Association of lipid droplet and hepatitis C virus proteins: insights for virus replication.

Hepatitis C virus (HCV) infection represents a major worldwide disease burden, with an estimated prevalence of >185 million people (1). Among the various comorbidities associated with chronic HCV infection, hepatic steatosis is a frequent complication, and its presence is a key prognostic indicator associated with progression to hepatic fibrosis and with the development of hepatocellular carcinoma (2). Several mechanisms have been proposed to account for the development of steatosis and fatty liver in the setting of HCV infection (3). HCV infection enhances lipogenesis, reduces secretion of VLDL, attenuates β-oxidation of lipids, and increases virus growth and replication through complex pathways that intersect via modulating host cell lipid metabolism (4). In addition, recent studies have implicated lipid droplet formation and turnover in the life cycle of HCV. Specifically, studies have identified a role for the triglyceride-synthesizing enzyme diacylglycerol acyltransferase 1 (DGAT1) in both the formation of lipid droplets within the bilayer of the endoplasmic reticulum (ER) and also in the formation of infectious virions (5). Those studies demonstrated that DGAT1 (but not DGAT2) physically interacts with components of the nucleocapsid core and in turn recruits the viral replication complex to the newly generated lipid droplets (5). In addition, more-recent studies have demonstrated that this physical interaction between DGAT1 and HCV core components then interferes with triglyceride lipolysis and lipid droplet turnover, an effect that further enhances hepatic steatosis (6). These findings collectively suggest that HCV infection impacts hepatic lipid metabolism over a broad range of pathways, including coopting elements of lipid droplet formation and turnover as well as modulating lipogenesis, FA oxidation, and VLDL secretion. However, despite the obvious public health impact and importance of HCV infection and its role in modulating hepatic lipid metabolism, there is still much to learn about the molecular and biochemical mechanisms by which the structural and nonstructural protein components of the HCV function within these pathways. This relative dearth of information is at least partially explained by the lack of preclinical experimental models of replicating HCV. Accordingly, most of the available information still derives from studies using transfected cell lines that support HCV genome replication, either as subgenomic or full-length constructs. With this background, the article from Tanaka et al. (7) in this issue of The Journal of Lipid Research provides new insights into the role of HCV NS4B (a nonstructural protein of the virus, known to be an integral ER membrane protein) and its association with lipid droplets. The authors demonstrated this association using state-of-the-art confocal imaging and biochemical fractionation of cell lines supporting HCV replication. Although the association of HCV with lipid droplets is well known (Moradpour et al., 1996; Miyanari et al., 2007, cited in the referenced article), the molecular and biochemical mechanisms that promote this interaction remain obscure. Among the key findings of this report is the identification of specific domains and potential clusters of amino acid residues that facilitate the interaction of NS4B with the lipid droplet. The authors found that the N-terminal domain interacts more strongly with lipid droplets compared with the C terminus, with almost 100% of the N-terminal domain demonstrating association compared with ~40% following transfection of the C-terminal domain. In addition, the authors identify a specific amino acid residue within the N-terminal α helix to be responsible for NS4B binding to lipid droplets. Mutating this key tryptophan residue to alanine (W43A) abrogated association of HCV NS4B with lipid droplets. This is an interesting and novel finding with respect to association of NS4B and lipid droplets and adds to our knowledge. Moving forward, it will be important to understand how a single amino acid residue change [both with nonpolar side chains (tryptophan and alanine)] abrogates the association of NS4B with the lipid droplet. Another key aspect of these observations is the identification of lipid droplet binding residues in NS4B that are important for HCV replication. Mutation of specific hydrophobic residue (W50A) resulted in defective virus replication and severely reduced release of infectious virus particles as compared with wild-type. It remains unclear whether the inhibition is at the level of HCV RNA replication or at the virus assembly stage. Additionally, whether mutation in NS4B within HCV affects accumulation of lipid droplets within hepatocytes remains unclear. In the current work, Tanaka et al. (7) used Huh7-derived cell lines supporting HCV RNA replication in order to study aspects of both protein localization and the interaction of NS4B and lipid droplets. They confirmed the results using confocal microscopy, biochemical fractionation of cells, and Western blot analyses. Although the authors found HCV core protein in the lipid droplets associated with the ER membrane, they did not demonstrate core protein in the purified lipid droplets. This observation contrasts with an earlier report (8), demonstrating HCV genotype 3 core protein binding to lipid droplets. In the current report, Tanaka et al. (7) used HCV genotype 1b (clone O) and genotype 2a (clone JFH1) in their experiments, and did not observe binding of the HCV core to lipid droplets. This observation raises the question as to whether the mechanism of HCV interaction with lipid droplets differs with respect to the genotype of the virus. It would be interesting to see if the proposed mechanisms for NS4B and lipid droplet interaction holds true for primary human hepatocytes as a natural host of HCV infection. Overall, this paper from Tanaka et al. (7) provides interesting observations regarding the molecular mechanism of HCV NS4B association with lipid droplets and that expand our understanding of the factors that influence HCV replication. These studies further highlight the importance of NS4B and its interactions with lipid droplets as a druggable target for inhibition of HCV growth across different genotypes, with potential for developing antiviral modalities. This implication is consistent with an earlier report suggesting that disrupting the amphipathic N-terminal helix of NS4B by site-directed mutagenesis abolished HCV RNA replication in a subgenomic replicon system from HCV genotype 1b (9). In addition, other studies demonstrated that residues in the C-terminus of HCV NS4B in HCV genotype 2a subgenomic replicon were critical for viral RNA replication (10). The same group of investigators suggested that the C-terminal domain of NS4B influences production of infectious virus. The present findings from Tanaka et al. (7) suggest that NS4B strongly associates with lipid droplets, principally through its N-terminal amphipathic helix. These findings raise the following questions: a) Is HCV RNA replication dependent upon association with lipid droplet? b) Does the lipid droplet act as a platform for RNA replication and/or virus assembly? c) How and in which step does the mutated NS4B impair HCV growth? d) Do lipid droplets accumulate when cells are infected with HCV harboring a mutated NS4B? Beyond these specific questions, however, there are more-general issues of relevance to our understanding of HCV infection and hepatic lipid metabolism. For example, given the requirement for the association of HCV with lipid droplets and the possible role of NS4B, what is the potential for impacting viral replication with pharmacologic manipulation of hepatic lipogenesis and FA oxidation, strategies that would presumably attenuate lipid droplet formation (11)? Alternatively, one might envision a scenario in which modulating VLDL secretion may alter infectious HCV production and release via apoE/apoB inhibition (12–14), and this approach may warrant testing to determine the effects in vivo. Further experimental observations will be required to more completely test the functional relevance of the current findings, but these studies point to an important intersection in lipid droplet biology and HCV replication.