Mechanism of Hepatitis C Virus secretion from Endoplasmic Reticulum Kiran Avula, Bharati Singh, Gulam H Syed Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India.
Abstract: Hepatitis C virus (HCV) affects ~300 million people worldwide and is most common etiological agent for hepatocellular carcinoma. Uniquely HCV is dependent on host lipids and lipid pathways for its propagation. HCV exists as a lipoprotein-virus hybrid lipoviral particle (LVP) both in cell culture and in patient sera. However, the understanding on how the LVP is formed and secreted is still rudimentary. In this study we trying to unravel the molecular mechanisms pertinent to HCV lipoviral particle (LVP) morphogenesis and secretion to identify crucial pro-viral host proteins that can serve as potential targets to develop antiviral strategies. The regular ER cargo exported from the ER to the Golgi is about 50-60nm in diameter, however VLDL and HCV-LVP are ≥ 100-120nm in diameter and represent atypical large cargo originating in the ER. Recent studies on export of large cargo from ER suggest the presence of protein machinery that can regulate the size of the COPII vesicle commensurate with the size of cargo. It is likely that HCV-LVP also exploits the same machinery to modulate the COPII vesicle size for it transport from ER-Golgi. Proteins like TANGO, TANGO-related protein and cTAGE were identified as key players in the export of large collagen oligomers and E3-ubiquitin ligases like KLHL12 and Cul3, regulate COPII vesicle size via ubiquitination of Sec proteins We are exploring if HCV exploits these machineries for its secretion and will also characterize the significance of ER exit sites and Golgi fragmentation in HCV egress. Objectives: Explore if HCV modulates COP-II vesicle size commensurate with size of HCV-LVP and determine the underlying mechanisms Determine the significance of ERGIC in facilitating the ER export of HCV-LVP. Evaluate if HCV remodels ER export sites to promote HCV-LVP secretion Elucidate the mechanisms associated with HCV-mediated Golgi fragmentation and determine its significance in HCV secretion
Fig 1: A) Model depicting the formation of HCV Lipoviral Particle (HCV-LVP) in the ER lumen. B) Model depicting regulation of COP-II vesicle size proportionate to the size of cargo
Fig 2:Determine the role of COPII specific proteins in HCV secretion. Huh7 cells were infected with HCV JC1 at 3 MOI and 48h post infection the cells were transfected with respective siRNAs.48 hours post transfection culture supernatants as well as cell lysates were collected for determination of intracellular as well as extracellular viral load by qRT-PCR (A). Efficiency of gene silencing was determine by Western blot analysis of respective proteins (B)
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Fig3:HCV promotes autophagy mediated degradation of ERES :(a) western blot data suggest HCV downregulates sec16a,ERESmaintenance protein,(B)addition of autophagy inhibitor baffalomycine A rescue it expression in infected cells suggest it undergoes autophagy degradation but no effect on ERGIC-53,(c) Immunofluorescence images shows HCV promotes ERES to become more constrictive pattern compared to mock infected cells wich shows regular reticulate manner , (c) percentage of cells showing constrictive ERES in HCV infected or mock infected cells, (d) number of cells having perinuclear region(exclusion of ERES)in HCV infected or Mock infected cells. 4a
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Fig4(a)HCV promotes both cis-trans Golgi fragmentation :Huh7 cells infected with HCV and Mock, after 60h post infection cells were fixed and stained with TGN46(trans Golgi network protein)and GM-130 (cis Golgi protein ), (b)quantitative analysis of different forms of Golgi network ( regular, fragmented and constrictive) showing with every 6hrs of post infection compared with mock infected cells. Preliminary Observations: HCV downregulates the expression of COP-II coat proteins suggesting a overall reduction in global secretion HCV secretion is not affected by the modest changes in expression of COP II coat proteins during infection. Known COPII vesicle size regulators,CUL3,KLHL12,Ctage5 mia3 have strong role HCV replication, morphogenesis and secretion. HCV promotes autophagy degradation of ERES maintenance protein called SEC16A,and also induce more constrictive pattern of ERES, knockdown of SEC16A shows effect on HCV secretion. HCV promotes both cis-trans Golgi fragmentation. Future Direction: • COP-II proteome analysis to identify proteins that can contribute in secretion of HCV-LVP. • Explore the significance of Golgi fragmentation and ER-secretion-autophagy crosstalk in HCV-LVP secretion. • Determine the significance of ERGIC in facilitating the ER export of HCV-LVP. We acknowledge the funds from DST-SERB and the intramural funds from ILS Bhubaneswar.