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Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication.

Jorba N, Coloma R, Ortín J - PLoS Pathog. (2009)

Bottom Line: We used efficient systems for recombinant RNP transcription/replication in vivo and well-defined polymerase mutants deficient in either RNA replication or transcription to address the roles of the polymerase complex present in the template RNP and newly synthesised polymerase complexes during replication and transcription.The results of trans-complementation experiments showed that soluble polymerase complexes can synthesise progeny RNA in trans and become incorporated into progeny vRNPs, but only transcription in cis could be detected.In contrast, transcription of the vRNP would occur in cis and the resident polymerase complex would be responsible for mRNA synthesis and polyadenylation.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias, Campus de Cantoblanco, Madrid, Spain.

ABSTRACT
The influenza A viruses genome comprises eight single-stranded RNA segments of negative polarity. Each one is included in a ribonucleoprotein particle (vRNP) containing the polymerase complex and a number of nucleoprotein (NP) monomers. Viral RNA replication proceeds by formation of a complementary RNP of positive polarity (cRNP) that serves as intermediate to generate many progeny vRNPs. Transcription initiation takes place by a cap-snatching mechanism whereby the polymerase steals a cellular capped oligonucleotide and uses it as primer to copy the vRNP template. Transcription termination occurs prematurely at the polyadenylation signal, which the polymerase copies repeatedly to generate a 3'-terminal polyA. Here we studied the mechanisms of the viral RNA replication and transcription. We used efficient systems for recombinant RNP transcription/replication in vivo and well-defined polymerase mutants deficient in either RNA replication or transcription to address the roles of the polymerase complex present in the template RNP and newly synthesised polymerase complexes during replication and transcription. The results of trans-complementation experiments showed that soluble polymerase complexes can synthesise progeny RNA in trans and become incorporated into progeny vRNPs, but only transcription in cis could be detected. These results are compatible with a new model for virus RNA replication, whereby a template RNP would be replicated in trans by a soluble polymerase complex and a polymerase complex distinct from the replicative enzyme would direct the encapsidation of progeny vRNA. In contrast, transcription of the vRNP would occur in cis and the resident polymerase complex would be responsible for mRNA synthesis and polyadenylation.

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Phenotype of trans-complemented RNPs.The purified RNP preparations presented in Fig. 1 were tested for in vitro transcription primed with either ApG (red) or β-globin mRNA (green). The data are presented as percent of maximal value and represent the averages and ranges of two independent complementation experiments. The transcription activities parallel the values of NP accumulation presented in Fig. 1 and show that the rescued RNPs have a wt cap-snatching phenotype.
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ppat-1000462-g002: Phenotype of trans-complemented RNPs.The purified RNP preparations presented in Fig. 1 were tested for in vitro transcription primed with either ApG (red) or β-globin mRNA (green). The data are presented as percent of maximal value and represent the averages and ranges of two independent complementation experiments. The transcription activities parallel the values of NP accumulation presented in Fig. 1 and show that the rescued RNPs have a wt cap-snatching phenotype.

Mentions: The progeny RNPs contained the replication-defective PB2 allele, since (i) they could be purified by Ni2+-NTA-agarose chromatography and (ii) the mobility of the PB2 subunit in the Western-blot assay corresponded to the His-tagged subunit and not to the untagged one. It is important to mention that only His-tagged PB2 protein was detected in the purified RNPs and not the untagged counterpart, indicating that no transcription-defective polymerase was co-purified (Fig. 1B). Furthermore, the phenotype of the rescued RNPs was tested by determination of their in vitro transcription activity (Fig. 2). Since the transcription-defective mutants had alterations in their cap-binding pocket, they show low in vitro transcription activity when a mRNA is used as a cap-donor, whereas cap-independent transcription is observed with a general primer as the dinucleotide ApG [20]. The transcription activity profile of rescued RNPs using ApG or β-globin mRNA as primers was identical to that of wt RNPs, as expected, and not to that of mutant E361A, that is unable to use β-globin as primer [20] (Fig. 2 and Fig. S3).


Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication.

