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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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Genetically distinct RNPs cannot transcribe reciprocally in vitro.(A) Recombinant RNPs were generated by in vivo amplification as described in Materials and Methods and the legend to Fig. 1, using either wt (WT) or transcription-defective (E361A) polymerase. Short (clone 23 -248 nt-), long (CAT -720 nt-) or no (CTRL) RNA replicons were used. The RNPs were purified by Ni2+-NTA-agarose chromatography and their transcription activity was determined. Top panel shows the phosphorimager data and bottom panel presents the quantisation, indicating the cap-snatching defective phenotype of RNPs containing the E361A mutation in PB2. (B) Purified wt (WT CAT) or E361A mutant RNPs (361 CAT) containing the cat gene were transcribed in vitro, using β-globin mRNA as primer, either alone or in a mixture with wt clone 23 RNPs (cl23). The transcription products were purified and analysed by polyacrylamide-urea denaturing gel electrophoresis. The mobility of molecular weight markers is shown to the left and the position of cat and clone 23 transcripts is indicated to the right.
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ppat-1000462-g006: Genetically distinct RNPs cannot transcribe reciprocally in vitro.(A) Recombinant RNPs were generated by in vivo amplification as described in Materials and Methods and the legend to Fig. 1, using either wt (WT) or transcription-defective (E361A) polymerase. Short (clone 23 -248 nt-), long (CAT -720 nt-) or no (CTRL) RNA replicons were used. The RNPs were purified by Ni2+-NTA-agarose chromatography and their transcription activity was determined. Top panel shows the phosphorimager data and bottom panel presents the quantisation, indicating the cap-snatching defective phenotype of RNPs containing the E361A mutation in PB2. (B) Purified wt (WT CAT) or E361A mutant RNPs (361 CAT) containing the cat gene were transcribed in vitro, using β-globin mRNA as primer, either alone or in a mixture with wt clone 23 RNPs (cl23). The transcription products were purified and analysed by polyacrylamide-urea denaturing gel electrophoresis. The mobility of molecular weight markers is shown to the left and the position of cat and clone 23 transcripts is indicated to the right.

Mentions: It is well established that vRNPs can transcribe mRNAs in the absence of any newly synthesised viral proteins (primary transcription) [39],[40] and highly purified recombinant RNPs can transcribe in vitro [18] (R. Coloma, unpublished results). However, it is not clear whether transcription takes place intramolecularly, i.e. in cis, or a RNP can transcribe another RNP. To test this possibility we reconstituted in vivo two genetically distinct RNPs, one containing a cat virus replicon (with the cat negative-polarity ORF flanked by the UTRs of the NS segment of influenza virus), the other one being the NS deletion mutant clone 23 [14],[15]. Both RNPs contained a His-tagged PB2 subunit to allow purification by affinity chromatography as indicated above but two PB2 alleles were used, either wt or mutant E361A, which is defective in the recognition of the cap structure [20]. Purified RNPs were used either separately or in combination for in vitro transcription with ApG or β-globin as primers and the transcription products were analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. The results are presented in Fig. 6. As expected, the purified wt RNPs were active, both when ApG or β-globin were used as primers (Fig. 6A). The RNPs containing the mutation PB2 E361A could transcribe mRNA with ApG as primer, but did so less efficiently when using β-globin mRNA as primer donor (Fig. 6A). These results allowed us to test whether a purified, wt clone 23 RNP could rescue the transcription activity of mutant E361A cat RNP in trans, since the mRNA products would be distinguishable by size (720 nt versus 248 nt). The wild-type cat RNPs could transcribe efficiently, both when incubated on their own and when mixed with clone 23 RNPs (Fig. 6B, middle panel). The cat RNPs containing PB2 E361A only produced background transcription levels and no increase in the amount of cat mRNA was observed when wt clone 23 RNP was co-transcribed (Fig. 6B, right panel). Quantisation of the relevant bands indicated that the increase in cat transcript in the co-transcription of clone 23 RNP+E361 cat RNP versus the transcription of E361 cat RNP was less than 3% of the cat transcript value obtained by co-transcription of clone 23 RNP+wt cat RNP. These results suggest that, at least in vitro, no transcription in trans among different RNPs takes place.


