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. 2009 Feb;83(3):1320-31.
doi: 10.1128/JVI.00977-08. Epub 2008 Nov 19.

Avian Influenza A virus polymerase association with nucleoprotein, but not polymerase assembly, is impaired in human cells during the course of infection

Affiliations

Avian Influenza A virus polymerase association with nucleoprotein, but not polymerase assembly, is impaired in human cells during the course of infection

Marie-Anne Rameix-Welti et al. J Virol. 2009 Feb.

Abstract

Strong determinants of the host range of influenza A viruses have been identified on the polymerase complex formed by the PB1, PB2, and PA subunits and on the nucleoprotein (NP). In the present study, molecular mechanisms that may involve these four core proteins and contribute to the restriction of avian influenza virus multiplication in human cells have been investigated. The efficiencies with which the polymerase complexes of a human and an avian influenza virus isolate assemble and interact with the viral NP and cellular RNA polymerase II proteins were compared in mammalian and in avian infected cells. To this end, recombinant influenza viruses expressing either human or avian-derived core proteins with a PB2 protein fused to the One-Strep purification tag at the N or C terminus were generated. Copurification experiments performed on infected cell extracts indicate that the avian-derived polymerase is assembled and interacts physically with the cellular RNA polymerase II at least as efficiently as does the human-derived polymerase in human as well as in avian cells. Restricted growth of the avian isolate in human cells correlates with low levels of the core proteins in infected cell extracts and with poor association of the NP with the polymerase compared to what is observed for the human isolate. The NP-polymerase association is restored by a Glu-to-Lys substitution at residue 627 of PB2. Overall, our data point to viral and cellular factors regulating the NP-polymerase interaction as key determinants of influenza A virus host range. Recombinant viruses expressing a tagged polymerase should prove useful for further studies of the molecular interactions between viral polymerase and host factors during the infection cycle.

