Date Published: January 4, 2016
Publisher: Public Library of Science
Author(s): Margarita Zayas, Gang Long, Vanesa Madan, Ralf Bartenschlager, Timothy L. Tellinghuisen.
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.
The hepatitis C virus (HCV) is a major causative agent of chronic liver diseases, affecting ~170 million people worldwide. HCV infection is frequently asymptomatic, however persistently infected people have a high risk to develop serious liver diseases including liver cirrhosis and hepatocellular carcinoma . HCV belongs to the genus Hepacivirus within the family Flaviviridae, which is a group of enveloped viruses with a single strand RNA genome of positive polarity. The HCV genome has a length of ~9,600 nucleotides and contains one long open reading frame that is flanked at the 5’ and 3’ end by non-translated regions required for RNA translation and replication, respectively . The open reading frame encodes a polyprotein precursor of ~3,000 amino acids that is translated at the rough endoplasmic reticulum (ER). Polyprotein cleavage occurs co- and post-translationally and is mediated by cellular and viral proteases giving rise to at least 10 different products: three structural proteins that build up the virus particle [core, envelope protein 1 (E1) and E2], and seven nonstructural proteins, i.e. p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. The viroporin p7 and the cysteine protease NS2 are not essential for RNA replication, but are required for the assembly of infectious HCV particles [3–6]. The remaining proteins NS3 to NS5B form a membrane-associated replicase complex catalyzing viral RNA replication .
Assembly of HCV particles requires a spatio-temporally coordinated association of the replicase, notably the NS3 helicase and NS5A, with the core protein to allow packaging of the RNA genome into the virion [11, 19]. We and others have earlier shown that NS5A plays a decisive role in this process. First, it interacts with core protein via NS5A DIII [30, 31]; second, NS5A is recruited to cLDs where core protein accumulates [9, 30, 31]; third, assembly requires the phosphorylation of a serine residue in NS5A DIII by casein kinase IIα ; fourth, NS5A facilitates the association of core protein with the viral RNA genome ( and this report); fifth, NS5A interacts with the p7-NS2 complex that is required for the envelopment of the HCV particles ; sixth, NS5A also interacts with apolipoprotein E that is incorporated into the virion [18, 48–52] and with Annexin A2, which is a host cell membrane sorting protein enhancing HCV assembly .