Role of the pyrimidine-rich tract on the translation of ‘chimeric’ polio-hepatitis A mRNAs with engineered 5′-terminal untranslated regions

Degener, Anna Marta; Carneiro, Jane Silveira; Cassetti, Cristina; Pierangeli, Alessandra; Pagnotti, P; Bucci, Mauro; Bercoff, R. Perez

doi:10.1016/0168-1702(95)00030-t

Cited by 4 publications

(7 citation statements)

References 25 publications

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“…The amount of GH present in the supernatants was determined by radioimmunoassay in triplicate 100 μl aliquots (Silveira Carneiro et al ., 1995 ). The cells were then detached from the plates, washed in ice-cold PBS, suspended in 300 μl 250 mM Tris–HCl (pH 8·3), frozen and thawed three times, and the CAT activity was assayed and quantified as described (Degener et al ., 1995 ; Silveira Carneiro et al ., 1995 ).…”

Section: Methodsmentioning

confidence: 99%

‘Primer alignment-and-extension’: a novel mechanism of viral RNA recombination responsible for the rescue of inactivated poliovirus cDNA clones

Pierangeli

Bucci

Forzan

et al. 1999

Journal of General Virology

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In the course of experiments designed to assess the potential role of alternative open reading frames (ORF) present in the 5h-terminal untranslated region (5h-UTR) of poliovirus type 1 (Mahoney strain) genomic RNA, we came across a double mutation that completely abrogated the infectivity of full-length cDNA clones. The infectivity was rescued in trans by cotransfecting COS-1 cells with short RNA transcripts of the wild-type 5h-UTR of poliovirus type 2 Lansing, provided a free 3h-OH was available. Direct sequencing of the viral RNA revealed that the infectious viruses recovered were recombinants Lansing/Mahoney, with variable points of ' crossing-over '. A novel mechanism of RNA-RNA recombination, which we propose to call ' primer alignment-and-extension ', is described that would explain the high rate of recombination of RNA viruses observed in natural conditions.

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Section: Methodsmentioning

confidence: 99%

‘Primer alignment-and-extension’: a novel mechanism of viral RNA recombination responsible for the rescue of inactivated poliovirus cDNA clones

Pierangeli

Bucci

Forzan

et al. 1999

Journal of General Virology

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“…The construction of bi‐cistronic plasmids containing cDNA representations of either the native or mutated 5′‐ECR of HAV RNA inserted between the genes encoding human GH and bacterial CAT has been described in detail [4, 10].…”

Section: Methodsmentioning

confidence: 99%

“…The genomic RNA of picornaviruses is exceptional among eukaryotic mRNAs in that it lacks a 5′‐terminal cap structure, and previous work from this laboratory provided the first experimental evidence supporting the contention that translation of picornavirus RNA involves a most efficient mechanism of internal (rather than 5′‐terminal) initiation that can by‐pass the need for either a cap or a 5′‐end [1–3]. Several lines of evidence indicate that this mechanism may not be confined to the RNA of picornaviruses, and that other eukaryotic mRNAs may use a similar mechanism of translation, an observation suggesting that the above findings may have broader biological implications than so far suspected ([4]and references therein).…”

Section: Introductionmentioning

confidence: 99%

“…Mutational analysis of this tract allowed us to identify four functional domains differently involved in securing the internal initiation of translation of a reporter gene in bi‐cistronic mRNAs [10]. Experiments designed to assess the role of the polypyrimidine sequences in translation driven by the 5′‐ECR of HAV RNA led us to postulate that the 5′‐PRT is indeed part of the cis ‐acting elements required to initiate translation, and that the 3′ one might play a merely vicarious role due, perhaps, to the too short distance intervening between this element and the downstream AUG codon [4]. This view was indirectly supported by the observation that the reduction of the spacer between the PRT and a downstream (silent) AUG triplet to less than 14 nt abrogated translation driven by poliovirus 5′‐ECR [11].…”

