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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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A model for influenza RNP replication and transcription.(A) Various steps in the process of RNP replication. The coloured NP indicates the polarity of the templates (brown: positive polarity; green: negative polarity). The parental polymerase complex is denoted by solid colours while the semi-transparent colouring indicates a newly synthesised complex. See text for details. (B) Various steps in the process of RNP transcription. The capped primer is depicted as a thick line with a red circle. See text for details.
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ppat-1000462-g008: A model for influenza RNP replication and transcription.(A) Various steps in the process of RNP replication. The coloured NP indicates the polarity of the templates (brown: positive polarity; green: negative polarity). The parental polymerase complex is denoted by solid colours while the semi-transparent colouring indicates a newly synthesised complex. See text for details. (B) Various steps in the process of RNP transcription. The capped primer is depicted as a thick line with a red circle. See text for details.

Mentions: The processes of virus RNA replication and transcription usually require the action of one to several virus-specific proteins, notably the RNA-dependent RNA polymerase (RdRp), and various host cell factors (for a review see [43]. To unravel the complex procedures involved, genetic experimental approaches have been particularly useful. For example, genetic data have strongly supported the requirement of RdRp oligomerisation for RNA replication in several virus groups, like poliovirus [44],[45], HCV [46],[47] and Sendai virus [48],[49]. Early studies on the dominance of RNA-synthesis negative ts mutants of VSV suggested that the oligomerisation of virus factors involved in RNA replication is an essential step in the process [50], a conclusion that could be also verified in the poliovirus system [51]. More generally, the multimeric nature of complex viral systems, as the virus particles, has profound consequences in the apparent phenotype observed [52],[53]. In the case of influenza, early data on the intragenic complementation of mutants affecting the PB1 and PA proteins suggested the potential role of virus polymerase interactions in the infectious cycle [54]–[56] and the recent biochemical evidence for virus polymerase oligomerisation supported such contention [57]. Here we have taken advantage of the availability of well-established recombinant systems for RNP replication and transcription and well-characterised polymerase mutants to address specific questions on the mechanisms of these processes. Due to the segmented nature of the influenza virus genome it is essential to use mutant polymerases having phenotypically distinct mutations in the same subunit, thus avoiding the problems of reassortment. Hence, we have used point mutants of polymerase PB2 subunit that abolish RNA replication but transcribe normally (R142A or F130A) [36] and/or mutants that are defective in cap-recognition and transcribe poorly, but replicate virus RNA normally (E361A among others) [20]. With these experimental tools we have asked whether the polymerase complex present in an RNP actually perform the replicative or transcriptional synthesis of RNA and whether the polymerase complex present in the progeny RNP is identical to that performing replicative synthesis of RNA. Our results will be discussed on the basis of the model presented in Fig. 8, in which only the replication step cRNP-to-vRNP is presented. The results shown in Figs. 1 and 2 indicated that two such phenotypically distinct mutant polymerases can complement to perform viral RNP replication in vivo and demonstrated that a replication-defective polymerase can be incorporated into progeny RNPs. These results are consistent with the model presented in Fig. 8A, step 4, that suggest that a polymerase complex distinct from that performing replicative synthesis is involved in the recognition of the 5′-end of the progeny vRNA. This model is also consistent with the results published earlier indicating that a pre-expressed polymerase can protect newly synthesised cRNA [13],[38]. The identity of the replicative polymerase complex could be tested by directly transfecting mutant RNPs as templates for the replication reaction and asking whether co-expressed replication-defective or transcription-defective polymerase complexes could carry out the replication process in trans. The results shown in Figs. 3 and 4 demonstrated that a polymerase complex genetically distinguishable from that present in the parental RNP was able to perform replication and became incorporated into the progeny RNPs. These results are compatible with the model presented in Fig. 8A, steps 2–4, whereby a soluble polymerase complex would interact with that resident in the parental RNP and gain access to the 3′-terminal sequence in the promoter. Such polymerase-polymerase interaction is supported by the genetic data presented here, by the intragenic complementation reported earlier [54],[55] and by the oligomerisation of influenza polymerase in vivo [57]. Although not shown in Fig. 8A, we can not exclude that a host factor(s) participate in the polymerase-polymerase interaction and in fact several nuclear factors have been described previously that could play such a role [25]–[30],[32]. The trans-replication model depicted in Fig. 8A, steps 2–4 relates to the cRNP-to-vRNP phase in replication. However, earlier data published on the protection of newly synthesised cRNA by pre-expressed polymerase would suggest that the vRNP-to-cRNA phase can occur in cis, since a pre-expressed, catalytically inactive polymerase allowed the accumulation of cRNA in cicloheximide-treated, virus-infected cells [13].


