Abstract
Coxsackievirus B3 (CVB3) is a principal viral cause of acute myocarditis in humans and has been implicated in the pathogenesis of dilated cardiomyopathy. The natural genetic determinants of cardiovirulence for CVB3 have not been identified, although using strains engineered in the laboratory, it has been demonstrated elsewhere that, for several wild-type CB3 strains, the primary molecular determinant of cardiovirulence phenotype localizes to the 5′ nontranslated region (5′NTR) and capsid. Stable RNA tetraloop motifs are found frequently in biologically active RNAs. These motifs carry out a wide variety of functions in RNA folding, in RNA–RNA and RNA–protein interactions. A great deal of knowledge about the structures and functions of tetraloop motifs has accumulated largely due to intensive theoretical, biochemical, and biophysical studies on one most frequently occurring family of tetraloop sequences, namely, the GNRA sequence, especially the GNAA sequence conserved in all enteroviruses. Here in this study, through construction of CVB3 chimeric mutants, the predicted stem loop (SL) V within the 5′NTR has been identified as important in determining viral cardiovirulence. Replication assays in HeLa cell monolayers revealed that wild-type CVB3 virus and two of the six mutants constructed here grow efficiently, whereas other mutant viruses replicate poorly. Furthermore, the in vitro translation products from these mutants and wild-type CVB3, demonstrated that the two mutants who replicate efficiently, translated at relatively equivalent amount than the wild-type. However, other mutants demonstrated a low efficiency in their production of protein when translated in a Rabbit Reticulocytes Lysats.
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References
Wimmer E, Hellen CU, Cao X (1993) Genetics of poliovirus. Annu Rev Genet 27:353–436
Takeda N, Kuhn RJ, Yang C-F, Takegami T, Wimmer E (1986) Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells. J Virol 60:43–53
Bienz K, Egger D, Pfister T, Troxler M (1990) Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region. J Virol 64:1156–1163
Bienz K, Egger D, Pfister T, Troxler M (1992) Structural and functional characterization of poliovirus RNA replication complex. J Virol 66:2740–2747
Chapman NM, Tu Z, Tracy S, Gauntt CJ (1994) An infectious cDNA copy of the genome of a non-cardiovirulent coxsackievirus B3 strain: its complete sequence analysis and comparison to the genomes of cardiovirulent coxsackieviruses. Arch Virol 135:115–130
Klump WM, Bergmann I, Muller BC, Ameis D, Kandolf R (1990) Complete nucleotide sequence of infectious coxsackievirus B3 cDNA: two initial 5′ uridine are regained during plus-strand RNA synthesis. J Virol 64:1573–1583
Lindberg AM, Stalhandske POK, Pettersson U (1987) Genome of coxsackievirus B3. Virology 156:50–63
Tracy S, Chapman NM, Tu Z (1992) Coxsackievirus B3 from an infectious cDNA copy of the genome is cardiovirulent in mice. Arch Virol 122:399–409
Rose JK, Trachsel H, Leong K, Baltimore D (1978) Inhibition of translation by poliovirus: inactivation of a specific initiation factor. Proc Natl Acad Sci USA 75:2732–2736
Sonenberg N (1990) Poliovirus translation. Curr Top Microbiol Immunol 161:23–47
Coller BA, Chapman NM, Beck MA, Pallansch MA Gauntt CJ, Tracy SM (1990) Echovirus 22 is an atypical enterovirus. J Virol 64:2692–2701
Pelletier J, Kaplan G, Racaniello VR, Sonenberg N (1988) Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5′ noncoding region. Mol Cell Biol 8:1103–1112
Pelletier J, Sonenberg N (1988) Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334:320–325
Chen CY, Sarnow P (1995) Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science 268:415–417
