apl. Prof. Dr. Volk­er Lohmann

volker.lohmann@med.uni-heidelberg.de

Phone: ++49 (0)6221–56 6449

Replication of positive strand RNA viruses and their recognition by the cell intrinsic innate immune response

Projects

We are inter­est­ed in dif­fer­ent aspects of Hepati­tis C virus (HCV) repli­ca­tion includ­ing the under­stand­ing of cell cul­ture adap­ta­tion, struc­ture-func­tion analy­ses on the HCV poly­merase and the iden­ti­fi­ca­tion of host cell fac­tors engaged in HCV repli­ca­tion, e.g. the cel­lu­lar lipid kinase PI4KA, Cyclophilins and microR­NA 122. One of our focus­es is a gen­er­al under­stand­ing of the bio­gen­e­sis of the mem­bra­nous repli­ca­tion organelles of pos­i­tive strand RNA virus­es, in par­tic­u­lar HCV and Norovirus. We are fur­ther­more inter­est­ed how HCV induces and coun­ter­acts innate and adap­tive immune respons­es to final­ly achieve per­sis­tence, in con­trast and com­par­i­son to hepati­tis A virus infec­tions, shar­ing a sim­i­lar repli­ca­tion strat­e­gy, but always being cleared.

1 | Regulation of lipid homeostasis at the HCV replication organelle and its role in genome replication and pathogenesis

Hepati­tis C virus (HCV) repli­ca­tion takes place at dis­tinct vesic­u­lar mem­brane struc­tures which are induced by a con­cert­ed action of the viral non­struc­tur­al pro­teins and host fac­tors. In pre­vi­ous stud­ies we have iden­ti­fied an inti­mate con­nec­tion of HCV repli­ca­tion and the cel­lu­lar lipid kinase Phos­phatidyli­nos­i­tol-4-kinase IIIa, (PI4KIIIa, PI4KA). PI4KA con­verts phos­phatidyli­nos­i­tol to phos­phatidyli­nos­i­tol 4‑phosphate (PI4P). The enzy­mat­ic activ­i­ty of PI4KA is acti­vat­ed by the HCV non­struc­tur­al pro­teins NS5A and NS5B, result­ing in ele­vat­ed intra­cel­lu­lar PI4P lev­els (Reiss et al., Cell Host & Microbe, 2011, Plos Path, 2013; Harak et al., JVI; 2014). This increased con­cen­tra­tion of PI4P has a cen­tral role for the bio­gen­e­sis of the viral repli­ca­tion organelle by shap­ing its lipid com­po­si­tion due to the action of lipid trans­port pro­teins like OSBP or FAPP2.  How­ev­er, acti­va­tion of PI4KA in Huh7 hepatoma cells is dele­te­ri­ous for most HCV wild­type iso­lates due to an excess of PI4P, explain­ing the inef­fi­cient repli­ca­tion of patient derived virus­es in cell cul­ture. Sur­pris­ing­ly, we could demon­strate that repli­ca­tion enhanc­ing adap­tive muta­tions, which are essen­tial to sup­port HCV repli­ca­tion in hepatoma cells,  in fact rely on a loss of func­tion mech­a­nism, abro­gat­ing acti­va­tion of PI4KA to com­pen­sate for high PI4KA expres­sion lev­els in hepatoma cells com­pared to pri­ma­ry hepa­to­cytes (Harak et al., Nature Micro­bi­ol­o­gy, 2016). Based on this find­ing we could estab­lish a reg­i­men based on PI4KA/Casein Kinase Iα (CKIα) inhibitors, allow­ing effi­cient repli­ca­tion of HCV wt iso­lates in cell cul­ture. Cur­rent­ly we are aim­ing at a com­pre­hen­sive under­stand­ing of the mech­a­nisms involved in shap­ing the lipid com­po­si­tion at the HCV repli­ca­tion organelle. We fur­ther inves­ti­gate the mech­a­nism of action of Sec14L2, a lipid trans­porter pro­tein expressed in hepa­to­cytes but not in Huh7. Sec14L2 was recent­ly iden­ti­fied by oth­ers to stim­u­late repli­ca­tion of HCV wt iso­lates, but the mech­a­nism is still poor­ly defined and the effect is very vari­able for dif­fer­ent clin­i­cal iso­lates (Cos­ta et al., J. Hepa­tol., 2018). We are cur­rent­ly test­ing the hypoth­e­sis that the lipid trans­port func­tion of Sec14L2 might link its mech­a­nism of action to PI4KA.

