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J Virol. Jan 1997; 71(1): 451–457.
PMCID: PMC191071

Internal entry of ribosomes is directed by the 5' noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon.

Abstract

Bicistronic RNAs containing the 373-nucleotide-long 5' nontranslated region (NTR) of the classical swine fever virus (CSFV) genome as intercistronic spacer were used to show the presence of an internal ribosome entry site (IRES) in the 5' end of the CSFV genome. By coexpression of the poliovirus 2A protease it was demonstrated that the CSFV 5' NTR-driven translation is independent of the presence of functional eukaryotic initiation factor eIF-4F. Deletion analysis indicated that the 5' border of the IRES is located between nucleotides 28 and 66. The role of a proposed pseudoknot structure at the 3' end of the CSFV 5' NTR in IRES-mediated translation was investigated by site-directed mutagenesis. Mutant RNAs that had lost the ability to base pair in stem II of the pseudoknot were translationally inactive. Translation to wild-type levels could be restored through the introduction of compensatory complementary base changes that repaired base pairing in stem II. In addition, we showed that the AUG codon, which is located 7 nucleotides upstream of the polyprotein initiation site and is conserved in pestiviruses, could not be used to initiate translation. Also, an AUG codon introduced downstream of the polyprotein initiation site was not recognized as an initiation site by ribosomes. These data suggest that after internal entry on the CSFV 5' NTR, ribosomal scanning for the initiation codon is limited to a small region.

