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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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Intracistronic polymerase complementation during influenza virus RNA replication.(A) Cultures of HEK293T cells were transfected with plasmids expressing a virus-like replicon of 248 nt, the NP and various combinations of the polymerase subunits as indicated in the diagram. The potential RNPs that could be generated are also depicted in the diagram, as well as the expected progeny RNPs, depending on the replication phenotype of the polymerase mutants used. (B) The progeny RNPs were purified from total cell extracts over Ni2+-NTA-agarose resin and analysed by Western-blot with anti-NP antibodies. The top panel presents the accumulation of NP in the total cell extract whereas the bottom panel shows the NP accumulation of purified RNPs. The integrity of the purified RNPs is verified by Western-blot using anti-PB2 and anti-PA antibodies. In the bottom graph the average NP accumulation and standard deviation of three independent complementation experiments are presented as percent of maximal value.
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ppat-1000462-g001: Intracistronic polymerase complementation during influenza virus RNA replication.(A) Cultures of HEK293T cells were transfected with plasmids expressing a virus-like replicon of 248 nt, the NP and various combinations of the polymerase subunits as indicated in the diagram. The potential RNPs that could be generated are also depicted in the diagram, as well as the expected progeny RNPs, depending on the replication phenotype of the polymerase mutants used. (B) The progeny RNPs were purified from total cell extracts over Ni2+-NTA-agarose resin and analysed by Western-blot with anti-NP antibodies. The top panel presents the accumulation of NP in the total cell extract whereas the bottom panel shows the NP accumulation of purified RNPs. The integrity of the purified RNPs is verified by Western-blot using anti-PB2 and anti-PA antibodies. In the bottom graph the average NP accumulation and standard deviation of three independent complementation experiments are presented as percent of maximal value.

Mentions: Using the approaches indicated above we first addressed the question whether the replication deficiency of point mutants within the N-terminus of PB2 [36] could be rescued in trans by co-expression of PB2 point mutants defective in cap-binding [20]. Cultures of HEK293T cells were co-transfected with plasmids encoding PB1, PA, NP and a deleted NS virus replicon (clone 23, 248 nt in length; [14],[15]). In addition, either PB2wt or PB2 mutants R142A or F130A (replication-defective) or mutant E361A (transcription-defective) were co-expressed. Alternatively, pair wise combinations of these PB2 mutants were co-expressed (R142A+E361A and F130A+E361A). Among the PB2 proteins expressed, either wt or the replication-defective mutants R142A or F130A were His-tagged at the C-terminus, a modification that does not alter their biological activity and allows the efficient purification of the in vivo RNP replication progeny [18]. The expression levels of all PB2 mutants were shown to be similar to that of PB2wt (Fig. S1) and the untagged PB2wt was used as a control for purification (see diagram of the experimental setting in Fig. 1A). After incubation, the cell extracts were used for Ni2+-NTA-agarose purification as described in Materials and Methods and the accumulation of progeny RNPs was determined by means of Western-blot assays using anti-NP sera. The purification of the complete RNPs was verified by Western-blot with antibodies specific for PB2 and PA (Fig. 1B). This strategy allows measuring the replication capacity of the RNPs formed in vivo, as omitting any RNP element or using a defective point mutant leads to undetectable RNP accumulation [15],[36],[37]. Amplification of virus RNPs was expected for wt and mutant polymerase containing transcription-defective PB2 (E361A), but not for those containing replication-defective PB2 (R142A and F130A). However, since no tag is present in the former mutant, only RNPs derived from cultures containing PB2His were expected in the Ni2+-NTA-agarose purified material. This was indeed the case, as shown in Fig. 1B. If the transcription-defective polymerase were able to rescue in trans the defect in replication of polymerase mutants R142A or F130A, one would expect the accumulation and purification of RNPs containing these mutant PB2. The results obtained by the co-expression of pairs of replication- and transcription-defective polymerases indicate that such prediction is hold (Fig. 1B). The transcription-defective mutant could rescue both R142A and F130A alleles and similar rescue was obtained when other transcription-defective mutants, like H357A, K370A, F404A [20] were used (Fig. S2).