Anoth­er inde­pen­dent project is ded­i­cat­ed to the impact of ele­vat­ed PI4P lev­els, which appear as a con­se­quence of PI4KA acti­va­tion by HCV, to viral patho­gen­e­sis. PI4P is a pre­cur­sor of oth­er phos­pho­inosi­tides involved in var­i­ous cel­lu­lar sig­nal­ing process­es, and by itself has cru­cial func­tions in intra­cel­lu­lar vesic­u­lar trans­port. In addi­tion, PI4KA expres­sion is upreg­u­lat­ed in hepatoma cells and there­fore might con­tribute to can­cer growth. We there­fore use dif­fer­ent cell based and ani­mal mod­els to under­stand the impact of PI4KA abun­dance and acti­va­tion on cel­lu­lar sig­nal­ing cas­cades, cell growth and mor­phol­o­gy to unrav­el poten­tial mech­a­nis­tic links to hepa­to­cel­lu­lar car­ci­no­ma.

Fig­ure 1 | Acti­va­tion of the cel­lu­lar lipid kinase PI4KIIIa (PI4KA) by the HCV non­struc­tur­al pro­teins NS5A and NS5B results in an intra­cel­lu­lar accu­mu­la­tion of PI4P, which is cru­cial for shap­ing the lipid com­po­si­tion of the viral repli­ca­tion organelle and affects sev­er­al cel­lu­lar process­es poten­tial­ly con­tribut­ing to viral patho­gen­e­sis.

2 | The role of miR-122 in HCV translation and replication

The liv­er spe­cif­ic microR­NA 122 (miR-122) rec­og­nizes two con­served sites at the 5’ end of the hepati­tis C virus (HCV) genome and con­tributes to sta­bil­i­ty, trans­la­tion and repli­ca­tion of the viral RNA. Stim­u­la­tion of the HCV inter­nal ribo­some entry site (IRES) by miR-122 is essen­tial for effi­cient viral repli­ca­tion. We have shown that the mech­a­nism relies on a dual func­tion of the 5’ ter­mi­nal pri­ma­ry sequence in the com­ple­men­tary pos­i­tive (IRES-medi­at­ed trans­la­tion) and neg­a­tive strand (RNA repli­ca­tion), requir­ing dif­fer­ent sec­ondary struc­tures. Our results sug­gest that miR-122 bind­ing assists the fold­ing of a func­tion­al IRES in an RNA chap­er­one-like man­ner by sup­press­ing ener­get­i­cal­ly favor­able alter­na­tive sec­ondary struc­tures (Schult et al., Nature Comm., 2018). We are cur­rent­ly assess­ing the quan­ti­ta­tive con­tri­bu­tion of the var­i­ous func­tions of miR-122 in HCV trans­la­tion, genome sta­bil­i­ty and repli­ca­tion in a math­e­mat­i­cal mod­el of HCV repli­ca­tion (Binder et al., Plos Path, 2013). We fur­ther aim to obtain a com­pre­hen­sive under­stand­ing of the spa­tio-tem­po­ral reg­u­la­tion of the inter­ac­tion between miR-122 and the viral genome.

Fig­ure 2 | Schemat­ic of the mech­a­nism how mir-122 con­tributes to the stim­u­la­tion of HCV trans­la­tion. Bind­ing of mir-122 to the 5’UTR of the HCV genome sta­bi­lizes the fold­ing of the inter­nal ribo­some entry site (IRES) by pre­vent­ing alter­na­tive struc­tures, which are ener­get­i­cal­ly favor­able due to the dual func­tion of the same sequence in trans­la­tion (pos­i­tive strand) and repli­ca­tion (neg­a­tive strand), requir­ing dif­fer­ent RNA sec­ondary struc­tures (Schult et al., Nat. Comm., 2018).

