Hepatitis C Virus RNA Codes for Proteins and Replicates: Does It also Trigger the Interferon Response?

ABSTRACT

Hepatitis C virus (HCV) is a positive sense virus with a genomic RNA molecule roughly 9,600 nucleotides in length. The single-stranded genomic RNA has a nontranslated region (NTR) at each end and a long open reading frame (coding region) in between. The 5′NTR and portions of the 3′NTR are the most conserved parts of HCV RNA. These conserved regions contain signals for replication and translation. Much of the 5′NTR is folded into a structure that binds ribosomes. This structure, an internal ribosome entry site, promotes the initiation of protein synthesis and is critical for HCV gene expression. The ribosome binding site may extend into the coding region; its exact boundaries are not known. The open reading frame encodes the HCV polyprotein, which is slightly more than 3,000 amino acids in length. The 3′NTR plays a key role in HCV replication and may also influence the rate of HCV protein synthesis. During replication, the genomic RNA is copied by virally encoded enzymes into a complementary antigenomic RNA, which itself is a template for the synthesis of progeny RNAs. At steady state, genomic strands outnumber antigenomic strands about 10 to 1. HCV RNA replication is thought to take place in the cytoplasm and is an error-prone process. It generates a mixed population of RNA sequences (quasispecies), including mutants that may be more fit than the parental type, less fit, or equally fit (but distinct). Natural selection acts upon the progeny RNAs, causing the population to change and drift. Over time, mutation, selection, and population bottlenecks led to the evolution of varied genotypes. The HCV replication complex is a potential source of double-stranded RNA, a powerful inducer of interferon. Thus, HCV-specific double-stranded RNA may trigger the first steps of innate immunity; however, for unknown reasons, the immune system often fails to clear the infection. The plasticity of the HCV genome and the low level of HCV gene expression may counterbalance any immunostimulatory effects of HCV RNA and allow the virus to escape specific immune responses. Antisense drugs and ribozymes directed against HCV RNA are under investigation. Future interventions may include nucleic acid drugs (antisense and ribozymes) and smaller pharmaceuticals that bind to intricate structures in HCV RNA and HCV-specific double-stranded RNA. Infectious clones of HCV RNA are available. These clones and other systems for expressing HCV proteins pave the way for vaccine development.

KEYWORD

internal ribosome entry site - double-stranded RNA - interferon - antisense

REFERENCES

• 1 Choo Q L, Kuo G, Weiner A J, Overby L R, Bradley D W, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.  Science . 1989;  244 359-362
• 2 Tanaka T, Kato N, Cho M J, Shimotohno K. A novel sequence found at the 3′terminus of hepatitis C virus genome.  Biochem Biophys Res Commun . 1995;  215 744-749
• 3 Blight K, Trowbridge R, Gowans E J. Absence of double-stranded replicative forms of HCV RNA in liver tissue from chronically infected patients.  J Viral Hepatitis . 1996;  3 29-36
• 4 Gowans E J. The distribution of markers of hepatitis C virus infection throughout the body.  Sem Liver Dis . 2000;  20 85-102
• 5 Martell M, Esteban J I, Quer J. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution.  J Virol . 1992;  66 3225-3229
• 6 Higashi Y, Kakumu S, Yoshioka K. Dynamics of genome change in the E2/NS1 region of hepatitis C virus in vivo.  Virology . 1993;  197 659-668
• 7 Trowbridge R, Gowans E J. Identification of novel sequences at the 5′terminus of the hepatitis C virus genome.  J Viral Hepatitis . 1998;  5 95-98
• 8 Carithers R L, Marquardt A, Gretch D R. Diagnostic testing for hepatitis C.  Sem Liver Dis 2000;20 (in press).
