## 5 The contribution of lipid droplets to production of infectious HCV  
### Lipid droplets are associated with HCV-induced membrane rearrangements  
It is widely accepted that the membranous web houses sites of HCV genome synthesis (see Section 1.2). In a recent study, a higher percentage of lipid droplets was observed in close proximity to modified ER membrane cisternae in Huh-7 cells that contained replicating full-length JFH-1 genomes compared to naive cells [73]. Specifically, rearranged ER membranes appeared to partially enclose lipid droplets in a configuration more reminiscent of the convoluted membrane structures observed in cells infected with Kunjin virus (a member of the _Flaviviridae_ family) [73, 89]. Core protein was observed mainly on the periphery of lipid droplets and although some NS5A protein was detected on the lipid droplet surface, the majority was distributed at sites distal to droplets [73]. Furthermore, 50 nm virus-like particles, which reacted with core- and E2-specific antibodies, were also observed in close proximity to lipid droplet-associated membranes, suggesting infectious HCV particles were generated from such a membranous environment [73]. In a separate study, the interaction of HCV-like particles with lipid droplets was assessed using three-dimensional reconstructions of serial ultrathin electron microscopy sections produced from cells producing HCV core protein [90]. The results also supported the notion that budding of virus is initiated from membranes closely associated with lipid droplets [90]. Virus production is dependent upon recruitment of replication complexes to lipid droplet-associated membranes  
The precise purpose for attachment of core to lipid droplets remains unknown and is currently an active area of study. Historically, the absence of tissue culture systems to propagate HCV meant that the mechanisms by which core-coated lipid droplets interacted with ER-resident replication complexes to facilitate virion assembly were not amenable to investigation. However, following the discovery that HCV strain JFH-1 genotype and chimeras derived from this strain could release infectious particles from cells [91-93], it has been established that the NS proteins localize to distinct foci juxtaposed to lipid droplets in cells producing progeny virus [21,73,94-96]. These specific lipid droplet-associated foci likely represent accumulations of replication complexes since negative-sense HCV RNA and virus-specific dsRNA replicative intermediates are detected within the foci [73,94]. Replication complexes do not localize to lipid droplet-associated regions of the ER in cells containing subgenomic HCV replicons, therefore, lipid droplets are presumably not required for HCV RNA replication per se [94]. Blocking attachment of core to lipid droplets in cells containing JFH-1 genomes, through mutations in either the D2 domain or the core-E1 signal sequence to impair signal peptide peptidase cleavage, prevents detection of HCV-induced dsRNA-containing foci and NS proteins in close proximity to lipid droplets [73,97]. Under these circumstances, HCV genome replication appeared unaffected but release of infectious virus was impaired [97]. Thus, recruitment of replication complexes to lipid droplet-associated regions of the ER membrane is a phenomenon likely required for the assembly of infectious virus progeny. There are at least two possible mechanisms in operation in HCV-infected cells, which serve to localize replication complexes to regions of the ER in close proximity to core-coated lipid droplets. The first centers on the capacity of core to induce lipid droplet redistribution [98,99]. In virus-infected cells, or cells expressing core protein alone, lipid droplets are redistributed from a diffuse cytoplasmic localization to the perinuclear region [98,99]. Lipid droplet redistribution coincides with release of infectious virus progeny in cells containing full-length JFH-1 genomes and redistribution is believed to be dependent upon the microtubule network [98].