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Selected References

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  • Berlioz C, Torrent C, Darlix JL. An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors. J Virol. 1995 Oct;69(10):6400–6407. [PMC free article] [PubMed]
  • Brown EA, Zhang H, Ping LH, Lemon SM. Secondary structure of the 5' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acids Res. 1992 Oct 11;20(19):5041–5045. [PMC free article] [PubMed]
  • Brown EA, Day SP, Jansen RW, Lemon SM. The 5' nontranslated region of hepatitis A virus RNA: secondary structure and elements required for translation in vitro. J Virol. 1991 Nov;65(11):5828–5838. [PMC free article] [PubMed]
  • Deng R, Brock KV. 5' and 3' untranslated regions of pestivirus genome: primary and secondary structure analyses. Nucleic Acids Res. 1993 Apr 25;21(8):1949–1957. [PMC free article] [PubMed]
  • Fuerst TR, Niles EG, Studier FW, Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. [PMC free article] [PubMed]
  • Gorman CM, Merlino GT, Willingham MC, Pastan I, Howard BH. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. [PMC free article] [PubMed]
  • Han JH, Shyamala V, Richman KH, Brauer MJ, Irvine B, Urdea MS, Tekamp-Olson P, Kuo G, Choo QL, Houghton M. Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5' untranslated region and poly(A) tails at the 3' end. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1711–1715. [PMC free article] [PubMed]
  • Jang SK, Kräusslich HG, Nicklin MJ, Duke GM, Palmenberg AC, Wimmer E. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988 Aug;62(8):2636–2643. [PMC free article] [PubMed]
  • Kaminski A, Howell MT, Jackson RJ. Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selected by a scanning mechanism. EMBO J. 1990 Nov;9(11):3753–3759. [PMC free article] [PubMed]
  • Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. [PMC free article] [PubMed]
  • Kühn R, Luz N, Beck E. Functional analysis of the internal translation initiation site of foot-and-mouth disease virus. J Virol. 1990 Oct;64(10):4625–4631. [PMC free article] [PubMed]
  • Le SY, Sonenberg N, Maizel JV., Jr Unusual folding regions and ribosome landing pad within hepatitis C virus and pestivirus RNAs. Gene. 1995 Mar 10;154(2):137–143. [PubMed]
  • Lloyd RE, Grubman MJ, Ehrenfeld E. Relationship of p220 cleavage during picornavirus infection to 2A proteinase sequencing. J Virol. 1988 Nov;62(11):4216–4223. [PMC free article] [PubMed]
  • Macejak DG, Sarnow P. Internal initiation of translation mediated by the 5' leader of a cellular mRNA. Nature. 1991 Sep 5;353(6339):90–94. [PubMed]
  • Meinkoth J, Wahl G. Hybridization of nucleic acids immobilized on solid supports. Anal Biochem. 1984 May 1;138(2):267–284. [PubMed]
  • Moormann RJ, van Gennip HG, Miedema GK, Hulst MM, van Rijn PA. Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus. J Virol. 1996 Feb;70(2):763–770. [PMC free article] [PubMed]
  • Oh SK, Scott MP, Sarnow P. Homeotic gene Antennapedia mRNA contains 5'-noncoding sequences that confer translational initiation by internal ribosome binding. Genes Dev. 1992 Sep;6(9):1643–1653. [PubMed]
  • Pelletier J, Sonenberg N. Internal binding of eucaryotic ribosomes on poliovirus RNA: translation in HeLa cell extracts. J Virol. 1989 Jan;63(1):441–444. [PMC free article] [PubMed]
  • Poole TL, Wang C, Popp RA, Potgieter LN, Siddiqui A, Collett MS. Pestivirus translation initiation occurs by internal ribosome entry. Virology. 1995 Jan 10;206(1):750–754. [PubMed]
  • Rijnbrand R, Bredenbeek P, van der Straaten T, Whetter L, Inchauspé G, Lemon S, Spaan W. Almost the entire 5' non-translated region of hepatitis C virus is required for cap-independent translation. FEBS Lett. 1995 May 29;365(2-3):115–119. [PubMed]
  • Rümenapf T, Unger G, Strauss JH, Thiel HJ. Processing of the envelope glycoproteins of pestiviruses. J Virol. 1993 Jun;67(6):3288–3294. [PMC free article] [PubMed]
  • Stark R, Meyers G, Rümenapf T, Thiel HJ. Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus. J Virol. 1993 Dec;67(12):7088–7095. [PMC free article] [PubMed]
  • Svitkin YV, Cammack N, Minor PD, Almond JW. Translation deficiency of the Sabin type 3 poliovirus genome: association with an attenuating mutation C472----U. Virology. 1990 Mar;175(1):103–109. [PubMed]
  • Thiel V, Siddell SG. Internal ribosome entry in the coding region of murine hepatitis virus mRNA 5. J Gen Virol. 1994 Nov;75(Pt 11):3041–3046. [PubMed]
  • Tsukiyama-Kohara K, Iizuka N, Kohara M, Nomoto A. Internal ribosome entry site within hepatitis C virus RNA. J Virol. 1992 Mar;66(3):1476–1483. [PMC free article] [PubMed]
  • Wang C, Le SY, Ali N, Siddiqui A. An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region. RNA. 1995 Jul;1(5):526–537. [PMC free article] [PubMed]
  • 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 Jun;67(6):3338–3344. [PMC free article] [PubMed]
  • Wang C, Sarnow P, Siddiqui A. A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA. J Virol. 1994 Nov;68(11):7301–7307. [PMC free article] [PubMed]
  • Whetter LE, Day SP, Elroy-Stein O, Brown EA, Lemon SM. Low efficiency of the 5' nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells. J Virol. 1994 Aug;68(8):5253–5263. [PMC free article] [PubMed]
  • Wiskerchen M, Belzer SK, Collett MS. Pestivirus gene expression: the first protein product of the bovine viral diarrhea virus large open reading frame, p20, possesses proteolytic activity. J Virol. 1991 Aug;65(8):4508–4514. [PMC free article] [PubMed]
  • Wiskerchen M, Collett MS. Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing. Virology. 1991 Sep;184(1):341–350. [PubMed]
  • Yu SF, Lloyd RE. Identification of essential amino acid residues in the functional activity of poliovirus 2A protease. Virology. 1991 Jun;182(2):615–625. [PubMed]

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