Final Report Summary - HCV HOST TRAFFICKING (Subversion of host trafficking machinery by hepatitis C virus to promote viral replication)
Summary description of the project objectives
The two main objectives of this proposal were to:
- determine the role of the NS5A-TBC1D20 interaction in establishment of the hepatitis C virus (HCV) replication complex;
- determine the role of Arf1 in HCV replication.
Description of the work performed since the beginning of the project
1. Determine the role of the NS5A-TBC1D20 interaction in establishment of the HCV replication complex
Towards accomplishing this aim we first characterised the interaction of fluorescently tagged NS5A with host cell membranes and binding partners was in living cells. These results were summarised in our recently published manuscript (Nevo-Yassaf et al., J. Virology, 2012). We found that NS5A bind both to the endoplasmic reticulum (ER) and to lipid droplets (LD). Its binding to LDs was found to be apparently irreversible, both in HCV-infected cells and when ectopically expressed. In HCV-infected cells, NS5A fluorescence was observed around the LDs and in perinuclear structures that were incorporated into a highly immobile platform superimposed over the Endoplasmic reticulum (ER) membrane. We have previously shown that interaction between HCV NS5A and the host protein, TBC1D20, is obligatory for efficient HCV replication. TBC1D20 is a Rab GTPase-activating protein (GAP) required for regulation of Rab, a small GTPase that plays a key role in Golgi biogenesis, ER-to-Golgi trafficking, and HCV replication. Our results show that TBC1D20 and its cognate GTPase Rab1 are recruited by NS5A to LDs. While overxpression of the Rab1 dominant negative (Rab1DN) GTPase mutant in cells abolished steady-state LDs. In infected cells, Rab1DN induced the elimination of NS5A from viral replication sites. Taken together, our results demonstrate the significance of the localisation of NS5A to LDs and support a model whereby its interaction with TBC1D20 and Rab1 affects LD metabolism to promote the viral life cycle. Insight at the molecular level of this phase in the viral life cycle will unravel potential new targets for antiviral therapy.
2. Determining the role of Arf1 in HCV replication
Using GAP assays, pharmacological inhibitors of Arf1 activation and colocalisation studies we identified a role for Arf1 in the viral life cycle (in collaboration with Dr Benny Aroeti, Hebrew University Jerusalem, Matto M. and Sklan E. H. et al., Journal of Virology, 2011).
We found that treatment of HCV infected cells with Brefeldin A (BFA) and Golgicide A (GCA), pharmacological inhibitors of Arf1 activation, decreased virus production. Similarly, down regulation of Arf1 expression using small interfereing ribonucleic acid (siRNA), significantly decreased HCV RNA replication and infectious viral particle production. Treatment of infected cells with BFA and GCA caused redistribution of the viral proteins to the surface of LDs. Dominant negative form of Arf1, co-localised with NS5A around LDs. LDs are thought to play a central role in assembly of the HCV virion, suggesting that Arf1 plays an important role in this process.
The expected final results and their potential impact and use
Our results lead to better understanding of the molecular virology of HCV that assists in identifying new targets for specific antiviral therapies.
Our results were published in the international scientific journals available for open access.
The two main objectives of this proposal were to:
- determine the role of the NS5A-TBC1D20 interaction in establishment of the hepatitis C virus (HCV) replication complex;
- determine the role of Arf1 in HCV replication.
Description of the work performed since the beginning of the project
1. Determine the role of the NS5A-TBC1D20 interaction in establishment of the HCV replication complex
Towards accomplishing this aim we first characterised the interaction of fluorescently tagged NS5A with host cell membranes and binding partners was in living cells. These results were summarised in our recently published manuscript (Nevo-Yassaf et al., J. Virology, 2012). We found that NS5A bind both to the endoplasmic reticulum (ER) and to lipid droplets (LD). Its binding to LDs was found to be apparently irreversible, both in HCV-infected cells and when ectopically expressed. In HCV-infected cells, NS5A fluorescence was observed around the LDs and in perinuclear structures that were incorporated into a highly immobile platform superimposed over the Endoplasmic reticulum (ER) membrane. We have previously shown that interaction between HCV NS5A and the host protein, TBC1D20, is obligatory for efficient HCV replication. TBC1D20 is a Rab GTPase-activating protein (GAP) required for regulation of Rab, a small GTPase that plays a key role in Golgi biogenesis, ER-to-Golgi trafficking, and HCV replication. Our results show that TBC1D20 and its cognate GTPase Rab1 are recruited by NS5A to LDs. While overxpression of the Rab1 dominant negative (Rab1DN) GTPase mutant in cells abolished steady-state LDs. In infected cells, Rab1DN induced the elimination of NS5A from viral replication sites. Taken together, our results demonstrate the significance of the localisation of NS5A to LDs and support a model whereby its interaction with TBC1D20 and Rab1 affects LD metabolism to promote the viral life cycle. Insight at the molecular level of this phase in the viral life cycle will unravel potential new targets for antiviral therapy.
2. Determining the role of Arf1 in HCV replication
Using GAP assays, pharmacological inhibitors of Arf1 activation and colocalisation studies we identified a role for Arf1 in the viral life cycle (in collaboration with Dr Benny Aroeti, Hebrew University Jerusalem, Matto M. and Sklan E. H. et al., Journal of Virology, 2011).
We found that treatment of HCV infected cells with Brefeldin A (BFA) and Golgicide A (GCA), pharmacological inhibitors of Arf1 activation, decreased virus production. Similarly, down regulation of Arf1 expression using small interfereing ribonucleic acid (siRNA), significantly decreased HCV RNA replication and infectious viral particle production. Treatment of infected cells with BFA and GCA caused redistribution of the viral proteins to the surface of LDs. Dominant negative form of Arf1, co-localised with NS5A around LDs. LDs are thought to play a central role in assembly of the HCV virion, suggesting that Arf1 plays an important role in this process.
The expected final results and their potential impact and use
Our results lead to better understanding of the molecular virology of HCV that assists in identifying new targets for specific antiviral therapies.
Our results were published in the international scientific journals available for open access.