Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

  • Phan T
  • Beran R
  • Peters C
 et al. 
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The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly. Hepatitis C virus (HCV) is a major cause of acute and chronic liver disease and contributes to the development of hepatocellular carcinoma. HCV is an enveloped, positive-strand RNA virus, the type member of the Hepacivirus genus in the family Flaviviridae (43). HCV exhibits high levels of se-quence diversity that cluster into seven major genotypes and numerous subtypes (21). HCV genomes are 9.6 kb and encode a single long open reading frame of ϳ3,011 codons (43). Translation of this ge-nome produces a large polyprotein that is co-and posttrans-lationally processed by viral and host proteases into 10 distinct products. The N-terminal one-third of the polyprotein encodes the structural proteins, which are thought to compose the virus particle. These include an RNA-binding nucleocapsid protein, core (C), and two viral envelope glycoproteins, E1 and E2. E1 and E2 are type I membrane proteins that coordinately fold into a heterodimer complex (36). The remainder of the ge-nome encodes the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B, which mediate the intracel-lular aspects of the viral life cycle. In addition, a small viro-porin-like protein, p7, resides between the structural and NS genes. HCV encodes two proteases, the NS2-NS3 cysteine autopro-tease and the NS3-NS4A serine protease. The only known substrate of the NS2-NS3 autoprotease is the NS2/3 junction. This enzyme is encoded by the C-terminal 121 amino acids (aa) of NS2, which forms a homodimer with twin composite active sites composed of two residues from one chain and one residue from the other (45). In addition, the serine protease domain of NS3 plays a noncatalytic role in stimulating NS2/3 cleavage (69). Upstream of the cysteine protease domain, the N-termi-nal hydrophobic region of NS2 mediates interaction with cel-lular membranes. While the membrane topology of NS2 is not yet fully known (67, 80), N-terminal cleavage by endoplasmic reticulum-resident signal peptidase and C-terminal cleavage by the cytosolic NS2-NS3 cysteine protease indicate that NS2 likely contains one or three transmembrane (TM) domains. The NS3-NS4A serine protease is encoded by the N-termi-nal domain of NS3 and is responsible for downstream cleav-ages at the NS3/4A, NS4A/B, NS4B/5A, and NS5A/B junc-tions. NS4A, a small (54-aa), membrane-anchored protein, acts as a cofactor for the serine protease activity by helping to complete the chymotrypsin-like fold of NS3 (14, 46). In addi-tion to polyprotein processing, NS3-NS4A serine protease helps to dampen the innate antiviral response by cleaving cel-lular proteins involved in signal transduction (65). The C-terminal region of NS3 encodes an RNA helicase/ NTPase activity that is essential for viral replication, although it is not yet clear which specific step(s) of the replication cycle requires this activity (29, 33). Interestingly, the NS3 serine protease and RNA helicase domains enhance each other's activities, suggesting that proteolysis and RNA replication may be functionally coordinated (5, 6). In addition, NS4A helps to promote RNA-stimulated ATP hydrolysis by the NS3 helicase domain (4). In addition to their role in polyprotein processing, emerging evidence indicates that NS2 and NS3-NS4A participate in virus particle assembly (52). Prior work showed that NS2 is not essential for RNA replication of subgenomic replicons engi-neered to express NS3 through NS5B (44). The first evidence

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  • T. Phan

  • R. K. F. Beran

  • C. Peters

  • I. C. Lorenz

  • B. D. Lindenbach

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