-polyso­mal mRNAs (Poor­ly or not trans­lat­ed mRNAs, sub-polyso­mal ribo­somes). Fla­vivirus­es reduce the bulk of active trans­lat­ing mRNAs in the course of the infec­tion.

4 | Role of TREX1 for the Innate Sensing of Retroviruses

As a neg­a­tive reg­u­la­tor of innate immu­ni­ty, Three Prime Repair Exonu­cle­ase 1 (TREX1) acts in a dual func­tion to pro­tect against autoim­mune phe­no­types, such as Aicar­di-Goutières syn­drome (AGS), famil­ial chilblain lupus (FCL), sys­temic lupus ery­the­mato­sus (SLE) and reti­nal vas­cu­lopa­thy with cere­bral leukody­s­tro­phy (RVCL). TREX1 is the most abun­dant 3’-5’ DNA exonu­cle­ase in mam­malian cells, which main func­tion as part of the SET com­plex in the nucle­u­sis to process aber­rant sin­gle- and dou­ble-strand­ed DNA repli­ca­tion inter­me­di­ates that accu­mu­late dur­ing DNA repli­ca­tion. On the oth­er hand, TREX1 also local­izes at the endo­plas­mic retic­u­lum where it sta­bi­lizes the cat­alyt­ic activ­i­ty of the oligosac­cha­ryl­trans­ferase (OST) com­plex. Recent­ly, TREX1 also shown to metab­o­lize exoge­nous retro­vi­ral reverse tran­scrip­tion (RT) prod­ucts in the cyto­plasm of mam­malian cells and there­fore sug­gest­ed to play a poten­tial role in the elim­i­na­tion of endoge­nous retro­virus RT prod­ucts. Unlike in mice, sev­er­al splice vari­ants of TREX1 are detect­ed in human cells. We are cur­rent­ly inves­ti­gat­ing TREX1 iso­form expres­sion and reg­u­la­tion in human periph­er­al blood-derived cells, as well as their local­iza­tion and exonu­cle­ase func­tion, par­tic­u­lar­ly in the con­text of retro­vi­ral infec­tion.

 

Fig­ure 4. The mul­ti­ple roles of TREX1 in mam­malian cells.

Selected Publications

Com­plete pub­li­ca­tion list (Orchid)

Rug­gieri, A. and Stoeck­lin, G. (2019). A Sig­nal to Con­dense. Nat Chem Biol. 15(1):5–6.

Schult, P., Roth H., Adams, R.L., Mas, C., Imbert, L., Orlik, C., Rug­gieri, A., Pyle, A.M., Lohmann, V. (2018). MicroR­NA-122 ampli­fies hepati­tis C virus trans­la­tion by shap­ing the struc­ture of the inter­nal ribo­so­mal entry site. Nat Com­mun. 9(1):2613.

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.

Bro­card, M., Rug­gieri, A., Lock­er, N. (2017). m6A RNA methy­la­tion, a new hall­mark in virus-host inter­ac­tions. J Gen Virol. 98(9):2207–2214.

Cortese, M., Goell­ner, S., Acos­ta, E.G., Neufeldt, C.J., Olek­siuk, O., Lampe, M., Hasel­mann, U., Funaya, C., Schieber, N., Ronchi, P., Schorb, M., Pru­un­sild, P., Schwab, Y., Cha­tel-Chaix, L., Rug­gieri, A., Barten­schlager, R. (2017). Ultra­struc­tur­al char­ac­ter­i­za­tion of Zika virus repli­ca­tion fac­to­ries. Cell Reports 18(9): 2113–2123.

Trautz, B., Wiede­mann, H., Lücht­en­borg, C., Pieri­ni, V., Kranich, J., Glass, B., Kräus­slich, H.-G., Brock­er, T., Piz­za­to, M., Rug­gieri, A., Brüg­ger, B., Fack­ler, O.T. (2017). The host-cell restric­tion fac­tor SERINC5 restricts HIV‑1 infec­tiv­i­ty with­out alter­ing the lipid com­po­si­tion and orga­ni­za­tion of viral par­ti­cles. J Biol Chem 292(33):13702–13713.

Cha­tel-Chaix, L., Cortese, M., Romero-Brey, I., Ben­der, S., Fis­chl, W., Scatur­ro, P., Fis­ch­er, B., Rug­gieri, A., Barten­schlager, R. (2016). Dengue virus mod­u­lates mito­chon­dr­i­al mor­pho­dy­nam­ics through the inhi­bi­tion of DRP‑1 for the ben­e­fit of viral repli­ca­tion. Cell Host Microbe 20(3):342–56.

Tro­tart, M., Tsopoulidis, N., Tibroni, N., Willem­sen, J., Binder, M., Rug­gieri, A., Fack­ler, O.T. (2015). Sens­ing of HIV‑1 infec­tion in Tzm-bl cells with recon­sti­tut­ed expres­sion of STING. J Virol. 90(4):2064–76.

Schmid, B., Rinas, M., Rug­gieri, A., Reuter, A., Fis­chl, W., Hard­er, N., Bergeest, J.-P., Floss­dorf, M., Rohr, K., Höfer, T., Barten­schlager, R. (2015). Live-cell analy­sis and mod­el­ing iden­ti­fy deter­mi­nants of atten­u­a­tion of Dengue virus 2‑O-methyl mutant. PLoS Pathog. 11(12):e1005345.

Grün­vo­gel, O., Ess­er-Nobis, K., Reustle, A., Schult, P., Müller, B., Metz, P., Trip­pler, M., Windisch, M.P., Frese, M., Binder, M., Fack­ler, O.T., Barten­schlager, R., Rug­gieri, A., Lohmann, V. (2015). Dead box heli­case 60-like (DDX60L) is an inter­fer­on stim­u­lat­ed gene restrict­ing hepati­tis C virus repli­ca­tion in cell cul­ture. J Virol. 89(20):10548–68.

Hiet, M.-S., Bauhofer, O., Zayas, M., Roth, H., Tana­ka, Y., Schir­ma­ch­er, P., Willem­sen, J., Grün­vo­gel, O., Ben­der, S., Binder, M., Lohmann, V., Lot­teau, V., Rug­gieri, A., Barten­schlager, R. (2015). Con­trol of tem­po­ral acti­va­tion of hepati­tis C virus-induced inter­fer­on response by domain 2 of non­struc­tur­al pro­tein 5A. J Hepa­tol. 63(4):829–37.

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.