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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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Analysis of the genomic RNA present in purified RNPs.Cultures of HEK293T cells were transfected with plasmids expressing the NP and various combinations of the polymerase subunits and purified RNPs containing either replication-deficient or transcription-deficient polymerase, as indicated in the diagram of Fig. 3A. The purified progeny RNPs were purified from total cell extracts by affinity chromatography over Ni2+-NTA-agarose and the RNA present in the purified RNPs was extracted as described under Materials and Methods. (A) Hybridisation controls. Dilutions of plasmid pHHΔNS clone 23, containing the sequence of the RNP replicons used (+), or total yeast RNA (−) were applied onto a nylon filter as hybridisation controls (from 103 to 100 ng, as indicated at the top of the figure). Hybridisation was performed using a positive-polarity or a negative-polarity probe comprising the full-length insert present in pHHΔNS clone 23, thereby detecting either vRNA or cRNA, respectively. (B) Aliquots of the RNA present in purified RNPs obtained from cultures transfected with the mixtures indicated at the bottom of the figure were hybridised in parallel to the hybridisation controls shown in (A) and the hybridisation signals were quantitated in a phosphorimager, using the signals in (A) to standardise the relative hybridisation efficiency of the positive- and negative-polarity probes. The results for vRNA (blue) and cRNA (orange) are presented as percent of the maximal signal and represent the averages and standard deviations of 4 quantisations.
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ppat-1000462-g004: Analysis of the genomic RNA present in purified RNPs.Cultures of HEK293T cells were transfected with plasmids expressing the NP and various combinations of the polymerase subunits and purified RNPs containing either replication-deficient or transcription-deficient polymerase, as indicated in the diagram of Fig. 3A. The purified progeny RNPs were purified from total cell extracts by affinity chromatography over Ni2+-NTA-agarose and the RNA present in the purified RNPs was extracted as described under Materials and Methods. (A) Hybridisation controls. Dilutions of plasmid pHHΔNS clone 23, containing the sequence of the RNP replicons used (+), or total yeast RNA (−) were applied onto a nylon filter as hybridisation controls (from 103 to 100 ng, as indicated at the top of the figure). Hybridisation was performed using a positive-polarity or a negative-polarity probe comprising the full-length insert present in pHHΔNS clone 23, thereby detecting either vRNA or cRNA, respectively. (B) Aliquots of the RNA present in purified RNPs obtained from cultures transfected with the mixtures indicated at the bottom of the figure were hybridised in parallel to the hybridisation controls shown in (A) and the hybridisation signals were quantitated in a phosphorimager, using the signals in (A) to standardise the relative hybridisation efficiency of the positive- and negative-polarity probes. The results for vRNA (blue) and cRNA (orange) are presented as percent of the maximal signal and represent the averages and standard deviations of 4 quantisations.