 

 

3 | Understanding the Toll-like receptor 3 response against HCV and HAV and its contribution to persistence and clearance of viral infections

Hepati­tis C virus (HCV, fam­i­ly Fla­viviri­dae) and hepati­tis A virus (HAV, fam­i­ly Picor­naviri­dae) are both pos­i­tive strand RNA virus­es with very sim­i­lar mech­a­nisms of RNA repli­ca­tion. How­ev­er, HAV infec­tions are always cleared where­as HCV estab­lish­es per­sis­tence in  ca. 70% of infect­ed indi­vid­u­als. Clear­ance cor­re­lates with low lev­els of innate immune respons­es in the liv­er in case of HAV, where­as per­sis­tence in case of HCV is asso­ci­at­ed with a long-last­ing induc­tion of a vari­ety of inter­fer­on-induced genes (ISGs), which main­ly orig­i­nate from infect­ed cells. Still, both virus­es acti­vate a sim­i­lar pan­el of pat­tern recog­ni­tion recep­tors and have evolved iden­ti­cal coun­ter­mea­sures involv­ing cleav­age of the same adap­tor mol­e­cules cru­cial for mount­ing intra­cel­lu­lar innate immune respons­es. Our pre­vi­ous work has estab­lished repli­ca­tion mod­els for both virus­es allow­ing a thor­ough side-by-side com­par­i­son (Ess­er-Nobis et al., Hepa­tol­ogy 2015). HAV, in stark con­trast to HCV, does not induce Toll-like recep­tor 3 (TLR3)-mediated innate immune respons­es, despite the fact that the sig­nal­ing path­way is not effi­cient­ly blocked in cells infect­ed with HAV. This result points to sub­stan­tial dif­fer­ences in the con­sti­tu­tion of the viral repli­ca­tion com­part­ment and the fate of viral repli­ca­tion inter­me­di­ates, reach­ing TLR3 in case of HCV, but not in case of HAV. How­ev­er, HCV has estab­lished an escape mech­a­nism to damp­en TLR3 respons­es by secre­tion of repli­ca­tion inter­me­di­ates in extra­cel­lu­lar vesi­cles, sug­gest­ing that a bal­anced TLR3 response might sup­port the estab­lish­ment of a per­sis­tent infec­tion (Grün­vo­gel et al., Gas­troen­terol­o­gy, 2018). We are cur­rent­ly aim­ing at a mol­e­c­u­lar under­stand­ing of the fate of the dou­ble strand­ed repli­ca­tion inter­me­di­ates of both virus­es. In addi­tion, we are inter­est­ed in gen­er­al aspects of the TLR3 response in hepa­to­cytes. To this end, we are ana­lyz­ing the func­tion of var­i­ous TLR3 poly­mor­phisms found in the human pop­u­la­tion. We are fur­ther­more ana­lyz­ing hits from a Cripr/Cas9 based knock­out screen on the TLR3 path­way to iden­ti­fy nov­el fac­tors involved in its reg­u­la­tion.

Fig­ure 3 | HCV par­tial­ly escapes recog­ni­tion by TLR3 upon secre­tion of dou­ble strand­ed repli­ca­tion inter­me­di­ates in exo­somes (Grün­vo­gel et al., Gas­troen­terol­o­gy, 2018).

