Regulation of HCV Cell-to-Cell Spread by ADP-ribosylation factor 1-Dependent Regulation of Cytosolic Lipid Droplet Homeostasis

• Author / Creator

  Awadh, Abdullah

• Hepatitis C virus (HCV) infects cells via cell-free virions by direct cell-to-cell transmission. Entry of cell-free HCV virions has been fairly well-characterized. Several host factors involved in this process have been identified, including the cell surface receptors, tight junction molecules, and lipid transport proteins. HCV cell-to-cell spread has not been equally characterized, beyond the identification of the involvement of a subset of the host factors involved. The relative importance of infection via cell-free virions or by direct cell-to-cell transmission remained unaddressed. Similarly, whether the release of virions into the extracellular space and the direct cell to cell transmission followed the same pathways or not was unknown. In collaboration with Dr. Novikov, we identified and characterized the mechanism of action of small molecule derivatives of a substituted uracil. An optimized derivative, Z390, inhibited HCV foci formation with no effect on a panel of related or unrelated viruses. Z390 inhibited HCV cell-to-cell spread without affecting HCV replication, assembly, or release of infectious virions, or susceptibility or permissivity of cells to HCV infection. Z390 altered the intracellular distribution of HCV core and NS5A proteins and the homeostasis of the cellular cytosolic lipid droplets (cLDs). Oleic acid (OA), which has the same effect as Z390 on cLDs homeostasis, also reproduced the effect of Z390 on HCV protein localization and HCV cell-to-cell spread. Knockdown of Arf1 had been shown before to mimic the phenotype of Z390 and OA on HCV proteins and cLDs. Knockdown of Arf1 inhibited HCV cell-to-cell spread, and expression of Arf1 non-cycling mutants mimicked the effects of Z390 on NS5A and core colocalization, on cLDs size and distribution and on cell-to-cell spread. Using Z390, we identified a mechanism of direct HCV cell-to-cell spread involving cellular cLDs and Arf1. We propose that regulation of cLD homeostasis by Arf1 cycling determines the preferred pathway of HCV spread, either via the release of cell-free virions or by direct cell-to-cell spread.

• Subjects / Keywords

  • HCV
  • cell-to-cell spread
  • lipid droplet
  • Arf1

• Graduation date

  Spring 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3154F431

• License

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