• 9 Tsukiyama Kohara K, Iizuka N, Kohara M, Nomoto A. Internal ribosome entry site within hepatitis C virus RNA.  J Virol. 1992;  66 1476-1483
• 10 Jang S K, Krausslich H G, Nicklin M JH, Duke G M, Palmenberg A C, Wimmer E. A segment of the 5′nontranslated region of encephalomyocarditis virus RNA directs internal entry ribosomes during in vitro translation.  J Virol . 1988;  62 2636-2643
• 11 Pelletier J, Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA.  Nature . 1988;  334 320-325
• 12 Lemon S M, Honda M. Internal ribosome entry sites within the RNA genomes of hepatitis C virus and other flaviviruses.  Semin Virol . 1997;  8 274-288
• 13 Wimmer E, Hellen C UT, Cao X. Genetics of poliovirus.  Annu Rev Genet . 1993;  27 353-436
• 14 Etchison D, Milburn S C, Edery I, Sonenberg N, Hershey J WB. Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex.  J Biol Chem . 1982;  257 14806-14810
• 15 Etchison D, Hansen J, Ehrenfeld E. Demonstration in vitro that eucaryotic initiation factor 3 is active but that a cap-binding protein complex is inactive in poliovirus infected HeLa cells.  J Virol . 1984;  51 832-837
• 16 Fields B N, Knipe D N. Fundamental Virology. New York: Raven Press,1991, pp.5-1021
• 17 Branch A D. Replication of hepatitis C virus: Catching it in the act.  Hepatology . 1996;  23 372-375
• 18 Honda M, Brown E A, Lemon S M. Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA.  RNA . 1996;  2 955-968
• 19 Honda M, Ping L H, Rijnbrand R CA. Structural requirements for initiation of translation by internal ribosome entry within genome length hepatitis C virus RNA.  Virology . 1996;  222 31-42
• 20 Brown E A, Zhang H, Ping L H, Lemon S M. Secondary structure of the 5′nontranslated regions of hepatitis C virus and pestivirus genomic RNAs.  Nucleic Acids Res . 1992;  20 5041-5045
• 21 Honda M, Beard M R, Ping L H, Lemon S M. A phylogenetically conserved stem-loop structure at the 5′border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation.  J Virol . 1999;  73 1165-1174
• 22 Wang C, Le S Y, Ali N, Siddiqui A. A pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5′noncoding region.  RNA . 1995;  1 526-537
• 23 Rijnbrand R, Bredenbeek P, van der Straaten T. Almost the entire 5′non-translated region of hepatitis C virus is required for cap-independent translation.  FEBS Lett . 1995;  365 115-119
• 24 Reynolds J E, Kaminski A, Kettinen H J. Unique features of internal initiation of hepatitis C virus RNA translation.  EMBO J . 1995;  14 6010-6020
• 25 Pestova T V, Shatsky I N, Fletcher S P, Jackson R J, Hellen C UT. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs.  Genes Dev . 1998;  12 67-83
• 26 Reynolds J E, Kaminski A, Carroll A R, Clarke B E, Rowlands D J, Jackson R J. Internal initiation of translation of hepatitis C virus RNA: The ribosome entry site is at the authentic initiation codon.  RNA . 1996;  2 867-878
• 27 Lu H H, Wimmer E. Poliovirus chimeras replicating under the translational control of genetic elements of hepatitis C virus reveal unusual properties of the internal ribosomal entry site of hepatitis C virus.  Proc Natl Acad Sci USA . 1996;  93 1412-1417
• 28 Wang C, Sarnow P, Siddiqui A. Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism.  J Virol . 1993;  67 3338-3344
• 29 Zhao W D, Wimmer E, Lahser F C. Poliovirus/hepatitis C virus (internal ribosomal entry site-core) chimeric viruses: Improved growth properties through modification of a proteolytic cleavage site and requirement for core RNA sequences but not for core-related polypeptides.  J Virol . 1999;  73 1546-1554
• 30 Cate J H, Gooding A R, Podell E. Crystal structure of a group I ribozyme domain: principles of RNA packing.  Science . 1996;  273 1678-1685
• 31 Ali N, Siddiqui A. The La antigen binds 5′noncoding region of the hepatitis C virus RNA in context of the initiator AUG codon and stimulates internal ribosome entry site-mediated translation.  Proc Natl Acad Sci USA . 1997;  94 2249-2254