Section: Introductionmentioning

confidence: 99%

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The distance between the 3′‐pyrimidine‐rich tract and the AUG codon modulates internal initiation of translation of hepatitis A virus RNA

et al. 1998

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Protein synthesis directed by hepatitis A virus (HAV) RNA is mediated by a mechanism involving the recognition of internal sequences. Two in-frame AUG codons initiate the long open reading frame (positions 734-736 and 740-742). The extra-cistronic region extending between the uncapped 5'-end and the ORF contains two pyrimidine-rich tracts (PRTs): one 12 nucleotides in length in the close vicinity of the initiator AUG, and a longer one between bases 94 and 140. In order to study the relative contribution of these elements to the process of internal initiation of translation, cDNA representations of the 5'-terminal extra-cistronic region of HAV RNA were inserted in the intergenic region of the bi-cistronic plasmid pSV-GH/CAT, between the genes encoding the human growth hormone (GH) and the bacterial enzyme chloramphenicol acetyltransferase (CAT), and following transfection of COS-1 cells, the transient expression of both genes was quantified. The importance of the 3'-PRT appeared to be strongly influenced by the length of the 'spacer' sequence extending between this structure and the translation initiation site: placed 45 nucleotides upstream from the initiator codon of a reporter gene, its integrity was stringently required for initiation to occur. Bringing the length of the 'spacer' back to its actual size in HAV RNA (i.e. 11 or 17 nt) reduced considerably the overall rate of internal initiation of translation, and the relative contribution to this process of the 3'-PRT became marginal. Concomitantly, the importance of the functional domains previously identified in the 5'-PRT fluctuated: while integrity of domain 100-106 was always stringently required for initiation to occur, the activity of domain 113-118 paralleled that of the 3'-PRT, and the opposite applied to domain 121-126, whose contribution became relevant only after switching off the 3'-PRT. Systematic mutations introduced in the 'spacer' sequences suggest that the length of this region may be responsible for the down regulation of translation of HAV RNA and, possibly, for its lengthy replication cycle.

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“…Simi-larly, HSP mRNAs are resistant to poliovirus-induced shutoff and it has been proposed that they may utilize cap-independent initiation (48). The limited secondary structure of the HSP 5ЈUTR, as well as the short and unstructured 5ЈUTR of rp-mRNAs, may determine a lower dependence on initiation factors compared to more-structured mRNAs (9,20,53). In line with this, it has been reported that the efficiency of translation of rp-mRNAs is regulated independently of the level or activity of eIF4E (51), whereas the selective translational repression of mRNAs bearing extensive secondary structure in the 5ЈUTR is relieved by the overexpression of this factor (27).…”

Section: Translation Of Rp-mrnas During Poliovirus Infectionmentioning

confidence: 99%

Resistance of Ribosomal Protein mRNA Translation to Protein Synthesis Shutoff Induced by Poliovirus

Cardinali

Fiore

Campioni

et al. 1999

J Virol

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Poliovirus infection induces an overall inhibition of host protein synthesis, although some mRNAs continue to be translated, suggesting different translation requirements for cellular mRNAs. It is known that ribosomal protein mRNAs are translationally regulated and that the phosphorylation of ribosomal protein S6 is involved in the regulation. Here, we report that the translation of ribosomal protein mRNAs resists poliovirus infection and correlates with an increase in p70s6k activity and phosphorylation of ribosomal protein S6.

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Role of the pyrimidine-rich tract on the translation of ‘chimeric’ polio-hepatitis A mRNAs with engineered 5′-terminal untranslated regions

Cited by 4 publications

References 25 publications

‘Primer alignment-and-extension’: a novel mechanism of viral RNA recombination responsible for the rescue of inactivated poliovirus cDNA clones

‘Primer alignment-and-extension’: a novel mechanism of viral RNA recombination responsible for the rescue of inactivated poliovirus cDNA clones

The distance between the 3′‐pyrimidine‐rich tract and the AUG codon modulates internal initiation of translation of hepatitis A virus RNA

Resistance of Ribosomal Protein mRNA Translation to Protein Synthesis Shutoff Induced by Poliovirus

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