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

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

Intracistronic polymerase complementation during influenza virus RNA replication.(A) Cultures of HEK293T cells were transfected with plasmids expressing a virus-like replicon of 248 nt, the NP and various combinations of the polymerase subunits as indicated in the diagram. The potential RNPs that could be generated are also depicted in the diagram, as well as the expected progeny RNPs, depending on the replication phenotype of the polymerase mutants used. (B) The progeny RNPs were purified from total cell extracts over Ni2+-NTA-agarose resin and analysed by Western-blot with anti-NP antibodies. The top panel presents the accumulation of NP in the total cell extract whereas the bottom panel shows the NP accumulation of purified RNPs. The integrity of the purified RNPs is verified by Western-blot using anti-PB2 and anti-PA antibodies. In the bottom graph the average NP accumulation and standard deviation of three independent complementation experiments are presented as percent of maximal value.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000462-g001: Intracistronic polymerase complementation during influenza virus RNA replication.(A) Cultures of HEK293T cells were transfected with plasmids expressing a virus-like replicon of 248 nt, the NP and various combinations of the polymerase subunits as indicated in the diagram. The potential RNPs that could be generated are also depicted in the diagram, as well as the expected progeny RNPs, depending on the replication phenotype of the polymerase mutants used. (B) The progeny RNPs were purified from total cell extracts over Ni2+-NTA-agarose resin and analysed by Western-blot with anti-NP antibodies. The top panel presents the accumulation of NP in the total cell extract whereas the bottom panel shows the NP accumulation of purified RNPs. The integrity of the purified RNPs is verified by Western-blot using anti-PB2 and anti-PA antibodies. In the bottom graph the average NP accumulation and standard deviation of three independent complementation experiments are presented as percent of maximal value.
Mentions: Using the approaches indicated above we first addressed the question whether the replication deficiency of point mutants within the N-terminus of PB2 [36] could be rescued in trans by co-expression of PB2 point mutants defective in cap-binding [20]. Cultures of HEK293T cells were co-transfected with plasmids encoding PB1, PA, NP and a deleted NS virus replicon (clone 23, 248 nt in length; [14],[15]). In addition, either PB2wt or PB2 mutants R142A or F130A (replication-defective) or mutant E361A (transcription-defective) were co-expressed. Alternatively, pair wise combinations of these PB2 mutants were co-expressed (R142A+E361A and F130A+E361A). Among the PB2 proteins expressed, either wt or the replication-defective mutants R142A or F130A were His-tagged at the C-terminus, a modification that does not alter their biological activity and allows the efficient purification of the in vivo RNP replication progeny [18]. The expression levels of all PB2 mutants were shown to be similar to that of PB2wt (Fig. S1) and the untagged PB2wt was used as a control for purification (see diagram of the experimental setting in Fig. 1A). After incubation, the cell extracts were used for Ni2+-NTA-agarose purification as described in Materials and Methods and the accumulation of progeny RNPs was determined by means of Western-blot assays using anti-NP sera. The purification of the complete RNPs was verified by Western-blot with antibodies specific for PB2 and PA (Fig. 1B). This strategy allows measuring the replication capacity of the RNPs formed in vivo, as omitting any RNP element or using a defective point mutant leads to undetectable RNP accumulation [15],[36],[37]. Amplification of virus RNPs was expected for wt and mutant polymerase containing transcription-defective PB2 (E361A), but not for those containing replication-defective PB2 (R142A and F130A). However, since no tag is present in the former mutant, only RNPs derived from cultures containing PB2His were expected in the Ni2+-NTA-agarose purified material. This was indeed the case, as shown in Fig. 1B. If the transcription-defective polymerase were able to rescue in trans the defect in replication of polymerase mutants R142A or F130A, one would expect the accumulation and purification of RNPs containing these mutant PB2. The results obtained by the co-expression of pairs of replication- and transcription-defective polymerases indicate that such prediction is hold (Fig. 1B). The transcription-defective mutant could rescue both R142A and F130A alleles and similar rescue was obtained when other transcription-defective mutants, like H357A, K370A, F404A [20] were used (Fig. S2).

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