HCV replication and assembly occur at the interface of LD and ER membranes (41
). However, the exact details of these processes are still obscure (see references 3, 40, and 47 for reviews). Here, we studied the relationship between NS5A and host ER and LD membranes, both alone and in the context of a full-length genome. When expressed alone, NS5A was evenly distributed and tightly bound to both host ER and LD membranes, with a stronger preference for the latter. The strong association of NS5A with LD membranes is supported by four independent observations: (i) the fluorescence intensity surrounding LDs is stronger than that surrounding the ER, (ii) LD-bound NS5A is more resistant to saponin treatment than ER, (iii) turnover of LD-bound NS5A-GFP is particularly slow, and (iv) recovery after photobleaching of ER-bound NS5A is mediated by lateral diffusion and not membrane turnover. A similar tight and irreversible interaction has been reported for the host LD-associated protein adipose triglyceride lipase (57
). In our experiments, NS5A distribution was unaffected by induction of LD biogenesis using OA. These experimental conditions were used on the basis of our view that the presence or absence of OA corresponds at least to some extent to physiologically relevant lipid-biosynthetic states of hepatocytes.
LD biogenesis experiments and time-lapse microscopy revealed that NS5A is found to be concentrated on LDs at the earliest detectable time. Furthermore, we followed NS5A-decorated LDs for several hours from the point of their generation at the cell periphery until their maturation to large perinuclear LDs. The growth of LDs occurred at least in part by homeotypic fusions (not shown), suggesting that NS5A does not interfere with this process. We also demonstrated that in the ER membrane, NS5A partitions into distinct domains that are associated with Bodipy-positive putative neutral lipid accumulation sites. Based on these observations, we postulated that NS5A is localized to the LD surface membrane prior to its budding from the ER. It was recently shown that the ER-localized triglyceride-synthesizing enzyme diacylglycerol acyltransferase-1 (DGAT1) interacts with core and is required for its targeting to lipid droplets (26
). A similar mechanism may be responsible for the observations reported in , where the partitioning and concentration of NS5A around forming LDs are mediated by similar interactions.
NS5A dynamics have been determined in cells containing subgenomic genotype 2a (30
) and genotype 1b (68
) genomes. Those studies showed the static nature of NS5A in what was shown to be replication complexes. Since the viral core protein plays a major role in viral assembly and LD binding (41
), we performed these experiments using the JC1/GFP full-length genome. We demonstrated that the apparent discrepancy in the distribution of NS5A when expressed alone versus within the replicating genome () does not arise from the different HCV genotypes or positions of the tag. We propose that an added complex array of competing interactions resulting from the presence of all HCV proteins and RNAs (41
) culminates in the restriction and alteration of the inherent characteristic intracellular distribution of NS5A. Moreover, a recent study has shown that formation of new replication complexes is abolished by several small-molecule HCV inhibitors that shift the localization of NS5A from the replication complexes to LDs (60
The viral replication platform is interlaced with LDs. We speculate that NS5A might be responsible for the recruitment of the LDs to this region. The most prominent feature of the dense replication region is its seemingly complete immobility, as revealed by failure to recover after photobleaching. Time-lapse images of Bodipy-labeled LDs demonstrated that they are fully confined to and immobilized within this perinuclear area. An analogous immobility of NS5A from subgenomic genotype 1b and genotype 2a genomes has been previously observed (30
). The strong association of NS5A with host membranes may provide, at least in part, a mechanism for this immobility. Thus, future experiments should address the link between immobility and viral subsistence.
High-resolution structural studies of NS5A have revealed that the protein forms dimers via contacts near its N terminus (62
). The positioning of NS5A in the interface of peripheral LDs suggests that its self-dimerization may contribute to recruitment of LDs to the replication zone. Site-directed mutagenesis of NS5A altering its LD-binding or homodimerization propensity is under way.
The significance of NS5A's localization to LDs is underscored by the fact that LDs are also the site of its interaction with key host factors. Here, we found that NS5A recruits TBC1D20 and its cognate GTPase Rab1 to the vicinity of LDs. Using immunofluorescence, we were unable to consistently demonstrate localization of endogenous Rab1 to LDs, presumably due to the dynamic nature of this GTPase. When coexpressed with NS5A, Rab1 clearly associated with LDs. In accord with these findings, Rab1 was reported to associate with LDs using proteomic analysis (4
). Using a dominant negative mutant of Rab1, we demonstrated that Rab1 GTPase activity is a prerequisite for LD homeostasis as well as for maintenance of viral replication complexes. These results are supported by previously published data showing that siRNA-mediated depletion of Rab1 significantly decreased HCV RNA levels (55
). Additional small GTPases associated with secretory transport were shown to be involved in LD metabolism as well: Rab18 was demonstrated to be recruited to LDs during lipolysis and to induce the apposition of LDs to the ER membranes (36
). Similarly, Arf1 has been shown to act on the LD surface to regulate droplet morphology and lipid utilization (21
). Arf1 inhibition was shown to inhibit HCV replication and to shift the localization of NS3 and NS5A from the ER to the LD surface (39
Infectious virus particles are exclusively those that are exported from the cell complexed with lipoproteins synthesized from LDs (1
). The data herein provide evidence linking the LD binding of NS5A to a proposed role in perturbing LD metabolism. It is tempting to hypothesize that NS5A binds to newly formed LDs and together with other viral LD-binding proteins, such as core, facilitates their incorporation into the perinuclear replication zone. Its interaction with TBC1D20 and, consequently, Rab1 at these sites might serve to recruit these host mediators. Our results confirm the independent localization of NS5A to LDs and support recent viral assembly models in which LD localization of both core and NS5A is pivotal for the assembly process (see reference 3 for updated models). Our data further confirm a role for the host factors TBC1D20 and Rab1 in HCV replication.