Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis

Abstract

Hepatitis C virus (HCV) is a unique enveloped virus that assembles as a hybrid lipoviral particle by tightly interacting with host lipoproteins. As a result, HCV virions display a characteristic low buoyant density and a deceiving coat, with host-derived apolipoproteins masking viral epitopes. We previously described methods to produce high-titer preparations of HCV particles with tagged envelope glycoproteins that enabled ultrastructural analysis of affinity-purified virions. Here, we performed proteomics studies of HCV isolated from culture media of infected hepatoma cells to define viral and host-encoded proteins associated with mature virions. Using two different affinity purification protocols, we detected four viral and 46 human cellular proteins specifically copurifying with extracellular HCV virions. We determined the C terminus of the mature capsid protein and reproducibly detected low levels of the viral nonstructural protein, NS3. Functional characterization of virion-associated host factors by RNAi identified cellular proteins with either proviral or antiviral roles. In particular, we discovered a novel interaction between HCV capsid protein and the nucleoporin Nup98 at cytosolic lipid droplets that is important for HCV propagation. These results provide the first comprehensive view to our knowledge of the protein composition of HCV and new insights into the complex virushost interactions underlying HCV infection. Hepatitis C virus (HCV) is a positive-sense ssRNA virus of the Flaviviridae family. This bloodborne pathogen causes chronic liver infection that develops into cirrhosis and hepatocellular carcinoma and is the leading indication for liver transplantation. Over 185 million people are chronically infected with HCV (2). Despite great advances in the ability to study this virus in vitro, significant gaps remain in our understanding of the infectious particle and the virus-host interactions required for HCV propagation.The protein composition of HCV is not known. Although the capsid protein, Core, and the E1 and E2 envelope glycoproteins are thought to be the major constituents of the virion, it remains to be determined if nonstructural viral proteins are packaged as well. A growing body of literature suggests that cellular proteins are important components of HCV. Indeed, this virus closely associates with LDL and very-LDL components, forming a chimeric lipoviral particle (LVP). Biochemical and ultrastructural studies demonstrated that infectious HCV particles are coated with endogenous apolipoproteins that play key roles in viral attachment and entry, explaining the higher infectivity of lipoprotein-associated HCV (2).The heterogeneous size and appearance of extracellular HCV, ranging from 40 to >100 nm in diameter, suggests that the set of associated proteins (both viral and cellular), as well as their stoichiometry, might vary across the virion population. Additionally, the specific infectivity of HCV changes according to the cell system/host that the virus is produced in, highlighting a stro...

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

“…The highly infectious purified particles have a diameter of 81 to 85 nm and display a complex internal structure (18). A proteomic analysis of cellular proteins associated with HCV virions purified by using neutralizing antibody AR3A showed that ApoE, ApoB-100, ApoA-II, ApoC-II, and ApoC-III are associated with purified HCV virions (48). Overall, these findings strongly support the notion that HCV virions are assembled and secreted as LVPs.…”

“…The interaction of the HCV envelope glycoproteins E1 and E2 with ApoB and ApoE at the ER hints that the glycoproteinapolipoprotein complex could initiate the formation of hybrid lipoviroparticles (50). The presence of ApoB, along with other exchangeable apolipoproteins such as ApoE, ApoA-II, ApoC-II, and ApoC-III, on HCV virions emphasizes its role in HCV maturation and egress (18,48). In correlation, immunogold electron microscopy clearly indicated the presence of ApoB, although in small amounts, and ApoE along with the HCV structural proteins in COPII vesicles, suggesting that the viral particles that bud from the ER are associated with ApoB and ApoE (Fig.…”

The roles of the nuclear pore complex in cellular dysfunction, aging and disease

“…The nuclear basket nucleoporin Nup153, which plays direct roles in importin alpha/beta mediated protein import [96], binds to the HIV capsid and knockdown of this nucleoporin reduces capsid entry [97]. In a proteomic analysis of HCV capsid interacting proteins, Nup98 was identified as critical for HCV entry into the nucleus and the subsequent propagation of the virus [98]. A nucleoporin found on both faces of the NPC, hCG1 [99], binds to the HIV protein Vpr and this interaction aids in the nuclear import of the viral protein [100].…”

Conversion of iPS derived hepatic progenitors into scalable, functional and developmentally relevant human organoids using an inverted colloidal crystal poly (ethylene glycol) scaffold engineered from collagen-coated pores of defined size

“…Knock down HCVcc is the corresponding polymerase-defective mutant that inhibits viral replication. Viral stocks were prepared using electroporation on Huh 7.5 cells as described [14]. Infectious titers of HCVcc inocula were determined using Huh 7.5 cells as described.…”