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

Significance Virion proteomics represents a powerful and unbiased approach to gain insights into the process of virus assembly and the host factors important for infection. For hepatitis C virus (HCV), it is known that infectious virions are assembled alongside endogenous lipoproteins to give rise to a chimeric lipoviral particle. Host-derived apolipoproteins coat the exterior of circulating particles, making for a veiled pathogen. The protein composition of HCV remains, however, undefined. Here, using MS, we identified novel viral and cellular proteins specifically associated with HCV virions and discovered an essential interaction between the capsid protein and the nucleoporin Nup98 that is required for virion biogenesis. 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 virus–host interactions underlying HCV infection.