• 32 Kaminski A, Hunt S L, Patton J G, Jackson R J. Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA.  RNA . 1995;  1 924-938
• 33 Hahm B, Kim Y K, Kim J H, Kim T Y, Jang S K. Hetergenous nuclear ribonucleoprotein L interacts with the 3′border of the internal ribosomal entry site of hepatitis C virus.  J Virol . 1998;  72 8782-8788
• 34 Krüger M, Berger C, Welch P J, Barber J, Wong-Staal F. Identification of cellular regulators of hepatitis C virus internal ribosome entry site (HCV-IRES)-mediated translation using a randomized hairpin ribozyme library.  Hepatology . 1999;  30 355A
• 35 Krüger M, Beger C, Wong-Staal F. Use of ribozymes to inhibit gene expression.  Methods Enzymol . 1999;  306 207-225
• 36 Blight K J, Rice C M. Secondary structure determination of the conserved 98-base sequence at the 3′terminus of hepatitis C virus genome RNA.  J Virol . 1997;  71 7345-7352
• 37 Ito T, Lai M MC. Determination of the secondary structure of and cellular protein binding to the 3′-untranslated region of the hepatitis C virus.  J Virol . 1997;  71 8698-8706
• 38 Ito T, Tahara S M, Lai M MC. The 3′-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site.  J Virol . 1998;  72 8789-8796
• 39 Gontarek R R, Gutshall L L, Herold K M. hnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3′NTR of the HCV RNA genome.  Nucleic Acids Res . 1999;  27 1457-1463
• 40 Oh J W, Ito T, Lai M MC. A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA.  J Virol . 1999;  73 7694-7702
• 41 Farci P, Purcell R H. Clinical significance of hepatitis C genotypes and quasispecies.  Semin Liver Dis . 2000;  20 103-126
• 42 Service R F. Microchip arrays put DNA on the spot.  Science . 1998;  282 396-399
• 43 Farci P, Munoz S J, Shimoda A. Experimental transmission of hepatitis C virus-associated fulminant hepatitis to a chimpanzee.  J Infect Dis . 1999;  179 1007-1011
• 44 Vargas H E, Laskus T, Wang L F. Outcome of liver transplantation in hepatitis C virus-infected patients who received hepatitis C virus-infected grafts.  Gastroenterology . 1999;  117 149-153
• 45 Frolov I, McBride S, Rice C M. cis-Acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5′nontranslated region chimeras.  RNA . 1998;  4 1418-1435
• 46 Macejak D J, Jensen K L, Bellon L, Pavco P A, Blatt L M. Inhibition of viral replication by nuclease resistant hammerhead ribozyme directed against hepatitis C virus RNA.  Hepatology . 1999;  30 409A
• 47 Lohmann V, Korner F, Koch J O, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.  Science . 1999;  285 110-113
• 48 Kolykhalov A A, Agapov E V, Blight K J, Mihalik K, Feinstone S M, Rice C M. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.  Science . 1997;  277 570-574
• 49 Yanagi M, Purcell R H, Emerson S U, Bukh J. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.  Proc Natl Acad Sci USA . 1997;  94 8738-8743
• 50 Yanagi M, St. Claire M, Emerson S U, Purcell R H, Buhk J. In vivo analysis of the 3′untranslated region of the haptitis C virus after in vitro mutagenesis of an infectious cDNA clone.  Proc Natl Acad Sci USA . 1999;  96 2291-2295
• 51 Major M E, Mihalik K, Fernandez J. Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus.  J Virol . 1999;  73 3317-3325
• 52 Shimizu YK Feinstone S M, Kohara M, Purcell R H, Yoshikura H. Hepatitis C virus: Detection by electron microscopy of intracellular virus particles.  Hepatology . 1996;  23 205-209
• 53 Kamimura T, Bonino F, Ponzetto A, Feinstone S M, Gerin J, Purcell R H. Cytoplasmic tubular structures in liver of HBsAg carrier chimpanzees infected with delta agent and comparison with cytoplasmic structures in non-A, non-B hepatitis.  Hepatology . 1983;  3 631-637
