Assembly of infectious hepatitis C virus particles Ralf Bartenschlager, Francois Penin, Volker Lohmann, Patrice André  Trends in Microbiology  Volume 19, Issue 2, Pages 95-103 (February 2011) DOI: 10.1016/j.tim.2010.11.005 Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 1 HCV genome structure and functions of viral proteins. Individual proteins within the polyprotein and their functions in the viral replication cycle are given. Proteases involved in polyprotein processing and cleavage sites are indicated in the top. The 5′ and 3′ NTRs are shown according to secondary structure predictions. For clarity, core protein derivatives generated by internal translation initiation or ribosomal frame shift are not shown ([88] and references cited therein). Abbreviations: SPP, signal peptide peptidase; SP, signal peptidase; cys., cysteine; membr., membranous. Trends in Microbiology 2011 19, 95-103DOI: (10.1016/j.tim.2010.11.005) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 2 Models of HCV assembly. The biogenesis of cytosolic LDs (cLDs) is thought to take place at distinct domains of the rER with the formation of an intra-ER membrane lens of apolar lipids that finally buds off towards the cytosol. (a) Continuity between ER membrane and cLDs should allow their ‘loading’ with core from the ER membrane (1). cLDs might associate with microtubule motors (MTs) and migrate to a specialized smooth ER (sER)-derived membranous web compartment. There, core-loaded cLDs might attach to the ER membrane via tethering proteins (e.g. Rab 18) at sites where luminal (lu) LDs form and in close proximity of ER-bound replicase (2). Nucleocapsid formation might occur by transfer of the RNA from the ER-resident replicase or (as shown here) from oligomeric complexes of NS5A [89] to the core protein that is released back from the cLD to the ER membrane. We suggest that the interface between cLDs and the ER membrane provides a site favoring the translocation of the hydrophobic core proteins to luLDs that are budding into the ER lumen. (b) In this model, cLDs are loaded with core and NS5A (1). ER-bound replication complexes could deliver HCV RNA to NS5A that can freely move onto cLDs, thus forming an NS5A–RNA array. As in the previous model, these cLDs could associate with MTs and move to specialized ER sites (2). Tethering factors might trap cLDs at the ER membrane where viral RNA is transferred from NS5A to core, thus triggering nucleocapsid formation. This step is linked to the delivery of the nucleocapsid into luLDs. It is also possible that nucleocapsids assemble and bud into cLDs at an early step prior to delivery to luLDs (not shown). Both models postulate that the hydrophobic core protein is pulled out of the cLD phospholipid monolayer and inserted into an ER membrane limiting lens of apolar lipids corresponding to nascent luLDs. This process would not require bending of membrane bilayer and thus only limited energy. Trends in Microbiology 2011 19, 95-103DOI: (10.1016/j.tim.2010.11.005) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 3 Models of structures of infectious HCV particles and their biogenesis. During translation at the rER, nascent apoB (blue line) is translocated into the ER lumen and loaded by MTP with phospholipids and triglycerides (left panel). This leads to the formation of a neutral lipid core that is converted into a spherical particle (VLDL2) acquiring exchangeable apoE and apoC (not shown for clarity). In the smooth ER (sER) or membranous web (m.w.), a second precursor (the luminal LD; luLD) is formed from the ER membrane and by MTP-mediated triglyceride enrichment (right panel). E1 and E2 retained at the ER membrane might slide onto this luLD prior to pinching-off [49]. The nucleocapsid would be inserted into the hydrophobic lipid core of the pinching-off luLD due to the hydrophobic nucleocapsid surface (formed by D2 of the core protein). In VLDL competent cells such as primary human hepatocytes, this precursor could fuse with VLDL2 to form the LVP. Alternatively in Huh-7 cells where VLDL1 formation is inefficient [49]. HCVcc is secreted predominantly as particles lacking apoB. Trends in Microbiology 2011 19, 95-103DOI: (10.1016/j.tim.2010.11.005) Copyright © 2010 Elsevier Ltd Terms and Conditions