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

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.

Figure 1. 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.

Figure 2. 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.

Figure 3. Replication of RNPs by a soluble polymerase complex in trans.

(A) Cultures of HEK293T cells were transfected with plasmids expressing the NP and various combinations of the polymerase subunits as indicated in the diagram. In some cases, purified RNPs containing replication-deficient or transcription-deficient polymerase were also transfected. The expected progeny RNPs are also depicted, depending on the replication phenotype of the polymerase mutants used. (B) Left panel: The amount of replication-deficient (R142) or transcription-deficient (R361) RNPs transfected was ascertained by Western-blot with anti-NP and anti-PB2 antibodies. The mobility of molecular weight markers is shown to the left and the position of PB2 and NP proteins is indicated to the right. Right panel: The transcription phenotype of the RNPs transfected was determined by in vitro transcription using ApG (ApG) or β-globin mRNA (β-glob) as primers. The panel shows the phosphorimager data. (C) 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 RNPs is verified by Western-blot using anti-PB2 and anti-PA antibodies. In the bottom graph the average NP accumulation and range of two independent complementation experiments are presented as percent of maximal value. The transfecting RNPs are denoted as RNP142 or RNP361. The genotypes of the transfected polymerases are indicated as Pol142 or Pol361. CMV indicates the transfection of empty pCMV plasmid.

Figure 4. 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.

Figure 5. Phenotype of trans-complemented RNPs.

The purified RNP preparations presented in Fig. 3 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 range of two independent complementation experiments. The transfecting RNPs are denoted as RNP142 or RNP361. The genotypes of the transfected polymerases are indicated as Pol142 or Pol361. CMV indicates the transfection of empty pCMV plasmid. The transcription activities parallel the values of NP accumulation presented in Fig. 3 and show that the rescued RNPs have a cap-snatching defective phenotype.

Figure 6. 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.

Figure 7. Lack of transcription of RNPs by a soluble polymerase complex in trans.

(A) Cultures of HEK293T cells were transfected with plasmids expressing various combinations of the polymerase subunits as indicated in the diagram. In some cases, purified cat RNPs containing wt or transcription-deficient polymerase were also transfected. (B) Left panel. The amount of wt (wt) or transcription deficient (361) RNPs transfected was ascertained by Western-blot with anti-NP and anti-PA antibodies. The mobility of molecular weight markers is shown to the left and the position of PA and NP proteins is indicated to the right. Right panel. The transcription phenotype of the RNPs transfected was determined by in vitro transcription using ApG (ApG) or β-globin mRNA (β-globin) as primers. The panel shows the phosphorimager data. (C) The amount of CAT protein accumulated in the cells was determined by ELISA. The data are presented as percent of the value obtained by transfection of purified wt RNPs and represent the average and standard deviation of six independent experiments. The 100% value represented corresponds to a concentration of 340 pg/ml of CAT protein.

Figure 8. 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.