Hepatitis C virus genomic RNA dimerization is mediated via a kissing complex intermediate

Abstract

With over 200 million people infected with hepatitis C virus (HCV) worldwide, there is a need for more effective and better-tolerated therapeutic strategies. The HCV genome is a positive-sense; single-stranded RNA encoding a large polyprotein cleaved at multiple sites to produce at least ten proteins, among them an error-prone RNA polymerase that confers a high mutation rate. Despite considerable overall sequence diversity, in the 3'-untranslated region of the HCV genomic RNA there is a 98-nucleotide (nt) sequence named X RNA, the first 55 nt of which (X55 RNA) are 100% conserved among all HCV strains. The X55 region has been suggested to be responsible for in vitro dimerization of the genomic RNA in the presence of the viral core protein, although the mechanism by which this occurs is unknown. In this study, we analyzed the X55 region and characterized the mechanism by which it mediates HCV genomic RNA dimerization. Similar to a mechanism proposed previously for the human immunodeficiency 1 virus (HIV-1) genome, we show that dimerization of the HCV genome involves formation of a kissing complex intermediate, which is converted to a more stable extended duplex conformation in the presence of the core protein. Mutations in the dimer linkage sequence loop sequence that prevent RNA dimerization in vitro significantly reduced but did not completely ablate the ability of HCV RNA to replicate or produce infectious virus in transfected cells.

FIGURE 1.

(A). Schematic representation of HCV genome. (B) Alternate predicted conformations of X RNA obtained by Mfold. The DLS region is highlighted in red. The mutations introduced in the X98_G30_31AP are shown in blue and red, respectively.

FIGURE 2.

Model for the kissing complex formed by the X55 RNA (top) and its proposed conversion into the more thermodynamically stable duplex conformation (bottom) in the presence of the core 2BD peptide. The two X55 RNA molecules are identical, but they are illustrated in different colors for clarity purposes.

FIGURE 3.

(A, right) TBM native gel of X55 RNA (20 μM) at different MgCl₂ concentrations: 1 mM (lane 1), 5 mM (lane 2), and 10 mM (lane 3). Both the gel and the running buffer contained 1 mM MgCl₂. (Left) TBE native gel with no magnesium: Abnova RNA marker R0001 (lane 1) and wild-type X55 RNA (lane 2). (B, right) TBM gel containing 10 mM MgCl₂. Both lanes contain X55 RNA annealed, treated with 10 mM MgCl₂ and incubated for 2.5 h at 55°C (lane 1) and 22°C (lane 2), respectively. (Left) TBE native gel with no magnesium. Both lanes contain X55 RNA annealed, treated with 10 mM MgCl₂ and incubated for 2.5 h at 55°C (lane 1) and 22°C (lane 2), respectively.

FIGURE 4.

(A) RNA samples used in the fluorescence spectroscopy assay to detect kissing complex formation (the samples were 55 nt, but only the DLS region with the indicated mutations highlighted in red, blue, and green is shown [top]) and their proposed kissing interactions (bottom). (B) TBM native gel showing that neither X55_G30_31AP RNA (lane 1) nor X55_C31 RNA (lane 2) is able to homodimerize in the presence of 10 mM MgCl₂, but they heterodimerize when mixed (lane 3), similar to the wild-type X55 RNA (lane 4). (C) Plots of the steady-state fluorescence of X55_G30_31AP (500 nM) in 1 mM cacodylic acid (pH 6.5) and 10 mM MgCl₂, titrated with nanomolar increments of X55_C31 RNA (blue triangles), with a mutant RNA X55_A30_A31, which contains a CAAG loop as opposed to the wild-type CUAG loop (black squares), or with a 21-nt fragment derived from EF1A mRNA (red circles). (D) Plot of the steady-state fluorescence of the full-length X98_G30_31AP RNA (500 nM) in 1 mM cacodylic acid (pH 6.5) and 10 mM MgCl₂, titrated with nanomolar increments of X55_C31. (E) Plot of the steady-state fluorescence of X55_G30_31AP (500 nM) in 1 mM cacodylic acid (pH 6.5) and 10 mM MgCl₂, titrated with nanomolar increments of DLS_C9 RNA.

FIGURE 5.

(A, right) TBM native gel: X55 RNA samples (20 μM) were annealed and treated with 10 mM MgCl₂, followed by incubation at 22°C (lane 1), incubation at 55°C (lane 2), and incubation at 22°C and treatment with 1:2 RNA: core 2BD peptide (lane 3). (Left) TBE native gel: X55 RNA samples (20 μM) were annealed and treated with 10 mM MgCl₂, followed by incubation at 22°C (lane 1), incubation at 22°C, and treatment with 1:2 RNA: core 2BD peptide (lane 2) and incubation at 55°C (lane 3). (B) RNA samples used in the fluorescence spectroscopy assay used to detect the kissing complex to duplex structural isomerization. The inserted 2AP and uracil are highlighted in red, and the loop mutations are highlighted in blue. (C) TBM native gel showing that neither X55_G30_52AP RNA (lane 1) nor X55_C31_U5 RNA (lane 2) is able to homodimerize, but they heterodimerize when mixed in equimolar ratio (lane 3), similar to the wild-type X55 RNA (lane 4). (D) Conversion of the kissing complex conformation to duplex by the 2BD core peptide (black triangles). The curve was fit with equation 2 (Materials and Methods) to determine the structural isomerization rate. Blue squares indicate that the addition of the 2BD core peptide in the absence of MgCl₂ does not quench the 2AP fluorescence. Red circles indicate that no structural isomerization occurs in the absence of the core 2BD peptide.

FIGURE 6.

(A) Native TBM gel showing that wild-type X55 RNA (lane 1) and its DLS loop mutated constructs: the double mutant (DLS_AU) with compensatory mutations can homodimerize (lane 2), whereas the single mutants—DLS_UU (lane 3) and DLS_AA (lane 4) —are not able to homodimerize due to their mismatched loops. (B, left) Normalized GLuc activity in culture supernatants collected at different times after transfection of cells with replication competent genome-length HCV RNA with a wild-type DLS (v3 WT, □), a negative-control RNA with defective RNA-dependent RNA polymerase activity (v3/AAG, ■), and related DLS-mutant RNAs: DLS_AA (○), DLS_UU (△), and DLS_AU (⋄). (Right) Yield of infectious virus released into supernatant fluids of cell cultures transfected with the H77Sv3DLS-UU viral RNA. Virus titers were determined by an infectious focus assay and are reported relative to that released by cells transfected in parallel with the wild-type H77Sv3 RNA (100%). Error bars, range of results obtained in replicate independent experiments.