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. 2000 Feb;74(4):2046-51.
doi: 10.1128/jvi.74.4.2046-2051.2000.

Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo

A A Kolykhalov  1 , K Mihalik, S M Feinstone, C M Rice
Affiliations

Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo

A A Kolykhalov et al. J Virol. 2000 Feb.

Abstract

Hepatitis C virus (HCV) infection is a widespread major human health concern. Significant obstacles in the study of this virus include the absence of a reliable tissue culture system and a small-animal model. Recently, we constructed full-length HCV cDNA clones and successfully initiated HCV infection in two chimpanzees by intrahepatic injection of in vitro-transcribed RNA (A. A. Kolykhalov et al., Science 277:570-574, 1997). In order to validate potential targets for development of anti-HCV therapeutics, we constructed six mutant derivatives of this prototype infectious clone. Four clones contained point mutations ablating the activity of the NS2-3 protease, the NS3-4A serine protease, the NS3 NTPase/helicase, and the NS5B polymerase. Two additional clones contained deletions encompassing all or part of the highly conserved 98-base sequence at the 3' terminus of the HCV genome RNA. The RNA transcript from each of the six clones was injected intrahepatically into a chimpanzee. No signs of HCV infection were detected in the 8 months following the injection. Inoculation of the same animal with nonmutant RNA transcripts resulted in productive HCV infection, as evidenced by viremia, elevated serum alanine aminotransferase, and HCV-specific seroconversion. These data suggest that these four HCV-encoded enzymatic activities and the conserved 3' terminal RNA element are essential for productive replication in vivo.

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Figures

FIG. 1
FIG. 1
Diagram of the HCV genome and mutant constructs. All mutant derivatives were constructed on the background of HCV FL, and their structures were verified by sequence analysis. Mutants are described by the nucleotide positions and substitutions (lowercase letters) relative to HCV FL (31); sequences of the oligonucleotides used for mutagenesis, plasmid manipulations, and complete sequence files are available upon request. (A) The HCV genome organization is shown at the top with 5′ and 3′ NTRs (solid lines), and the ORF (open box) and the polyprotein cleavage products are indicated. Mutant full-length clones are shown below, highlighting the regions encoding the four enzymatic activities (shadowed), the positions of the mutations (asterisks), and the construct names (at the left). HCV FL(2-3pro−) contains the amino acid substitutions H952A (3195 to 3200; gcgtTa) and C993A (3318 to 3319; gc). HCV FL(3pro−) contains the substitutions D1107A (3660 to 3664; gcctt) and S1165A (3831 to 3836; agCgCt). HCV FL(hel−) contains the substitutions D1316A (4286 to 4289; cGca) and E1317A (4291 to 4292; ca). HCV FL(pol−) contains the substitutions G2737A (8551 to 8552; cg), D2738A (8554; c), and D2739G (8557 to 8559; gCc). (B) Organization of the 3′ portion of HCV genome RNA showing (5′ to 3′) the C-terminal part of the ORF (open box), the polyprotein translation termination codon (UGA), the variable part of the 3′ NTR (solid straight line), the poly(U/UC) tract, the highly conserved 52-base sequence (curved line), and the 3′ terminal 46-base stem-loop structure (SL I). Mutant clones are shown below with their corresponding names to the right. HCV FL(3′Δ52) is identical to HCV FL, except for an internal deletion encompassing the 5′ 52 bases of the 3′ terminal 98-base sequence. For HCV FL(3′Δ98), the entire 3′ 98-base sequence was deleted. A novel restriction site (NsiI) distinguishing HCV FL(3′Δ52) from HCV FL(3′Δ98) is indicated. nucl., nucleotides.
FIG. 2
FIG. 2
Polyprotein processing by HCV FL and mutant derivatives. Transfection of HCV FL (wt) or mutant plasmid DNAs (indicated at the top), protein labeling, immunoprecipitation of 35S-labeled proteins with HCV-specific sera, and the analysis of the immune complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were conducted as described previously (32). pGEM3Zf(+) was transfected as a negative control (mock). Products were immunoprecipitated with patient serum JHF recognizing NS3, NS4A, NS4B, and NS5A (A and B) or rabbit anti-NS5B (C); separated by sodium dodecyl sulfate–12 or 9% polyacrylamide gel electrophoresis, respectively; and visualized by autoradiography. The positions of molecular weight markers are shown on the left (in kilodaltons); HCV-specific polyproteins and cleavage products are identified on the right. The ∼21-kDa species which is observed in the HCV FL(3pro−)-NS3181 cotransfection is an N terminally truncated form of the NS4B protein (32). The values on the left are molecular sizes in kilodaltons.
FIG. 3
FIG. 3
Analysis of Ch1552 samples. RNA transcription and inoculation of RNAs into the liver were performed as described previously (31). For each RNA, approximately 150 μg of RNA in phosphate-buffered saline (PBS) was injected into two separate sites, and 1 μg of an RNA-Lipofectin-PBS mixture was also injected at two separate sites. Ch1552 was challenged at week 32 with infectious HCV FL RNA transcripts using nonsurgical procedures. One hundred micrograms of RNA in 1 ml of PBS was injected into the liver percutaneously through a biopsy needle. Three additional intrahepatic injections of 100 μg of RNA in 1 ml of PBS per injection were administered with a lumbar puncture needle. A fifth lumbar puncture needle injection was performed with 3 μg of RNA mixed with 30 μl of Lipofectin and PBS in a total volume of 0.5 ml. (A) Serum ALT, HCV RNA (molecules per milliliter), and HCV-specific antibodies (Ab; as measured by HCV ELISA 3.0). (B) Detection of HCV-specific antibodies by Ortho HCV version 3.0 ELISA and by Chiron RIBA 2.0. For the RIBA 2.0, the open box indicates HCV-seronegative serum samples and the solid bar (beginning at week 41) indicates positive samples. OD490, optical density at 490 nm.
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