Abstract
Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7.
Highlights
160 million people are chronically infected with Hepatitis C virus (HCV) [1] and viral persistence is associated with severe liver diseases such as steatosis, cirrhosis and hepatocellular carcinoma [2]
In case of HCV, p7 function is essential for production of infectious progeny and its ion channel activity is well documented in vitro and in cell-based systems
Recent evidence indicated that p7 channel activity dissipates the low pH of the cellular secretory compartment protecting the viral glycoproteins from low pH induced misfolding and inactivation
Summary
160 million people are chronically infected with HCV [1] and viral persistence is associated with severe liver diseases such as steatosis, cirrhosis and hepatocellular carcinoma [2]. HCV is an enveloped virus with a positive-strand RNA genome that encodes a polyprotein of about 3,000 amino acids. The polyprotein is cleaved co- and post-translationally by cellular and viral proteases into 10 mature proteins. The structural proteins are resident in the N-terminal portion of the polyprotein and include the core protein and the envelope glycoproteins E1 and E2.The nonstructural proteins NS3, 4A, 4B, 5A and 5B are encoded on the C-terminal part of the polyprotein, and are components of the viral replicase complex [3]. The p7 and NS2 proteins reside in between structural proteins and NS3-5B. Both are dispensable for replication but necessary for the production of progeny viruses [4,5,6]
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