Mentions: The rescue of viral RNPs containing the mutant R142A polymerase complex, as described above, enabled us to purify these RNPs and use them as templates for a second in vivo reconstitution experiment in which instead of a template RNA we introduced the rescued and purified R142A mutant RNPs in the system. This strategy ensured that only replication-defective RNPs are used as templates for in vivo replication and allowed us to ask whether the resident polymerase complex or a distinct, soluble polymerase is responsible for replication of RNPs in vivo. The concentration and biological activity of these purified RNPs was first controlled by Western-blot and in vitro transcription. The results are presented in Fig. 3B and show that higher yields were obtained for RNPs containing the E361A mutation in PB2 than those containing the R142A mutation. This was expected, as the latter could only be amplified by trans-complementation (see Fig. 1 above). The transcription phenotype of these purified RNPs was in agreement with the mutations present in PB2 (Fig. 3B, right panel). Therefore, cultures of HEK293T cells were co-transfected with purified RNPs containing either the R142A mutation or the E361A mutation in PB2, plasmids encoding PB1, PA, NP and a plasmid encoding either PB2-His R142A (replication-defective) or PB2-His E361A (transcription-defective) (see Fig. 3A for a diagram of the experimental setting). As controls, the RNPs were co-transfected with empty pCMV vector or the expression plasmids were transfected in the absence of template RNPs. The intracellular accumulation of progeny RNPs was determined by Ni2+-NTA-agarose purification, Western-blot and in vitro transcription as indicated above and the results are presented in Fig. 3C and Fig. 4. The cultures co-transfected with RNPs E361A and plasmids including PB2 E361A (Fig. 3C; RNP361-Pol361) served as positive control and, indeed gave rise to the accumulation of RNPs to levels similar to the standard, wt system (see Fig. 1B, HisPB2). No background was observed when template RNPs were transfected (Fig. 3C; RNP142/CMV, RNP361/CMV). A fraction of the NP expressed was retained in the Ni2+-NTA-agarose resin (Fig. 3C; Pol142, Pol361) and defined the background level of the purification protocol (but see Fig. 4 below). The co-transfection of RNPs containing mutation PB2 R142A and the same mutant plasmids yielded no increase above background in the level of purified RNPs (Fig. 3C; RNP142/Pol142) but the mixed transfection of RNPs with the mutation PB2 R142A and the expression plasmids with mutation PB2 E361A led to a high level of replication (around 80% of control values) (Fig. 3C; RNP142/Pol361). To verify these results and to determine the polarity of the progeny RNA, similar experiments were carried out and the RNA present in the purified his-RNPs was analysed by hybridisation with positive- and negative-polarity RNA probes comprising the NS sequence. The results reinforced the data obtained by Western-blot and indicated that most of the progeny RNPs are vRNPs (Fig. 4), as previously reported [15]. The accumulation and phenotype of the progeny RNPs was also verified by in vitro transcription using either ApG or β-globin as primers (Fig. 5). The accumulations observed paralleled those shown in Fig. 3 but the background levels from samples Pol142, Pol361 and RNP142-Pol142 were negligible. Much higher activity levels were obtained with ApG primer, indicating that the progeny RNPs contained PB2 with mutation E361A. The results presented in Figs. 3 and 4 indicate that a polymerase complex distinct from that present in the template RNP can perform the replicative synthesis of viral RNA. The high level of replication detected by trans-complementation suggests that virus RNA replication mostly occurs in trans. It could be argued that the mutation R142A in PB2 might destabilise the polymerase-promoter complex, allowing the efficient replacement by a polymerase complex containing the E361A mutation. However, RNPs containing the R142A mutation are as efficient in transcription as wt RNPs, suggesting that they are not affected in promoter recognition.


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

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

Analysis of the genomic RNA present in purified RNPs.Cultures of HEK293T cells were transfected with plasmids expressing the NP and various combinations of the polymerase subunits and purified RNPs containing either replication-deficient or transcription-deficient polymerase, as indicated in the diagram of Fig. 3A. The purified progeny RNPs were purified from total cell extracts by affinity chromatography over Ni2+-NTA-agarose and the RNA present in the purified RNPs was extracted as described under Materials and Methods. (A) Hybridisation controls. Dilutions of plasmid pHHΔNS clone 23, containing the sequence of the RNP replicons used (+), or total yeast RNA (−) were applied onto a nylon filter as hybridisation controls (from 103 to 100 ng, as indicated at the top of the figure). Hybridisation was performed using a positive-polarity or a negative-polarity probe comprising the full-length insert present in pHHΔNS clone 23, thereby detecting either vRNA or cRNA, respectively. (B) Aliquots of the RNA present in purified RNPs obtained from cultures transfected with the mixtures indicated at the bottom of the figure were hybridised in parallel to the hybridisation controls shown in (A) and the hybridisation signals were quantitated in a phosphorimager, using the signals in (A) to standardise the relative hybridisation efficiency of the positive- and negative-polarity probes. The results for vRNA (blue) and cRNA (orange) are presented as percent of the maximal signal and represent the averages and standard deviations of 4 quantisations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682650&req=5

