A chimeric plasmid from cDNA clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive.

Semler, Bert L.; Johnson, Victoria H.; Tracy, Steven

doi:10.1073/pnas.83.6.1777

Cited by 63 publications

(44 citation statements)

References 42 publications

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“…Because many of the predicted higher-order structures of enteroviral 5Ј NTR RNAs are well conserved (1,12,34,40,49), the 30% nucleotide nonidentity between the CVB3 and PV1 5Ј NTR sequences (51) likely plays some role in attenuating the progeny virus. That the chimera CPV/49 replicated similarly to the parental CVB3 strain in HeLa cells demonstrates that the PV1 5Ј NTR is a near-normal functional substitute for that of the related but nonidentical CVB3 in the HeLa cell environment, despite the difference in 5Ј NTR primary structures, confirming and extending work done in the PV system (29,47). Even within the diverse cell cultures studied here, the CPV/49 chimera replicated within 10-fold of the yield of parental virus, demonstrating the functional conservation of the 5Ј NTR among divergent enteroviruses.…”

Section: Discussionsupporting

confidence: 63%

“…Somewhat greater nucleotide nonidentity can occur between genomes of groups, such as between PV and CVB (26,42). Notwithstanding an overall 30% nonidentity at the primary structure level, the 5Ј NTR of CVB3 was shown to be able to functionally replace that of poliovirus type 1 (PV1) (29,47), a finding that was confirmed later by others (57). Replacement of a PV 5Ј NTR with that from a human rhinovirus also produced infectious virus (24).…”

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confidence: 83%

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A Group B Coxsackievirus/Poliovirus 5′ Nontranslated Region Chimera Can Act as an Attenuated Vaccine Strain in Mice

Chapman

Ragland

Leser

et al. 2000

J Virol

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The linear, single-stranded enterovirus RNA genome is flanked at either end with a nontranslated region (NTR). By replacing the entire 5 NTR of coxsackievirus B3 (CVB3) with that from type 1 poliovirus, a progeny virus was obtained following transfection of HeLa cells. The chimeric virus, CPV/49, replicates like the parental CVB3 strain in HeLa cells but is attenuated for replication and yield in primary human coronary artery endothelial cell cultures, in a human pancreas tumor cell line, and in primary murine heart fibroblast cultures. Western blotting analyses of CPV/49 replication in murine heart fibroblast cultures demonstrate that synthesis of CPV/49 proteins is significantly slower than that of the parental CVB3 strain. CPV/49 replicates in murine hearts and pancreata, causing no disease in hearts and a minor pancreatic inflammation in some mice that resolves by 28 days postinoculation. A single inoculation with CPV/49 induces protective anti-CVB3 neutralizing antibody titers that completely protect mice from both heart and pancreatic disease when mice are challenged 28 days p.i. with genetically diverse virulent strains of CVB3. That a chimeric CVB3 strain, created from sequences of two virulent viruses, is sufficiently attenuated to act as an avirulent, protective vaccine strain in mice suggests that chimeric genome technology merits further evaluation for the development of new nonpoliovirus enteroviral vectors.The six serotypes of the group B coxsackieviruses (CVB1 to CVB6) are enteroviruses in the picornavirus family (17). The enterovirus genus also includes the group A coxsackieviruses, polioviruses (PVs), echoviruses, and several other numbered serotypes. Very similar to PV, the prototype enterovirus, in nucleotide sequence and identical in gene order, the CVB genome is 7,400 nucleotides (nt) in length. The single open reading frame encodes 11 proteins and is flanked by nontranslated regions (NTR). The 5Ј-terminal nucleotide is linked to a virus-encoded protein, and the 3Ј terminus is completed with a polyadenosine tail. The CVB are etiologically linked to a wide range of human diseases ranging from mild, common cold-like symptoms through serious and life-threatening illnesses such as meningitis and inflammatory heart disease (reviewed in reference 38).The enteroviral 5Ј NTR, at about 740 nt in length, represents 10% of the viral genome. The 5Ј NTR nucleotide sequence is well maintained among certain enterovirus groups; within the CVB, for example, primary structure is 73% conserved overall, with sequences of individual strains within serotypes much more highly conserved (44). Somewhat greater nucleotide nonidentity can occur between genomes of groups, such as between PV and CVB (26,42). Notwithstanding an overall 30% nonidentity at the primary structure level, the 5Ј NTR of CVB3 was shown to be able to functionally replace that of poliovirus type 1 (PV1) (29, 47), a finding that was confirmed later by others (57). Replacement of a PV 5Ј NTR with that from a human rhinovirus also produced infectious...