Jorba N, Coloma R, Ortín J - PLoS Pathog. (2009)

Phenotype of trans-complemented RNPs.The purified RNP preparations presented in Fig. 1 were tested for in vitro transcription primed with either ApG (red) or β-globin mRNA (green). The data are presented as percent of maximal value and represent the averages and ranges of two independent complementation experiments. The transcription activities parallel the values of NP accumulation presented in Fig. 1 and show that the rescued RNPs have a wt cap-snatching phenotype.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682650&req=5

ppat-1000462-g002: Phenotype of trans-complemented RNPs.The purified RNP preparations presented in Fig. 1 were tested for in vitro transcription primed with either ApG (red) or β-globin mRNA (green). The data are presented as percent of maximal value and represent the averages and ranges of two independent complementation experiments. The transcription activities parallel the values of NP accumulation presented in Fig. 1 and show that the rescued RNPs have a wt cap-snatching phenotype.
Mentions: The progeny RNPs contained the replication-defective PB2 allele, since (i) they could be purified by Ni2+-NTA-agarose chromatography and (ii) the mobility of the PB2 subunit in the Western-blot assay corresponded to the His-tagged subunit and not to the untagged one. It is important to mention that only His-tagged PB2 protein was detected in the purified RNPs and not the untagged counterpart, indicating that no transcription-defective polymerase was co-purified (Fig. 1B). Furthermore, the phenotype of the rescued RNPs was tested by determination of their in vitro transcription activity (Fig. 2). Since the transcription-defective mutants had alterations in their cap-binding pocket, they show low in vitro transcription activity when a mRNA is used as a cap-donor, whereas cap-independent transcription is observed with a general primer as the dinucleotide ApG [20]. The transcription activity profile of rescued RNPs using ApG or β-globin mRNA as primers was identical to that of wt RNPs, as expected, and not to that of mutant E361A, that is unable to use β-globin as primer [20] (Fig. 2 and Fig. S3).

Bottom Line: We used efficient systems for recombinant RNP transcription/replication in vivo and well-defined polymerase mutants deficient in either RNA replication or transcription to address the roles of the polymerase complex present in the template RNP and newly synthesised polymerase complexes during replication and transcription.The results of trans-complementation experiments showed that soluble polymerase complexes can synthesise progeny RNA in trans and become incorporated into progeny vRNPs, but only transcription in cis could be detected.In contrast, transcription of the vRNP would occur in cis and the resident polymerase complex would be responsible for mRNA synthesis and polyadenylation.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias, Campus de Cantoblanco, Madrid, Spain.

ABSTRACT
The influenza A viruses genome comprises eight single-stranded RNA segments of negative polarity. Each one is included in a ribonucleoprotein particle (vRNP) containing the polymerase complex and a number of nucleoprotein (NP) monomers. Viral RNA replication proceeds by formation of a complementary RNP of positive polarity (cRNP) that serves as intermediate to generate many progeny vRNPs. Transcription initiation takes place by a cap-snatching mechanism whereby the polymerase steals a cellular capped oligonucleotide and uses it as primer to copy the vRNP template. Transcription termination occurs prematurely at the polyadenylation signal, which the polymerase copies repeatedly to generate a 3'-terminal polyA. Here we studied the mechanisms of the viral RNA replication and transcription. We used efficient systems for recombinant RNP transcription/replication in vivo and well-defined polymerase mutants deficient in either RNA replication or transcription to address the roles of the polymerase complex present in the template RNP and newly synthesised polymerase complexes during replication and transcription. The results of trans-complementation experiments showed that soluble polymerase complexes can synthesise progeny RNA in trans and become incorporated into progeny vRNPs, but only transcription in cis could be detected. These results are compatible with a new model for virus RNA replication, whereby a template RNP would be replicated in trans by a soluble polymerase complex and a polymerase complex distinct from the replicative enzyme would direct the encapsidation of progeny vRNA. In contrast, transcription of the vRNP would occur in cis and the resident polymerase complex would be responsible for mRNA synthesis and polyadenylation.

Show MeSH
Related in: MedlinePlus