Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

Abstract

Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.

Spatiotemporal Coupling of the Hepatitis C Virus Replication Cycle by Creating a Lipid Droplet- Proximal Membranous Replication Compartment

“…processes; therefore, one would not expect a distinct nucleocapsid-like structure (Zayas et al, 2016). We employed several other approaches, including electron tomography of Jc1 wildtype infected cells, cells treated with brefeldin A to allow accumulation of intracellular viral particles (Gastaminza et al, 2008), and immuno-EM.…”

“…Quantification of virus titers and HCV core protein amounts Virus titers were determined by limiting-dilution assay using Huh7.5 target cells (Blight et al, 2002) and staining of the NS5A protein with the 9E10 monoclonal antibody as described elsewhere (Zayas et al, 2016). Titers of infectious virus are expressed as 50% tissue culture infective dose (TCID 50 ) per mL.…”

“…Quantitative detection of HCV RNA by RT-qPCR Total RNA was extracted from suspensions of transfected cells using a NucleoSpin RNA extraction kit (Macherey-Nagel) according to the instructions of the manufacturer. Amplifications were performed in triplicate with a BioRad CFX96TM Real-Time System/ C1000 Thermal cycler using a One Step RT-PCR Kit, primers, 6FAM-and TAMRA-labeled HCV-specific probes (TIB Molbiol, Berlin, Germany) and a VIC-and TAMRA-labeled probe for the house keeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Applied Biosystems) as described recently (Zayas et al, 2016). Numbers of HCV RNA copies were calculated by comparison to serially diluted in vitro transcripts included in parallel in the RT-qPCR analysis.…”

The history of hepatitis C virus (HCV): Basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control

“…Overall, it has been found that the amphipathic alpha-helix and domains I and II are essential for HCV RNA replication [224,225,230,231]; a recent study showed a critical role for generation of double-membrane vesicles associated with replication [232]. In addition, domain III has a primary role in production of infectious particles with a direct role in coordinating viral assembly [233][234][235].…”

Perilipin-2 is critical for efficient lipoprotein and hepatitis C virus particle production

“…Hindering trafficking of core or NS5A to LDs either by mutation or by inhibition of critical host factors results in decreased infectious particle production Herker et al, 2010;Liefhebber et al, 2014;Menzel et al, 2012;Miyanari et al, 2007). Vice versa, cells infected with mutant HCV viruses defective in virion morphogenesis often accumulate core at LDs (Gentzsch et al, 2013;Zayas et al, 2016). To investigate whether LD localization of viral proteins is affected by PLIN2 deficiency, we first analyzed levels of core protein in LD fractions from HCV-infected cells.…”