4 | Function of norovirus nonstructural proteins 

Human norovirus­es (huNoV) are the most fre­quent cause of non-bac­te­r­i­al acute gas­troen­teri­tis world­wide, par­tic­u­lar­ly genogroup II geno­type 4 (GII.4) vari­ants. The viral non­struc­tur­al (NS) pro­teins encod­ed by the ORF1 polypro­tein induce vesi­cal clus­ters har­bor­ing the viral repli­ca­tion sites. In a pre­vi­ous study we com­pared the ultra­struc­tur­al changes induced by expres­sion of norovirus ORF1 polypro­teins with those induced upon infec­tion with murine norovirus (MNV). Char­ac­ter­is­tic mem­brane alter­ations induced by ORF1 expres­sion resem­bled those found in MNV infect­ed cells, con­sist­ing of vesi­cle accu­mu­la­tions like­ly built from the endo­plas­mic retic­u­lum (ER) which includ­ed sin­gle mem­brane vesi­cles (SMVs), dou­ble mem­brane vesi­cles (DMVs) and mul­ti mem­brane vesi­cles (MMVs). Expres­sion of GII.4 NS1‑2, NS3 and NS4 fused to GFP each revealed dis­tinct mem­brane alter­ations. Inter­est­ing­ly, NS4 was the only GII.4 pro­tein capa­ble of induc­ing SMV and DMV for­ma­tion when expressed indi­vid­u­al­ly.  We there­by iden­ti­fied NS4 as a key fac­tor in the for­ma­tion of mem­brane alter­ations of huNoV and pro­vid­ed mod­els of the puta­tive mem­brane topolo­gies of NS1‑2, NS3 and NS4 to guide future stud­ies. Cur­rent­ly  we are ana­lyz­ing the spe­cif­ic func­tions of these pro­teins in the bio­gen­e­sis of the human norovirus repli­ca­tion organelle. We are fur­ther aim­ing at the estab­lish­ment of robust cell cul­ture mod­els for human norovirus­es.

Fig­ure 4 | Repli­ca­tion of norovirus­es is linked to mem­brane alter­ations induced by the non­struc­tur­al pro­teins. The main dri­vers of this process are NS1‑2, NS3 and NS4. The upper pan­el pro­vides a ten­ta­tive view on the pro­posed struc­ture of these pro­teins, the low­er pan­els a cor­rel­a­tive-light and elec­tron microscopy study to iden­ti­fy dis­tinct mem­brane alter­ations induced by the indi­vid­u­al­ly expressed pro­teins (Doer­flinger et al., Plos Path, 2017).

Fund­ing

5 Major Publications

Grün­vo­gel O, Colas­an­ti O, Lee JY, Klöss V, Belouzard S, Reustle A, Esser-Nobis K, Hesebeck-Brinckmann J, Mutz P, Hoff­mann K, Mehra­bi A, Koschny R, Von­dran FWR, Got­thardt D, Schnit­zler P, Neumann-Haefelin C, Thimme R, Binder M, Barten­schlager R, Dubuis­son J,Dalpke AH, Lohmann V: Secre­tion of Hepati­tis C Virus Repli­ca­tion Inter­me­di­ates Reduces Acti­va­tion of Toll-Like Recep­tor 3 in Hepa­to­cytes. Gas­troen­terol­o­gy 2018; 154(8):2237‐2251.

Schult P, Roth H, Adams RL, Mas C, Imbert L, Orlik C, Rug­gieri A, Pyle AM, Lohmann V: microRNA-122 ampli­fies hepati­tis C virus trans­la­tion by shap­ing the struc­ture of the inter­nal ribo­so­mal entry site. Nat Com­mun 2018; 4; 9(1):2613.

Doer­flinger SY, Cortese M, Romero-Brey I, Menne Z, Tubiana T, Schenk C, White PA, Barten­schlager R, Bres­sanel­li S, Hans­man GS, Lohmann V: Mem­brane alter­ations induced by non­struc­tur­al pro­teins of human norovirus. PLoS Pathog 2017; 27;13(10).

Harak C, Meyrath M, Romero-Brey I, Schenk C, Gondeau C, Schult P, Esser-Nobis K, Saeed M, Ned­der­mann P, Schnit­zler P, Got­thardt D, Perez Del-Pulgar S, Neumann-Haefelin C, Thimme R, Meule­man P, Von­dran FW, Francesco R, Rice CM, Barten­schlager R, Lohmann V:Tuning a cel­lu­lar lipid kinase activ­i­ty adapts hepati­tis C virus to repli­ca­tion in cell cul­ture. Nat Micro­bi­ol. 2016 Dec 19;2:16247.

Lohmann V., Körn­er F, Koch JO, Her­ian U, Theil­mann L and Barten­schlager R.: Repli­ca­tion of subge­nom­ic hepati­tis C virus RNAs in a hepatoma cell line. Sci­ence 1999; 285: 110‐113.