## 5 The contribution of lipid droplets to production of infectious HCV
### Lipid droplets are associated with HCV-induced membrane rearrangements
Furthermore, in nocodazole-treated cells in which lipid droplet redistribution has been inhibited, virus release is impaired [98]. Aggregation of lipid droplets at the perinuclear region increases the level of colocalization observed between core-coated lipid droplets and ER-resident replication complexes and may effectively serve to concentrate core at sites of replication to increase the likelihood of virus assembly [94,98]. Another mechanism that could facilitate the congregation of core-coated lipid droplets and replication complexes involves the HCV-encoded NS5A protein. NS5A is a component of the HCV replication complex and is essential for viral genome replication but its precise function in the HCV life cycle remains unknown [68-71,100-102]. However, several lines of evidence exist to support a role for NS5A in the recruitment of replication complexes to core-coated lipid droplets. Firstly, NS5A exhibits an inherent capacity to localize to the surface of droplets [72]. Furthermore, variants of NS5A containing mutations in domain DI have impaired capacity for lipid droplet-association, and also prevent recruitment of other NS proteins and viral RNA to lipid droplets leading to diminished virus production [73]. Deletions in domain DIII have also been shown to disrupt the colocalization of NS5A with core-coated lipid droplets resulting in the abrogation of infectious particle release [21]. Additionally, DIII facilitates an interaction between NS5A and core, suggesting a mechanism whereby NS5A can target replication complexes to lipid droplets via a direct interaction with core protein on the droplet surface [103]. One other option is that cellular cyclophilins, which share peptidyl-prolyl cis-trans-isomerases activity and play an important role in HCV RNA replication [104-106], may influence the mechanism for directing replication complexes to core-coated lipid droplets although this possibility has not been tested. Taken together, the combined actions of core and NS5A in virus-infected cells likely play pivotal roles in the accumulation of replication complexes at lipid droplet-associated regions of the ER membrane. Rationale for targeting replication complexes to lipid droplet-associated regions of the ER membrane  
The requirement for replicated genomes to be available at sites of virus assembly to initiate virion morphogenesis may serve to explain the close proximity of replication complexes with lipid droplets coated with core protein [73,97]. The mechanism for transfer of replicated genomes from replication complexes to core on the surface of lipid droplets is unknown, but NS5A has several specific biological properties that appear particularly suited to such a role. Firstly, NS5A binds to the 3' positive-sense polypyrimidine tract of HCV genomes with high affinity, a function believed to be mediated following the dimerization of the protein, which creates a putative RNA-binding cleft in the DI domains of the dimer [69,102,107]. Secondly, replicons encoding defects in NS5A can be rescued by trans-complementation with wt replicons suggesting NS5A is able to transfer between individual replication complexes [21,108,109]. This finding indicates NS5A may be a more flexible, rather than fixed, component of the replication complex, a property suited to a role in the transfer of replicated genomes from replication complexes to core at the surface of lipid droplets. Thirdly, NS5A has long been known to exist as differentially phosphorylated species in cells replicating HCV RNA [110-112], and phosphorylation of NS5A is believed to act as a regulator of events in the HCV life cycle [24,66,113]. Indeed, inhibition of NS5A hyperphosphorylation elevates levels of HCV genome replication [66,100,114,115]. Recently, it was established that phosphorylation of a serine residue located in a casein kinase II consensus motif in the DIII domain of JFH-1 NS5A was required for virion production [24]. Abrogating phosphorylation at this site perturbed production of infectious virus particles without affecting RNA replication [24].