Baboonian C, Davies MJ, Booth JC, McKenna WJ (1997) Coxsackie B viruses and human heart disease. In: Tracy S, Chapman NM, Mahy BWJ (eds) The coxsackie B viruses. Springer-Verlag, Berlin, pp 31–52
Archard LC, Bowles NE, Cunningham L (1993) Enteroviruses RNA sequences in hearts with dilated cardiomyopathy: a pathogenic link between virus infection and dilated cardiomyopathy. In: Baroldi G, Camerini F, Goodwin JF (eds) Advances in cardiomyopathies. Springer-Verlag, Berlin, pp 194–198
Kandolf R (1988) The impact of recombinant DNA technology on the study of enteroviral heart disease. In: Bendinelli M, Friedman H (eds) Coxsackieviruses-a general update, Plenum, New York, pp 292–318
Martino TA, Liu P, Sole MJ (1994) Viral infection and the pathologenesis of dilated cardiomyopathy. Circ Res 74:182–188
Tracy S, Chapman NM, McManus BM, Pallansch MA, Beck MA, Carstens J (1990) A molecular and serological evaluation of enteroviral involvement in human myocarditis. J Mol Cell Cardiol 22:403–414
Why HJ, Meany BT, Richardson PT, Olsen EG, Bowles NE, Cunninghamm L, Freeke CA, Archard LC (1994) Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy. Circulation 89:2582–2589
Almond JW (1987) The attenuation of poliovirus neurovirulence. Annu Rev Microbiol 41:153–180
Stanway G (1990) Structure, function and evolution of picornaviruses. J Gen Virol 71:2483–2501
Agol VI, Drozdov SG, Kolesnikova MS, Korolev MB, Tolskaya EA (1989) Restricted growth of attenuated poliovirus strains in cultured cells of a human neuroblastoma. J Virol 63:4034–4038
La Monica N, Racaniello VR (1989) Differences in replication of attenuated and neurovirulent polioviruses in human neuroblastoma cell line SH-SY5Y. J Virol 63:2357–2560
Zhang H, Yousef G, Cunningham L (1993) Attenuation of a reactivated cardiovirulent coxsackievirus B3: the 5′-nontranslated region does not contain major attenuation determinants. J Med Virol 41:129–137
Gutell R, Weiser R, Woese CR, Noller HF (1985) Comparative anatomy of 16 S-like ribosomal RNA. Prog Nucl Acids Res Mol Biol 32:155–216
Woeses C, Winker R, Gutell RR (1990) Architecture of ribosomal RNA-constraints on the sequence of tetra-loops. Proc Natl Acad Sci USA 87:8467–8471
Tuerk C, Gauss P, Thermes C, Groebe DR, Gayle M, Guild N, Stomo G, Daubentoncarafa Y, Uhlenbeck OC, Tinoco I Jr, Brody EN, Gold L (1988) CUUCGG hairpins-extraordinarily stable RNA secondary structures associated with various biochemical processes. Proc Natl Acad Sci USA 85:1364–1368
Uhlenbeck OC (1990) Nucleic-acid structure-tetraloops and RNA folding. Nature 346:613–614
Michel F, Westof E (1990) Modeling of the three-dimensional architecture of group-I catalytic introns based on comparative sequence analysis. J Mol Biol 216:585–610
Pley H, Flaherty KM, McKay DB (1994) Model for an RNA tertiary interaction from the structural of an intermolecular complex between a GAAA tetraloop and an RNA helix. Nature 372:111–113
Gluck A, Endo Y, Wool IG (1992) Ribosomal-RNA identity elements for ricin A-chain recognition and catalysis-analysis with tetraloop mutants. J Mol Biol 226:411–424
Teterina NL, Kean MK, Gorbalenya AE, Agol VI, Girard M (1992) Analysis of the functional significance of amino acid residues in the putative NTP-binding pattern of the poliovirus 2C protein. J Gen Virol 73:1977–1986
Gharbi J, El Hiar R, Ben M’hadheb M, Jaïdane H, Bouslama L, N’saïbia S, Aouni M (2006) Nucleotide sequences of IRES domains IV and V of natural ECHO virus type 11 isolates with different replication capacity phenotypes. Virus Genes 32:269–276
Jackson RJ, Hunt T (1983) Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol 96:50–74
Borman AM, Deliat FG, Kean MK (1994) Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis. EMBO J 13:3149–3159