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Section: Discussionsupporting

confidence: 63%

mentioning

confidence: 83%

A Group B Coxsackievirus/Poliovirus 5′ Nontranslated Region Chimera Can Act as an Attenuated Vaccine Strain in Mice

Chapman

Ragland

Leser

et al. 2000

J Virol

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“…A previously reported chimera between PV1 and CBV3 has been shown to exhibit a temperature-sensitive (ts) phenotype (50). Specifically, this chimera replicates more efficiently at temperatures slightly lower than physiological temperature.…”

Section: Ecv12(5ntr)cbv3 In Vitro Replication Kineticsmentioning

confidence: 91%

A Predicted Secondary Structural Domain within the Internal Ribosome Entry Site of Echovirus 12 Mediates a Cell-Type-Specific Block to Viral Replication

Bradrick

Lieben

Carden

et al. 2001

J Virol

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The enterovirus 5 nontranslated region (NTR) contains an internal ribosome entry site (IRES), which facilitates translation initiation of the viral open reading frame in a 5 (m 7 GpppN) cap-independent manner, and cis-acting signals for positive-strand RNA replication. For several enteroviruses, the 5 NTR has been shown to determine the virulence phenotype. We have constructed a chimera consisting of the putative IRES element from the Travis strain of echovirus 12 (ECV12), a wild-type, relatively nonvirulent human enterovirus, exchanged with the homologous region of a full-length infectious clone of coxsackievirus B3 (CBV3). The resulting chimera, known as ECV12(5NTR)CBV3, replicates similarly to CBV3 in human and simian cell lines yet, unlike CBV3, is completely restricted for growth on two primary murine cell lines at 37°C. By utilizing a reverse-genetics approach, the growth restriction phenotype was localized to the predicted stem-loop II within the IRES of ECV12. In addition, a revertant of ECV12(5NTR)CBV3 was isolated which possessed three transition mutations and had restored capability for replication in the utilized murine cell lines. Assays for cardiovirulence indicated that the ECV12 IRES is responsible for a noncardiovirulent phenotype in a murine model for acute myocarditis. The results indicate that the 5 NTRs of ECV12 and CBV3 exhibit variable intracellular requirements for function and serve as secondary determinants of tissue or species tropism.The Enterovirus genus within the family Picornaviridae contains human pathogens responsible for clinical syndromes involving multiple tissues and organ systems (reviewed in reference 37). The enteroviruses, like all members of the family Picornaviridae, are positive-stranded, nonenveloped RNA viruses which possess a characteristic icosahedral capsid. This genus is subdivided into five groups: the polioviruses, coxsackievirus groups A and B, echoviruses, and the numbered enteroviruses. The ϳ7.4-kb enterovirus genome is structurally similar to eukaryotic mRNA in several respects: a single open reading frame encoding structural and nonstructural proteins is flanked by 5Ј and 3Ј nontranslated regions (NTRs) and a 3Ј polyadenylated tail. The 5Ј NTR is highly structured, containing six predicted secondary structural domains, or stem-loops (SLs) (44, 52), and carries out functions crucial to the life cycle of the virus (2, 42). In addition, the 5Ј NTR has been shown to influence the virulence phenotypes of several enteroviruses (16,20,32,41). SL I, also known as the cloverleaf, within the poliovirus (PV) 5Ј NTR is a cis element essential for stimulation of positivesense RNA transcription (2, 3). A discontinuous region encompassing SLII through SLVI constitutes a complex element known as the internal ribosome entry site (IRES) (42). The IRES stimulates translation initiation of the open reading frame by a mechanism that is unlike that utilized by the majority of cellular mRNAs. Specifically, the enterovirus genome lacks a 5Ј (m 7 GpppN) cap structure (26, 40) whi...

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“…Therefore, it has been difficult to characterize very early events in infected cells, such as the translation of the infecting RNA molecules and the synthesis of the first negative strands. With the discovery that a cloned cDNA copy of the viral genome is infectious upon transfection into mammalian cells (6,7), it was possible to construct defined viral mutants that have defects in viral RNA amplification (8)(9)(10)(11)(12) or in the inhibition of cellular protein biosynthesis (13,14).…”

mentioning

confidence: 99%

Translation of glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA is increased in poliovirus-infected cells at a time when cap-dependent translation of cellular mRNAs is inhibited.

Sarnow

1989

Proc. Natl. Acad. Sci. U.S.A.

124

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All cellular cytoplasmic mRNAs carry a 7-methylguanylate cap attached to their 5' ends. This cap structure is recognized by cap-binding proteins that then direct the binding of ribosomal subunits to this 5'-end complex. Poliovirus, a plus-stranded RNA virus, interferes with this cellular translation process by proteolytically inactivating the capbinding protein complex. Subsequently the viral mRNA can be translated by an initiation process in which ribosomes bind internally to the mRNA [Pelletier, J. & Sonenberg, N. (1988) Nature (London) 334, 320-325], obviating cap-dependent translation. At least one cellular mRNA, encoding a heat shock-like protein, glucose-regulated protein 78/immunoglobulin heavy-chain binding protein, has been discovered to be translated at an increased rate in poliovirus-infected cells at a time when the translation of other cellular mRNAs is inhibited.The glucose-regulated protein 78/immunoglobulin heavychain binding protein mRNA thus exemplifies a cellular mRNA that is translated at a specifically enhanced rate by an asyet-unresolved cap-independent initiation process in cells when the cap-binding protein complex is not functional.

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A chimeric plasmid from cDNA clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive.

Cited by 63 publications

References 42 publications

A Group B Coxsackievirus/Poliovirus 5′ Nontranslated Region Chimera Can Act as an Attenuated Vaccine Strain in Mice

A Group B Coxsackievirus/Poliovirus 5′ Nontranslated Region Chimera Can Act as an Attenuated Vaccine Strain in Mice

A Predicted Secondary Structural Domain within the Internal Ribosome Entry Site of Echovirus 12 Mediates a Cell-Type-Specific Block to Viral Replication

Translation of glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA is increased in poliovirus-infected cells at a time when cap-dependent translation of cellular mRNAs is inhibited.

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