• 54 Yu S H, Enomoto N, Nagayama K, Kurosaki M, Sato C. Overexpression of dsRNA-activated protein kinase (PKR) in the liver with chronic hepatitis C.  Hepatology . 1999;  30 359A
• 55 Thimme R L, Guidotti G, Bukh J, Koch R, Pemberton J, Purcell R H, Chisari F V. Different intrahepatic cytokine profiles during acute HBV and HCV infection in the same chimpanzees.  Hepatology . 1999;  30 411A
• 56 Muller U, Steinhoff U, Reis L F. Functional role of type I and type II interferons in antiviral defense.  Science . 1994;  264 1918-1921
• 57 Marcus P I, Sekellick M J. Defective interfering particles with covalently linked [±/-]RNA induce interferon.  Nature . 1977;  266 815-819
• 58 Jacobs B L, Langland J O. When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA.  Virology . 1996;  219 339-349
• 59 Gomatos P J, Tamm I. The secondary structure of reovirus RNA.  Proc Natl Acad Sci USA . 1963;  49 707-714
• 60 Magliano D, Marshall J A, Bowden D S, Vardaxis N, Meanger J, Lee J Y. Rubella virus replication complexes are virus-modified lysosomes.  Virology . 1998;  240 57-63
• 61 Enomoto N, Sakuma I, Asahina Y. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1B infection.  N Engl J Med . 1996;  334 77-81
• 62 Gale M, Blakely C M, Kwieciszewski B. Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation.  Mol Cell Biol . 1998;  18 5208-5218
• 63 Jeffrey I W, Kadereit S, Meurs E F. Nuclear localization of the interferon-inducible protein kinase PKR in human cells and transfected mouse cells.  Exp Cell Res . 1995;  218 17-27
• 64 De Francesco R, Neddermann P, Tomei L, Steinkühler C, Gallinari P, Folgori A. Biochemical and immunological properties of the nonstructural proteins of the hepatitis C virus: implications for development of antiviral agents and vaccines.  Semin Liver Dis . 2000;  20 69-84
• 65 Gribaudo G, Lembo D, Cavallo G, Landolfo S, Lengyel P. Interferon action: binding of viral RNA to the 40-kilodalton 2′-5′-oligoadenylate synthetase in interferon-treated HeLa cells infected with encephalomyocarditis virus.  J Virol . 1991;  65 1748-1757
• 66 Desai S Y, Patel R C, Sen G C, Malhotra P, Ghadge G D, Thimmapaya B. Activation of interferon-inducible 2′-5′oligoadenylate synthetase by adenoviral VAI RNA.  J Biol Chem . 1995;  270 3454-3461
• 67 Nilsen T W, Maroney P A, Robertson H D, Baglioni C. Heterogeneous nuclear RNA promotes synthesis of (2′,5′)oligoadenylate and is cleaved by the (2′,5′)oligoadenylate-activated endoribonuclease.  Mol Cell Biol . 1982;  2 154-160
• 68 Lei M, Liu Y, Samuel C E. Adenovirus VAI RNA antagonizes the RNA-editing activity of the ADAR adenosine deaminase.  Virology . 1998;  245 188-196
• 69 Scadden A D, Smith C W. A ribonuclease specific for inosine-containing RNA: A potential role in antiviral defence?.  EMBO J . 1997;  16 2140-2149
• 70 Zhou A, Paranjape J M, Der S D, Williams B R, Silverman R H. Interferon action in triply deficient mice reveals the existence of alternative antiviral pathways.  Virology . 1999;  258 435-440
• 71 O'Farrelly C, Crispe I N. Prometheus through the looking glass: Reflections on the hepatic immune system.  Immunol Today . 1999;  20 394-398
• 72 Rehermann B. Interaction between HCV and the immune system.  Semin Liver Dis . 2000;  20 137
• 73 Wakita T, Wands J R. Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotides.  In vitro model for selection of target sequence. J Biol Chem . 1994;  269 14205-14210
• 74 Mizutani T, Kato N, Hirota M, Sugiyama K, Murakami A, Shimotohno K. Inhibition of hepatitis C virus replication by antisense oligonucleotide in culture cells.  Biochem Biophys Res Commun . 1995;  212 906-911
• 75 Alt M, Renz R, Hofschneider P H, Paumgartner G, Caselmann W H. Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides.  Hepatology . 1995;  22 707-717
• 76 Hanecak R, Brown-Driver V, Fox M C. Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.  J Virol . 1996;  70 5203-5212