ppat-1000462-g004: Analysis of the genomic RNA present in purified RNPs.Cultures of HEK293T cells were transfected with plasmids expressing the NP and various combinations of the polymerase subunits and purified RNPs containing either replication-deficient or transcription-deficient polymerase, as indicated in the diagram of Fig. 3A. The purified progeny RNPs were purified from total cell extracts by affinity chromatography over Ni2+-NTA-agarose and the RNA present in the purified RNPs was extracted as described under Materials and Methods. (A) Hybridisation controls. Dilutions of plasmid pHHΔNS clone 23, containing the sequence of the RNP replicons used (+), or total yeast RNA (−) were applied onto a nylon filter as hybridisation controls (from 103 to 100 ng, as indicated at the top of the figure). Hybridisation was performed using a positive-polarity or a negative-polarity probe comprising the full-length insert present in pHHΔNS clone 23, thereby detecting either vRNA or cRNA, respectively. (B) Aliquots of the RNA present in purified RNPs obtained from cultures transfected with the mixtures indicated at the bottom of the figure were hybridised in parallel to the hybridisation controls shown in (A) and the hybridisation signals were quantitated in a phosphorimager, using the signals in (A) to standardise the relative hybridisation efficiency of the positive- and negative-polarity probes. The results for vRNA (blue) and cRNA (orange) are presented as percent of the maximal signal and represent the averages and standard deviations of 4 quantisations.
Mentions: The rescue of viral RNPs containing the mutant R142A polymerase complex, as described above, enabled us to purify these RNPs and use them as templates for a second in vivo reconstitution experiment in which instead of a template RNA we introduced the rescued and purified R142A mutant RNPs in the system. This strategy ensured that only replication-defective RNPs are used as templates for in vivo replication and allowed us to ask whether the resident polymerase complex or a distinct, soluble polymerase is responsible for replication of RNPs in vivo. The concentration and biological activity of these purified RNPs was first controlled by Western-blot and in vitro transcription. The results are presented in Fig. 3B and show that higher yields were obtained for RNPs containing the E361A mutation in PB2 than those containing the R142A mutation. This was expected, as the latter could only be amplified by trans-complementation (see Fig. 1 above). The transcription phenotype of these purified RNPs was in agreement with the mutations present in PB2 (Fig. 3B, right panel). Therefore, cultures of HEK293T cells were co-transfected with purified RNPs containing either the R142A mutation or the E361A mutation in PB2, plasmids encoding PB1, PA, NP and a plasmid encoding either PB2-His R142A (replication-defective) or PB2-His E361A (transcription-defective) (see Fig. 3A for a diagram of the experimental setting). As controls, the RNPs were co-transfected with empty pCMV vector or the expression plasmids were transfected in the absence of template RNPs. The intracellular accumulation of progeny RNPs was determined by Ni2+-NTA-agarose purification, Western-blot and in vitro transcription as indicated above and the results are presented in Fig. 3C and Fig. 4. The cultures co-transfected with RNPs E361A and plasmids including PB2 E361A (Fig. 3C; RNP361-Pol361) served as positive control and, indeed gave rise to the accumulation of RNPs to levels similar to the standard, wt system (see Fig. 1B, HisPB2). No background was observed when template RNPs were transfected (Fig. 3C; RNP142/CMV, RNP361/CMV). A fraction of the NP expressed was retained in the Ni2+-NTA-agarose resin (Fig. 3C; Pol142, Pol361) and defined the background level of the purification protocol (but see Fig. 4 below). The co-transfection of RNPs containing mutation PB2 R142A and the same mutant plasmids yielded no increase above background in the level of purified RNPs (Fig. 3C; RNP142/Pol142) but the mixed transfection of RNPs with the mutation PB2 R142A and the expression plasmids with mutation PB2 E361A led to a high level of replication (around 80% of control values) (Fig. 3C; RNP142/Pol361). To verify these results and to determine the polarity of the progeny RNA, similar experiments were carried out and the RNA present in the purified his-RNPs was analysed by hybridisation with positive- and negative-polarity RNA probes comprising the NS sequence. The results reinforced the data obtained by Western-blot and indicated that most of the progeny RNPs are vRNPs (Fig. 4), as previously reported [15]. The accumulation and phenotype of the progeny RNPs was also verified by in vitro transcription using either ApG or β-globin as primers (Fig. 5). The accumulations observed paralleled those shown in Fig. 3 but the background levels from samples Pol142, Pol361 and RNP142-Pol142 were negligible. Much higher activity levels were obtained with ApG primer, indicating that the progeny RNPs contained PB2 with mutation E361A. The results presented in Figs. 3 and 4 indicate that a polymerase complex distinct from that present in the template RNP can perform the replicative synthesis of viral RNA. The high level of replication detected by trans-complementation suggests that virus RNA replication mostly occurs in trans. It could be argued that the mutation R142A in PB2 might destabilise the polymerase-promoter complex, allowing the efficient replacement by a polymerase complex containing the E361A mutation. However, RNPs containing the R142A mutation are as efficient in transcription as wt RNPs, suggesting that they are not affected in promoter recognition.

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