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

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

Genetically distinct RNPs cannot transcribe reciprocally in vitro.(A) Recombinant RNPs were generated by in vivo amplification as described in Materials and Methods and the legend to Fig. 1, using either wt (WT) or transcription-defective (E361A) polymerase. Short (clone 23 -248 nt-), long (CAT -720 nt-) or no (CTRL) RNA replicons were used. The RNPs were purified by Ni2+-NTA-agarose chromatography and their transcription activity was determined. Top panel shows the phosphorimager data and bottom panel presents the quantisation, indicating the cap-snatching defective phenotype of RNPs containing the E361A mutation in PB2. (B) Purified wt (WT CAT) or E361A mutant RNPs (361 CAT) containing the cat gene were transcribed in vitro, using β-globin mRNA as primer, either alone or in a mixture with wt clone 23 RNPs (cl23). The transcription products were purified and analysed by polyacrylamide-urea denaturing gel electrophoresis. The mobility of molecular weight markers is shown to the left and the position of cat and clone 23 transcripts is indicated to the right.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682650&req=5

ppat-1000462-g006: Genetically distinct RNPs cannot transcribe reciprocally in vitro.(A) Recombinant RNPs were generated by in vivo amplification as described in Materials and Methods and the legend to Fig. 1, using either wt (WT) or transcription-defective (E361A) polymerase. Short (clone 23 -248 nt-), long (CAT -720 nt-) or no (CTRL) RNA replicons were used. The RNPs were purified by Ni2+-NTA-agarose chromatography and their transcription activity was determined. Top panel shows the phosphorimager data and bottom panel presents the quantisation, indicating the cap-snatching defective phenotype of RNPs containing the E361A mutation in PB2. (B) Purified wt (WT CAT) or E361A mutant RNPs (361 CAT) containing the cat gene were transcribed in vitro, using β-globin mRNA as primer, either alone or in a mixture with wt clone 23 RNPs (cl23). The transcription products were purified and analysed by polyacrylamide-urea denaturing gel electrophoresis. The mobility of molecular weight markers is shown to the left and the position of cat and clone 23 transcripts is indicated to the right.
Mentions: It is well established that vRNPs can transcribe mRNAs in the absence of any newly synthesised viral proteins (primary transcription) [39],[40] and highly purified recombinant RNPs can transcribe in vitro [18] (R. Coloma, unpublished results). However, it is not clear whether transcription takes place intramolecularly, i.e. in cis, or a RNP can transcribe another RNP. To test this possibility we reconstituted in vivo two genetically distinct RNPs, one containing a cat virus replicon (with the cat negative-polarity ORF flanked by the UTRs of the NS segment of influenza virus), the other one being the NS deletion mutant clone 23 [14],[15]. Both RNPs contained a His-tagged PB2 subunit to allow purification by affinity chromatography as indicated above but two PB2 alleles were used, either wt or mutant E361A, which is defective in the recognition of the cap structure [20]. Purified RNPs were used either separately or in combination for in vitro transcription with ApG or β-globin as primers and the transcription products were analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. The results are presented in Fig. 6. As expected, the purified wt RNPs were active, both when ApG or β-globin were used as primers (Fig. 6A). The RNPs containing the mutation PB2 E361A could transcribe mRNA with ApG as primer, but did so less efficiently when using β-globin mRNA as primer donor (Fig. 6A). These results allowed us to test whether a purified, wt clone 23 RNP could rescue the transcription activity of mutant E361A cat RNP in trans, since the mRNA products would be distinguishable by size (720 nt versus 248 nt). The wild-type cat RNPs could transcribe efficiently, both when incubated on their own and when mixed with clone 23 RNPs (Fig. 6B, middle panel). The cat RNPs containing PB2 E361A only produced background transcription levels and no increase in the amount of cat mRNA was observed when wt clone 23 RNP was co-transcribed (Fig. 6B, right panel). Quantisation of the relevant bands indicated that the increase in cat transcript in the co-transcription of clone 23 RNP+E361 cat RNP versus the transcription of E361 cat RNP was less than 3% of the cat transcript value obtained by co-transcription of clone 23 RNP+wt cat RNP. These results suggest that, at least in vitro, no transcription in trans among different RNPs takes place.

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