Hepatitis C virus NS3 RNA helicase activity is modulated by the two domains of NS3 and NS4A

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169

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, positivestrand RNA virus, the type member of the Hepacivirus genus in the family Flaviviridae (43). HCV exhibits high levels of sequence 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 genome produces a large polyprotein that is co-and posttranslationally 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 genome encodes the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B, which mediate the intracellular aspects of the viral life cycle. In addition, a small viroporin-like protein, p7, resides between the structural and NS genes.HCV encodes two proteases, the NS2-NS3 cysteine autoprotease 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-terminal hydrophobic region of NS2 mediates interaction with cellular membranes. While the membrane topology of NS2 is not yet f...

Section: Resultsmentioning

confidence: 99%

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

Beran

Peters

et al. 2009

117

169

“…NS3 is important for RNA replication (27,31), modulating innate antiviral defenses (33,36,42,47), and has been implicated in virus particle assembly (45,55). NS4A is a small protein that binds NS3, functions as a cofactor for NS3 serine protease and RNA helicase activities, and anchors the NS3-4A enzyme complex to cellular membranes (6,13,28,73). NS4B is a multispanning membrane protein that is important for organizing the membrane-bound viral RNA replication machinery (reviewed in reference 17).…”

mentioning

confidence: 99%

Hepatitis C Virus NS2 Coordinates Virus Particle Assembly through Physical Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

Stapleford

Lindenbach

2011

122

132

The hepatitis C virus (HCV) NS2 protein is essential for particle assembly, but its function in this process is unknown. We previously identified critical genetic interactions between NS2 and the viral E1-E2 glycoprotein and NS3-NS4A enzyme complexes. Based on these data, we hypothesized that interactions between these viral proteins are essential for HCV particle assembly. To identify interaction partners of NS2, we developed methods to site-specifically biotinylate NS2 in vivo and affinity capture NS2-containing protein complexes from virus-producing cells with streptavidin magnetic beads. By using these methods, we confirmed that NS2 physically interacts with E1, E2, and NS3 but did not stably interact with viral core or NS5A proteins. We further characterized these protein complexes by blue native polyacrylamide gel electrophoresis and identified Ϸ520-kDa and Ϸ680-kDa complexes containing E2, NS2, and NS3. The formation of NS2 protein complexes was dependent on coexpression of the viral p7 protein and enhanced by cotranslation of viral proteins as a polyprotein. Further characterization indicated that the glycoprotein complex interacts with NS2 via E2, and the pattern of N-linked glycosylation on E1 and E2 suggested that these interactions occur in the early secretory pathway. Importantly, several mutations that inhibited virus assembly were shown to inhibit NS2 protein complex formation, and NS2 was essential for mediating the interaction between E2 and NS3. These studies demonstrate that NS2 plays a central organizing role in HCV particle assembly by bringing together viral structural and nonstructural proteins.

“…HCV helicase is composed of the C-terminal two-thirds of NS3. Although the N-terminal protease region, which is responsible for processing HCV proteins NS3 through NS5B, is not absolutely required for unwinding, it facilitates the interaction of NS3 and RNA and accelerates helicase action (6,8,15,36). Structurally (3,12,34,35), HCV helicase is a three-domain protein that shares several conserved motifs with other related cellular and viral helicases and helicase-like motor proteins, all of which are located in two N-terminal helicase domains (domains 1 and 2).…”

mentioning

confidence: 99%

Hepatitis C Virus Subgenomic Replicon Requires an Active NS3 RNA Helicase

Lam

Frick

2006

108

Mutations were introduced into the NS3 helicase region of a hepatitis C virus (HCV) Con1 subgenomic replicon to ascertain the role of the helicase in viral replication. One new replicon lacked two-thirds of the NS3 helicase (⌬hel), and six others contained one of the following six amino acid substitutions in NS3: R393A, F438A, T450I, E493K, W501A, and W501F. It has been previously reported that purified R393A, F438A, and W501A HCV helicase proteins do not unwind RNA but unwind DNA, bind RNA, and hydrolyze ATP. On the other hand, previous data suggest that a W501F protein retains most of its unwinding abilities and that purified T450I and E493K HCV helicase proteins have enhanced unwinding abilities. In a hepatoma cell line that has been cured of HCV replicons using interferon, the T450I and W501F replicons synthesized both negative-sense and positive-sense viral RNA and formed colonies after selection with similar efficiencies as the parent replicon. However, the ⌬hel, R393A, F438A, and W501A replicons encoded and processed an HCV polyprotein but did not synthesize additional viral RNA or form colonies. Surprisingly the same phenotype was seen for the E493K replicon. The inability of the E493K replicon to replicate might point to a role of pH in viral replication because a previous analysis has shown that, unlike the wild-type NS3 protein, the helicase activity of an E493K protein is not sensitive to pH changes. These results demonstrate that the RNA-unwinding activity of the HCV NS3 helicase is needed for RNA replication.Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus with a 9.6-kilobase genome that encodes one long polypeptide, which is processed into at least 10 structural and nonstructural (NS) proteins. The structural proteins form the viral capsid and its glycoproteins, while the NS proteins are responsible for the replication of the viral genome. Among the HCV replicative proteins, the NS3 protease/helicase is one of the best characterized. However, the biological role that the helicase portion of NS3 plays during the replication cycle of the virus still remains largely unclear. HCV helicase is composed of the C-terminal two-thirds of NS3. Although the N-terminal protease region, which is responsible for processing HCV proteins NS3 through NS5B, is not absolutely required for unwinding, it facilitates the interaction of NS3 and RNA and accelerates helicase action (6,8,15,36). Structurally (3,12,34,35), HCV helicase is a three-domain protein that shares several conserved motifs with other related cellular and viral helicases and helicase-like motor proteins, all of which are located in two N-terminal helicase domains (domains 1 and 2). The C-terminal domain (domain 3) contains no motifs conserved with other helicases, and structurally similar domains have not been seen in related cellular proteins. One strand of nucleic acid binds in the cleft between domain 3 and the first two domains, and ATP likely binds in the cleft separating domains 1 and 2. It is not clear where the complementa...