PhD, La Trobe University
Lipid droplets act as signalling platforms which alter the early innate antiviral response
The early innate host response to viral infection, consists of the activation of multiple networked pathways. This produces an orchestrated anti-viral response, which ultimately determines the fate of the infected cell, and potentially the outcome of the viral infection on the host. This response is pivotal in determining how a host cell responds to viral infection, and this particular response is the focus of a new era of anti-viral drug treatments, attempting to artificially drive these pathways to cure multiple acute and chronic infections for which their currently exists no cure, such as HIV, Hepatitis B Virus, Zika, Dengue, West Nile Virus and Chikungunya virus.
However, although these new treatments have shown some promise, they still lack effectiveness, and require further tailoring. An improved knowledge of the host cell factors involved in regulating the host response pathways that control this anti-viral response will better inform the tailoring of these new treatment regimes.
More recently, a number of molecules have been described that reside on certain host organelles, to assist in the activation and driving of the early innate host response pathways to viral infection, most notably, STING on the mitochondria, and the peroxisome, and Viperin on the lipid droplet.
However we still do not have a full picture of the molecules involved in augmenting these early anti-viral response pathways, nor the localisations many of the known major adaptor proteins driving these pathways.
I have shown lipid droplets to be an important player in the antiviral response. With a lower lipid droplet content, cells are not able to overcome viral infection as well as cells with larger amounts of lipid droplets. If these lipid droplets are creating regions of high immune signalling activity, it is possible they will create a region desirable to target antiviral therapeutics to.
Abstract: The interferon-stimulated gene, viperin, has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here, we demonstrate that viperin plays an integral part in the ability of interferon to limit the replication of cell-culture-derived HCV (JFH-1) that accurately reflects the complete viral life cycle. Using confocal microscopy and fluorescence resonance energy transfer (FRET) analysis, we demonstrate that viperin localizes and interacts with HCV nonstructural protein 5A (NS5A) at the lipid-droplet (LD) interface. In addition, viperin also associates with NS5A and the proviral cellular factor, human vesicle-associated membrane protein-associated protein subtype A (VAP-A), at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C-terminus, as well as an N-terminal amphipathic helix. Removal of the amphipathic helix-redirected viperin from the cytosolic face of the endoplasmic reticulum and the LD to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C-terminal viperin mutants still localized to the LD interface and replication complexes, but did not interact with NS5A proteins, as determined by FRET analysis.In conclusion, we propose that viperin interacts with NS5A and the host factor, VAP-A, to limit HCV replication at the replication complex. This highlights the complexity of the host control of viral replication by interferon-stimulated gene expression.
Pub.: 03 Nov '11, Pinned: 28 Jul '17
Abstract: Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and relatively static NS5A-positive foci are enriched with host factors VAP-A and Rab5A. Pulse-chase imaging revealed that newly synthesized NS5A foci are small and distinct from aged foci, while further studies using a unique dual fluorescently tagged infectious HCV chimera showed a relatively stable association of NS5A foci with core-capped LDs. These results reveal new details about the dynamics and maturation of NS5A and the nature of potential sites of convergence of HCV replication and assembly pathways.Hepatitis C virus (HCV) is a major cause of serious liver disease worldwide. An improved understanding of the HCV replication cycle will enable development of novel and improved antiviral strategies. Here we have developed complementary fluorescent labeling and imaging approaches to investigate the localization, traffic and interactions of the HCV NS5A protein in living, virus-producing cells. These studies reveal new details as to the traffic, composition and biogenesis of NS5A foci and the nature of their association with putative sites of virus assembly.
Pub.: 17 Jan '14, Pinned: 28 Jul '17
Abstract: Zika virus (ZIKV) infection has emerged as a global health threat and infection of pregnant women causes intrauterine growth restriction, spontaneous abortion and microcephaly in newborns. Here we show using biologically relevant cells of neural and placental origin that following ZIKV infection, there is attenuation of the cellular innate response characterised by reduced expression of IFN-β and associated interferon stimulated genes (ISGs). One such ISG is viperin that has well documented antiviral activity against a wide range of viruses. Expression of viperin in cultured cells resulted in significant impairment of ZIKV replication, while MEFs derived from CRISPR/Cas9 derived viperin(-/-) mice replicated ZIKV to higher titers compared to their WT counterparts. These results suggest that ZIKV can attenuate ISG expression to avoid the cellular antiviral innate response, thus allowing the virus to replicate unchecked. Moreover, we have identified that the ISG viperin has significant anti-ZIKV activity. Further understanding of how ZIKV perturbs the ISG response and the molecular mechanisms utilised by viperin to suppress ZIKV replication will aid in our understanding of ZIKV biology, pathogenesis and possible design of novel antiviral strategies.
Pub.: 02 Jul '17, Pinned: 28 Jul '17
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