Hepatitis C virus subgenomic replicon requires an active NS3 RNA helicase

Abstract

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 (Deltahel), 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 Deltahel, 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.

FIG. 1.

(A) Schematic representation of the Con1 strain blasticidin-selectable subgenomic replicon S2204I-Bsd. The HCV 5′ UTR is fused to the HCV IRES sequence, followed by the blasticidin S deaminase gene, the encephalomyocarditis virus (EMCV) IRES, the HCV NS3 to NS5B coding region, and the HCV 3′ UTR. The GDD⁻ variant with a lethal mutation in the NS5B active site (G2737A, D2738A, or D2739G) was used as a negative control. All replicons contain the adaptive mutation (S2204I) within the NS5A region. Site-directed mutagenesis was used to engineer mutations (R393A, F438A, T450I, E493K, W501A, and W501F) into the NS3 helicase region, and their locations relative to the conserved superfamily 2 helicase motifs are indicated. (B) In vitro-transcribed HCV RNA replicons analyzed on a 1% agarose gel with λ DNA digested with HindIII as markers (lane m). Lane 1, S2204I-Bsd; lane 2, GDD⁻; lane 3, Δhel; lane 4, R393A; lane 5, F438A; lane 6, T450I; lane 7, E493K; lane 8, W501A; lane 9, W501F.

FIG. 2.

Colony formation efficiencies of cells transfected with replicons containing either wild-type NS3 helicase or its mutants. (A) Huh-7.5 cells containing S2204I-Bsd, Δhel, GDD⁻, R393A, F438A, T450I, E493K, W501A, or W501F replicons. Cells were initially plated at a density of 6 × 10⁵ cells/dish. (B) Huh-7.5 cells containing S2204I-Bsd, T450I, and W501F replicons were diluted with cells containing GDD⁻ replicons 10-fold (6 × 10⁴ cells plus 5.4 × 10⁵ GDD⁻ cells), 100-fold (6 × 10³ cells plus 5.94 × 10⁵ GDD⁻ cells), and 1,000-fold (6 × 10² cells plus 5.994 × 10⁵ GDD⁻ cells). Blasticidin-resistant colonies were stained with crystal violet after 3 weeks of antibiotic selection.

FIG. 3.

Intracellular HCV RNA. Total RNA was extracted from cells at the indicated time points and reverse transcribed into cDNA using primers annealing either to the plus or minus strand of the HCV 5′-UTR region. Quantification of HCV RNA was determined by using real-time PCR. (A) HCV plus-strand RNA in cells containing either wild-type helicase or its mutants. (B) HCV minus-strand RNA in cells containing either wild-type helicase or its mutants. RT-PCR was repeated three times.

FIG. 4.

HCV NS3 and NS5A proteins in Huh-7.5 cells containing replicons. Twenty micrograms of HCV RNA was electroporated into Huh-7.5 cells. At 4 h, cells were washed with PBS, scraped off the plates, lysed with 5% SDS, and analyzed with a 10% SDS gel. Antibodies against either (A) NS3 or (B) NS5A proteins were used for immunoblotting. Blots were stripped and reprobed with a β-actin antibody.

FIG. 5.

Long-term effects of the T450I and W501F mutations. (A) Amount of plus-strand HCV RNA detected per microgram of RNA isolated at different times from cells transfected with S2204I-Bsd, T450I, or W501F replicons. (B) Western blot analysis of NS3 and NS5A protein expression in Huh-7.5 cells 25 days after electroporation. In both panels Huh-7.5 cells without replicons that were grown over the same period of time (without blasticidin) are shown for comparison.