Tu Z, Chapman NM, Hufnagel G, Tracy S, Romero JR, Barry WH, Zhao L, Currey K, Shapiro B (1995) The cardiovirulent phenotype of coxsackievirus B3 is determined at a single site in the genomic 5′ nontranslated region. J Virol 69:4607–4618
Agol VI (1991) The 5′-untranslated region of picornaviral genomes. Adv Virus Res 40:103–180
Evans DM, Dunn G, Minor PD, Schild GC, Cann AJ, Stanway G, Almond JW, Currey K, Maizel JV (1985) Increased neurovirulence associated with a single nucleotide change in a noncoding region of the Sabin type 3 poliovaccine genome. Nature 314:548–550
Macadam AJ, Ferguson G, Burlison J, Stone D, Skuce R, Almond JW, Minor PD (1992) Correlation of RNA secondary structure and attenuation of Sabin vaccine strains of poliovirus in tissue culture. Virology 189:415–422
Skinner MA, Racaniello VR, Dunn G, Cooper J, Minor PD, Almond JW (1989) New model for the secondary structure of the 5′ non-coding RNA of poliovirus is supported by biochemical and genetic data that also show that RNA secondary structure is important in neurovirulence. J Mol Biol 207:379–392
Svitkin YV, Pestova AE, Maslova SV, Agol VI (1988) Point mutations modify the response of poliovirus RNA to a translation initiation factor: a comparison of neurovirulent and attenuated strains. Virology 166:394–404
Svitkin YV, Cammack N, Minor PD, Almond JW (1990) Translation deficiency of the Sabin type 3 poliovirus genome: association with an attenuating mutation C472-U. Virology 175:103–109
Duke GM, Hoffman MA, Palmenberg AC (1992) Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation. J Virol 66:1602–1609
Dunn JJ, Chapman NM, Tracy S, Romero J (2000) Genomic determinants of cardiovirulence in coxsackievirus B3 clinical isolates: localization to the 5′ nontranslated region. J Virol 74:4787–4794
Lee C, Maull E, Chapman N, Tracy S, Gauntt C (1997) Genomic regions of coxsackievirus B3 associated with cardiovirulence. J Med Virol 52:341–347
Henke A, Wagner E, Whitton JL, Zell R, Stelzner A (1998) Protection of mice against lethal coxsackievirus B3 infection by using DNA immunization. J Virol 72:8327–8331
Kandolf R, Klingel K, Zell R et al (1993) Molecular pathogenesis of enterovirus-induced myocarditis: virus persistence and chronic inflammation. Intervirol 35:140–151
Dunn JJ, Bradrick SS, Chapman MN, Tracy S, Romero RJ (2003) The stem loop II within the 5′ nontranslated region of clinical coxsackievirus B3 genomes determines cardiovirulence phenotype in a murine model. J Infec Dis 187:1552–1561
Knowlton KU, Jeon ES, Berkley N, Wessely R, Huber S (1996) A mutation of the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the woodruff variant of coxsackievirus B3. J Virol 70:7811–7818
Hellen CU, Wimmer E (1995) Enterovirus genetics. In: Rotbart HA (ed) Human enterovirus infection. American Society for Microbiology, Washington, pp 25–72
Agol VI (2002) Picornavirus genome: an overview. In: Semler BL, Wimmer E (eds) Molecular biology of picornaviruses. ASM Press, Washington DC, pp 127–148
Bhattacharyya S, Das S (2004) Mapping of secondary structure of the spacer region within the 5′-untranslated region of the coxsackievirus B3 RNA: possible role of an apical GAGA loop in binding La protein and influencing internal initiation of translation. Virus Res 108:89–100
Dalldorf G (1955) The coxsackie viruses. Annu Rev Microbiol 9:277–296
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M’hadheb-Gharbi, M.B., Kean, K.M. & Gharbi, J. Molecular analysis of the role of IRES stem-loop V in replicative capacities and translation efficiencies of Coxsackievirus B3 mutants. Mol Biol Rep 36, 255–262 (2009). https://doi.org/10.1007/s11033-007-9174-3
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DOI: https://doi.org/10.1007/s11033-007-9174-3