• 77 Mizutani T, Kato N, Saito S, Ikeda M, Sugiyama K, Shimotohno K. Characterization of hepatitis C virus replication in cloned cells obtained from a human T-cell leukemia virus Type 1-infected cell line.  J Virol . 1996;  70 7219-7223
• 78 Eisenhardt S, Samstag W, Jahn-Homann K. Comparison of the inhibitory effect of different chemically modified antisense oligodeoxynucleotides on hepatitis C viral gene expression.  Hepatology . 1996;  24 396A
• 79 Lima W F, Brown-Driver V, Fox M, Hanecak R, Bruice T W. Combinatorial screening and rational optimization for hybridization to folded hepatitis C virus RNA of oligonucleotides with biological antisense activity.  J Biol Chem . 1997;  272 626-638
• 80 Alt M, Renz R, Hofschneider P H, Caselmann W H. Core specific antisense phosphorothioate oligodeoxynucleotides as potent and specific inhibitors of hepatitis C viral translation.  Arch Virol . 1997;  142 589-599
• 81 Wu C H, Wu G Y. Targeted inhibition of hepatitis C virus-directed gene expression in human hepatoma cell lines.  Gastroenterology . 1998;  114 1304-1312
• 82 Zhang H, Hanecak R, Brown-Driver V. Antisense oligonucleotide inhibition of hepatitis C virus (HCV) gene expression in livers of mice infected with and HCV-vaccinia virus recombinant.  Antimicrob Agents Chemother . 1999;  43 347-353
• 83 Brown-Driver V, Eto T, Lesnik E, Anderson K P, Hanecak R C. Inhibition of translation of hepatitis C virus RNA by 2′-modified antisense oligonucleotides.  Antisense Nucleic Acid Drug Dev . 1999;  9 145-154
• 84 Lieber A, He C Y, Polyak S J, Gretch D R, Barr D, Kay M A. Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes.  J Virol . 1996;  70 8782-8791
• 85 Sakamoto N, Wu C H, Wu G Y. Intracellular inhibition of HCV protein synthesis by hammerhead ribozyme expression vectors.  Hepatology . 1996;  24 357A
• 86 Ohkawa K, Yuki N, Kanazawa K. Comparison of three different hammerhead ribozymes for cleavage efficiency of hepatitis C virus RNA in vitro.  Hepatology . 1996;  24 396A
• 87 Alt M, Schussler S, Steigerwald R, Hofschneider P H, Caselmann W H. Hepatitis C virus-specific hammerhead ribozymes cleave target RNA efficiently and inhibit viral gene expression.  J Hepatol . 1996;  25(Suppl 1) 73
• 88 Sakamoto N, Wu C H, Wu G Y. Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein translation by hammerhead ribozymes.  J Clin Invest . 1996;  98 2720-2728
• 89 Branch A D. Instructive surprises from antisense treatments for viral hepatitis and other diseases.  Proc VIII Intl Symp Viral Hepatits . 1998;  (Suppl 1) 33-39
• 90 Gura T. Anti sense has growing pains.  Science . 1995;  270 575-577
• 91 Stein C A. Phosphorothioate antisense oligodeoxynucleotides: Questions of specificity.  TIBTECH . 1996;  14 147-149
• 92 Crooke S T, Bennett C F. Progress in antisense oligonucleotide therapeutics.  Annu Rev Pharmacol Toxicol . 1996;  36 107-129
• 93 Branch A D. A hitchhiker's guide to antisense and nonantisense biochemical pathways.  Hepatology . 1996;  24 1517-1529
• 94 Branch A D. A good antisense molecule is hard to find.  Trends Biochem Sci . 1998;  23 45-50
• 95 Iizuka A, Watanabe T, Kubo T. M13 bacteriophage DNA inhibits duck hepatitis B virus during acute infection.  Hepatology . 1994;  19 1079-1087
• 96 Krieg A M, Yi A K, Matson S. CpG motifs in bacterial DNA trigger direct B-cell activation.  Nature . 1995;  374 546-549
• 97 Stein C A, Krieg A M. Problems in interpretation of data derived from in vitro and in vivo use of antisense oligodeoxynucleotides.  Antisense Res Dev . 1994;  4 67-69
• 98 Moazed D, Noller H F. Interaction of antibiotics with functional sites in 16S ribosomal RNA.  Nature . 1987;  327 389-394
• 99 Gravel M, Melancon P, Brakier Gingras L. Cross-linking of streptomycin to the 16S ribosomal RNA of Escherichia coli Biochemistry .  1987;  26 6227-6232