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. 2001 Mar;75(5):2119-29.
doi: 10.1128/JVI.75.5.2119-2129.2001.

Self-assembly of nucleocapsid-like particles from recombinant hepatitis C virus core protein

M Kunkel  1 , M Lorinczi, R Rijnbrand, S M Lemon, S J Watowich
Affiliations

Self-assembly of nucleocapsid-like particles from recombinant hepatitis C virus core protein

M Kunkel et al. J Virol. 2001 Mar.

Abstract

Little is known about the assembly pathway and structure of hepatitis C virus (HCV) since insufficient quantities of purified virus are available for detailed biophysical and structural studies. Here, we show that bacterially expressed HCV core proteins can efficiently self-assemble in vitro into nucleocapsid-like particles. These particles have a regular, spherical morphology with a modal distribution of diameters of approximately 60 nm. Self-assembly of nucleocapsid-like particles requires structured RNA molecules. The 124 N-terminal residues of the core protein are sufficient for self-assembly into nucleocapsid-like particles. Inclusion of the carboxy-terminal domain of the core protein modifies the core assembly pathway such that the resultant particles have an irregular outline. However, these particles are similar in size and shape to those assembled from the 124 N-terminal residues of the core protein. These results provide novel opportunities to delineate protein-protein and protein-RNA interactions critical for HCV assembly, to study the molecular details of HCV assembly, and for performing high-throughput screening of assembly inhibitors.

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Figures

FIG. 1
FIG. 1
Sequence analysis of HCV-1a core protein. (A) Amino acid sequence of full-length core protein, highlighting conserved basic, positively charged clusters (∗) at residues 2 to 23, 39 to 74, and 101 to 121. (B) Kyte-Doolittle hydrophobicity plot of core residues 1 to 191, using ProtScale tool (24). (C) Schematic of HCV core constructs HCVC 124 and HCVC 179 with predicted secondary structure determined using the PHD method (33, 34). □, random coil; ■, extended strand; formula image, alpha helix.
FIG. 2
FIG. 2
HCV core expression and purification. All samples were resolved on an SDS–16% polyacrylamide gel under reducing conditions and visualized by Coomassie blue stain. Lanes: A, expression of HCVC 124 in lysed bacterial cell pellets; B, purified HCVC 124; C, expression of HCVC 179 in lysed bacterial cell pellets; D, purified HCVC 179. Sizes are indicated in kilodaltons.
FIG. 3
FIG. 3
Sequences and secondary structure of nucleic acids used for assembly. (A) Secondary structure within the complete 5′UTR of HCV and immediately downstream open reading frame (20). Numbered nucleotides indicated boundaries of the RNA constructs used in the assembly reactions. (B) Linear representation showing extents and locations of the RNA constructs used in the assembly reactions. (C) Sequences of ssDNA oligonucleotides used in the assembly reactions.
FIG. 4
FIG. 4
Two percent agarose gel showing folded or unfolded state of nucleic acids. Samples were loaded with 10 μg of ethidium bromide/ml in gel loading buffer. Marker hatches on left are in kilobases. Lanes: A, folded tRNA in H2O; B, unfolded tRNA in 100 mM EDTA; C, folded 5′UTR II in H2O; D, unfolded 5′UTR II in 100 mM EDTA.
FIG. 5
FIG. 5
Micrographs of negatively stained samples. All samples were incubated as described in Materials and Methods. (A) HCVC 124; (B) tRNA; (C) HCVC 124 and tRNA in 10:1 ratio; (D) immunogold-labeled HCVC 124 and tRNA particles from 10:1 ratio sample; (E) HCVC 124 and 5′UTR II/III/IV in 10:1 ratio; (F) HCVC 124 and 5′UTR in 10:1 ratio. Bars, 100 nm.
FIG. 6
FIG. 6
Histograms representing the frequency distribution of particle diameters in assembly experiments of HCVC 124 with various nucleic acids. (A) 5′UTR HCV; (B) 5′UTR HCV II/III/IV; (C) 5′UTR HCV II; (D) 5′UTR HCV III/IV; (E) tRNA.
FIG. 7
FIG. 7
Micrographs of negatively stained samples of HCVC 179 incubated as described in Materials and Methods. (A) HCVC 179; (B) HCVC 179 and tRNA in 10:1 ratio, showing irregular particle formation. Bars, 100 nm.
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