Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles

Abstract

Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP).In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogoldlabeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route.IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV virions with host lipoproteins occurs. Using immunoprecipitation of COPII vesicles and immunogold electron microscopy (EM), we characterize the existence of LVPs that cofractionate with lipoproteins, viral proteins, RNA, and vesicular components. Our results show that this assembly occurs in the ER, and LVPs thus formed are carried through the Golgi network by vesicular transport. This work provides a unique insight into the HCV LVP assembly process within infected cells and offers opportunities for designing antiviral therapeutic cellular targets.

lncRNA HULC facilitates efficient loading of HCV‐core protein onto lipid droplets and subsequent virus‐particle release

“…This indicated that HULC plays an important role in regulating the efficient release of HCV particles. We additionally analysed the viral RNA using quantitative reverse transcription polymerase chain reaction (RT‐qPCR) method as explained earlier (Syed, Khan, Yang, & Siddiqui, ), to reaffirm that the effect observed in the infectivity assay amounts for the effect on HCV release. In support to our hypothesis, we found significant effect on the intracellular and extracellular viral RNA titre, which was in concert with the infectivity assay (Figure S2).…”

Hepatitis B subviral envelope particles use the COPII machinery for intracellular transport via selective exploitation of Sec24A and Sec23B

“…This study is the first to document an essential role of the COPII machinery in HBV's pathogenic life cycle. HBV is yet another example of COPII cargo adaptor takeover by viral pathogens, encompassing VSV (Nishimura & Balch, ), the turnip mosaic virus (Jiang, Patarroyo, Garcia Cabanillas, Zheng, & Laliberte, ), HCV (Syed et al, ), and the Ebola virus (Yamayoshi et al, ). Together, those reports and our study add clues for pathogen‐specific exploitation of special COPII coats for host cell exit with potential implications for cell biology, virology, and antiviral drug designs.…”

“…Pathogens like the hepatitis C virus (HCV) and Ebola virus have been shown to utilize COPII as a transport mechanism for viral proteins en route to viral assembly and budding sites (Syed, Khan, Yang, & Siddiqui, ; Yamayoshi et al, ). Hence, the COPII machinery can become a distinct ally regarding pathogenic morphogenesis and release.…”

Rtn4a promotes exocytosis in mammalian cells while ER morphology does not necessarily affect exocytosis and translation

“…24 h post-nucleofection, medium was removed and cells were scraped into 2.5 ml ice cold PBS (without Ca2+ and Mg2+) supplemented with 10 µg/ml each of leupeptin, pepstatin, and chymostatin. Cells were spun down at 2,000 rpm for 3 min at 4°C and the pellets were resuspended in 1.5 ml ice cold Homogenization Buffer (250 mM Sucrose, 25 mM KCl, 10 mM HEPES pH=7.2, 1 mM EGTA, and 1x Sigma Fast Protease Inhibitor cocktail), as previously described (Syed et al, 2017). Cell suspensions were homogenized by passing through a 25-gauge syringe needle (10 times) and homogenates were centrifuged (600 g for 5 min) twice at 4°C.…”