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

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

A model for influenza RNP replication and transcription.(A) Various steps in the process of RNP replication. The coloured NP indicates the polarity of the templates (brown: positive polarity; green: negative polarity). The parental polymerase complex is denoted by solid colours while the semi-transparent colouring indicates a newly synthesised complex. See text for details. (B) Various steps in the process of RNP transcription. The capped primer is depicted as a thick line with a red circle. See text for details.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000462-g008: A model for influenza RNP replication and transcription.(A) Various steps in the process of RNP replication. The coloured NP indicates the polarity of the templates (brown: positive polarity; green: negative polarity). The parental polymerase complex is denoted by solid colours while the semi-transparent colouring indicates a newly synthesised complex. See text for details. (B) Various steps in the process of RNP transcription. The capped primer is depicted as a thick line with a red circle. See text for details.
Mentions: The processes of virus RNA replication and transcription usually require the action of one to several virus-specific proteins, notably the RNA-dependent RNA polymerase (RdRp), and various host cell factors (for a review see [43]. To unravel the complex procedures involved, genetic experimental approaches have been particularly useful. For example, genetic data have strongly supported the requirement of RdRp oligomerisation for RNA replication in several virus groups, like poliovirus [44],[45], HCV [46],[47] and Sendai virus [48],[49]. Early studies on the dominance of RNA-synthesis negative ts mutants of VSV suggested that the oligomerisation of virus factors involved in RNA replication is an essential step in the process [50], a conclusion that could be also verified in the poliovirus system [51]. More generally, the multimeric nature of complex viral systems, as the virus particles, has profound consequences in the apparent phenotype observed [52],[53]. In the case of influenza, early data on the intragenic complementation of mutants affecting the PB1 and PA proteins suggested the potential role of virus polymerase interactions in the infectious cycle [54]–[56] and the recent biochemical evidence for virus polymerase oligomerisation supported such contention [57]. Here we have taken advantage of the availability of well-established recombinant systems for RNP replication and transcription and well-characterised polymerase mutants to address specific questions on the mechanisms of these processes. Due to the segmented nature of the influenza virus genome it is essential to use mutant polymerases having phenotypically distinct mutations in the same subunit, thus avoiding the problems of reassortment. Hence, we have used point mutants of polymerase PB2 subunit that abolish RNA replication but transcribe normally (R142A or F130A) [36] and/or mutants that are defective in cap-recognition and transcribe poorly, but replicate virus RNA normally (E361A among others) [20]. With these experimental tools we have asked whether the polymerase complex present in an RNP actually perform the replicative or transcriptional synthesis of RNA and whether the polymerase complex present in the progeny RNP is identical to that performing replicative synthesis of RNA. Our results will be discussed on the basis of the model presented in Fig. 8, in which only the replication step cRNP-to-vRNP is presented. The results shown in Figs. 1 and 2 indicated that two such phenotypically distinct mutant polymerases can complement to perform viral RNP replication in vivo and demonstrated that a replication-defective polymerase can be incorporated into progeny RNPs. These results are consistent with the model presented in Fig. 8A, step 4, that suggest that a polymerase complex distinct from that performing replicative synthesis is involved in the recognition of the 5′-end of the progeny vRNA. This model is also consistent with the results published earlier indicating that a pre-expressed polymerase can protect newly synthesised cRNA [13],[38]. The identity of the replicative polymerase complex could be tested by directly transfecting mutant RNPs as templates for the replication reaction and asking whether co-expressed replication-defective or transcription-defective polymerase complexes could carry out the replication process in trans. The results shown in Figs. 3 and 4 demonstrated that a polymerase complex genetically distinguishable from that present in the parental RNP was able to perform replication and became incorporated into the progeny RNPs. These results are compatible with the model presented in Fig. 8A, steps 2–4, whereby a soluble polymerase complex would interact with that resident in the parental RNP and gain access to the 3′-terminal sequence in the promoter. Such polymerase-polymerase interaction is supported by the genetic data presented here, by the intragenic complementation reported earlier [54],[55] and by the oligomerisation of influenza polymerase in vivo [57]. Although not shown in Fig. 8A, we can not exclude that a host factor(s) participate in the polymerase-polymerase interaction and in fact several nuclear factors have been described previously that could play such a role [25]–[30],[32]. The trans-replication model depicted in Fig. 8A, steps 2–4 relates to the cRNP-to-vRNP phase in replication. However, earlier data published on the protection of newly synthesised cRNA by pre-expressed polymerase would suggest that the vRNP-to-cRNA phase can occur in cis, since a pre-expressed, catalytically inactive polymerase allowed the accumulation of cRNA in cicloheximide-treated, virus-infected cells [13].

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