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Figures

FIG. 1.
FIG. 1.
Growth properties of recombinant influenza viruses expressing core proteins derived from the avian MZ or human P908 influenza virus isolate in the WSN background. (A) Growth curves of the parental P908 and reassortant P908-WSN viruses on MDCK cells. Cell monolayers were infected at an MOI of 0.001 and incubated for 72 h at 37, 35, or 33°C. At the indicated time points, the supernatants were harvested, and virus titers were determined by plaque assays on MDCK cells. (B) Growth curves of the parental MZ and reassortant MZ-WSN viruses on MDCK cells. Experimental conditions were as described in panel A, except that virus titers were determined on QT6 cells. (C to F) Plaque phenotype of the P908 and reassortant P908-WSN viruses (C and E) or MZ and reassortant MZ-WSN viruses (D and F) assayed on MDCK or QT6 cells, as described in Materials and Methods. Cell monolayers were stained with crystal violet after 72 h of incubation at 37 or 33°C. Representative data are shown. The dilutions of the viral stock used for infection are indicated.
FIG. 2.
FIG. 2.
Characterization of recombinant MZ-WSN and P908-WSN influenza viruses with a PB2 segment encoding a PB2-Strep fusion protein. (A and B) Schematic representation of the PB2 vRNAs expressed in plasmid-based reverse genetics experiments to produce recombinant viruses expressing a PB2 protein fused to the One-Strep tag at the C terminus (A) or at the N terminus (B). The PB2-ORF (hatched box) is fused to the sequence encoding the One-Strep tag (gray box) and flanked by the 3′ and 5′ NCRs (black lines). In panel A the duplicated PB2-ORF sequences are represented as white boxes, and the sequence, which is identical to the wt PB2 vRNA at the 5′ end, is indicated by a double-headed arrow. The stop codons are indicated by black arrowheads. (C to E) Growth curves of the P908-WSN and MZ-WSN viruses compared to the P908-Cstrep-WSN and MZ-Cstrep-WSN viruses on MDCK cells (C) or on QT6 cells (E) and compared to the P908-Nstrep-WSN and MZ-Nstrep-WSN viruses on MDCK cells (D). Cell monolayers were infected at an MOI of 0.001 and incubated for 72 h at 37°C. At the indicated time points, the supernatants were harvested, and virus titers were determined by plaque assays on MDCK cells (for P908-WSN, P908-Cstrep-WSN and P908-Nstrep-WSN) or on QT6 cells (for MZ-WSN, MZ-Cstrep-WSN and MZ-Nstrep-WSN). nt, nucleotides.
FIG. 3.
FIG. 3.
Western blot analysis of the steady-state levels of viral proteins in cells infected with recombinant MZ-WSN and P908-WSN influenza viruses expressing a PB2-Strep fusion protein. Whole-cell lysates were prepared from MDCK or QT6 cells at 6 h p.i. with the P908-WSN or MZ-WSN viruses expressing either a PB2-wt (−), PB2-Cstrep (C), or PB2-Nstrep (N) protein, as indicated. The samples were loaded on a 4 to 12% polyacrylamide gel and analyzed by Western blotting as described in Materials and Methods, using either a polyclonal antibody directed against the PB2 or PB1 protein, a polyclonal serum recognizing the NP and M1 proteins, or a monoclonal antibody specific for the One-Strep epitope. The positions of the PB2 and PB1 proteins (filled arrowheads) and of cellular proteins recognized nonspecifically by the anti-PB2 and anti-PB1 antibodies (open arrowheads) are indicated.
FIG. 4.
FIG. 4.
Intracellular distribution of PB2 and NP in 293T cells infected with a P908-WSN or MZ-WSN virus expressing a PB2-HA fusion protein. 293T cells were infected at an MOI of 2 with the P908-HA-WSN or MZ-HA-WSN virus expressing a PB2-HA fusion protein and incubated at 37°C. At 2 and 6 h p.i., immunofluorescence assays were performed as described in Materials and Methods, using either an anti-HA (red staining) or an anti-NP (green staining) monoclonal antibody. In columns A and B, distinct settings had to be used because of the low signal levels in MZ-WSN samples. The same settings were used for all data shown in columns C and D. Columns B and D represent merges between the anti-HA or anti-NP stainings shown in columns A and C, respectively, and the DAPI (4′,6′-diamidino-2-phenylindole) nucleic acid staining of the same fields (blue staining).
FIG. 5.
FIG. 5.
Western blot analysis of P908-Cstrep-WSN- or MZ-Cstrep-WSN-infected cell lysates before and after purification on Strep-tactin beads. (A and B) Analysis of protein complexes eluted from Strep-tactin beads. 293T or DF1 cells were infected at an MOI of 2 with MZ-WSN (MZ), MZ-Cstrep-WSN (MZ-Cstrep), P908-WSN (P908), or P908-Cstrep-WSN (P908-Cstrep) virus and incubated at 37 or 33°C for 6 h. Whole-cell lysates were prepared, and a fraction was incubated with Strep-tactin beads as described in Materials and Methods. Protein complexes were eluted and serially diluted in Laemmli buffer, loaded on a 4 to 12% sodium dodecyl sulfate-polyacrylamide gel, and analyzed by Western blotting as described in Materials and Methods, using either polyclonal antibodies directed against the PB2, PB1, PA, or NP protein or a monoclonal antibody specific for the cellular RNA Pol II, as indicated. (C and D) Analysis of the levels of NP protein before and after purification on Strep-tactin beads. The whole-cell lysates and the Strep-tactin eluates prepared from infected 293T or DF1 cells were serially diluted in whole-cell lysates prepared from uninfected cells and in Laemmli buffer, respectively, loaded on a 4 to 12% sodium dodecyl sulfate-polyacrylamide gel, and analyzed by Western blotting, as described in Materials and Methods, using a polyclonal antibody recognizing the NP protein. The samples are from the same experiment as shown in panels A and B, respectively. The sample dilution factors are indicated using the following notation: 1, undiluted; 1/2, diluted twofold, 1/4, diluted fourfold, and so on. (E to H) Graphic representation of the results of Western blot quantification. After the membranes were scanned using a G-Box (SynGene), the signals corresponding to the PB2, PB1, PA, NP, or RNA Pol II proteins were quantified using the GeneTools software (SynGene). The signals measured in samples derived from MZ-Cstrep-WSN-infected cells were compared to those measured in samples derived from P908-Cstrep-WSN-infected cells, taking into account the sample dilution factor. The MZ/P908 ratios measured in total cell extracts (black bars) and in Strep-tactin eluates (gray bars) are expressed as percentages. The percentage indicated by the asterisk is overestimated due to the fact that the MZ-NP signal was at background levels. The results of one representative experiment out of three are shown.
FIG. 6.
FIG. 6.
Western blot analysis of P908-Nstrep-WSN- or MZ-Nstrep-WSN-infected cell lysates before and after purification on Strep-tactin beads. 293T cells were infected at an MOI of 2 with MZ-WSN, MZ-Cstrep-WSN, P908-WSN, or P908-Cstrep-WSN virus in one experiment and with MZ-WSN, MZ-Nstrep-WSN, P908-WSN, or P908-Nstrep-WSN virus in an independent experiment and incubated at 37°C for 6 h. Whole-cell lysates were prepared, a fraction was incubated with Strep-tactin beads, and eluted protein complexes were analyzed using polyclonal antibodies directed against the PB2, PB1, or NP protein, as described in the legend of Fig. 5. The signals measured in samples derived from MZ-Cstrep-WSN- and MZ-Nstrep-WSN-infected cells were compared to those measured in samples derived from P908-Cstrep-WSN- and P908-Nstrep-WSN-infected cells, respectively, taking into account the sample dilution factor. The MZ/P908 ratios measured in total cell extracts (black bars) and in Strep-tactin eluates (gray bars) are expressed as percentages. The percentage indicated by the asterisk is overestimated because the MZ-NP signal was at background levels. The results of one representative experiment out of three (Cstrep viruses) or out of two (Nstrep viruses) are shown.
FIG. 7.
FIG. 7.
Effect of the Glu-Lys substitution at PB2 residue 627 on the phenotype of the avian-like MZ-WSN viruses. (A) Western blot analysis of mutant or wt MZ-Cstrep-WSN-infected cell lysates before and after purification on Strep-tactin beads. 293T cells were infected at an MOI of 2 with the P908-WSN, MZ-WSN, MZ-627K-WSN, P908-Cstrep-WSN, MZ-Cstrep-WSN, or MZ-627K-Cstrep-WSN virus and incubated at 37°C for 6 h. Whole-cell lysates were prepared, a fraction was incubated with Strep-tactin beads, and eluted protein complexes were analyzed using polyclonal antibodies directed against the PB2, PB1, or NP protein, as described in the legend to Fig. 5. The signals measured in samples derived from MZ-Cstrep-WSN- and MZ-627K-Cstrep-WSN-infected cells were compared to those measured in samples derived from P908-Cstrep-WSN-infected cells, taking into account the sample dilution factor. The MZ/P908 ratios measured in total cell extracts (black bars) and in Strep-tactin eluates (gray bars) are expressed as percentages. (B) Growth curves of the 627K mutant compared to the wt MZ-WSN and MZ-Cstrep-WSN viruses on MDCK cells. Cell monolayers were infected at an MOI of 0.001 and incubated for 72 h at 37°C. At the indicated time points, the supernatants were harvested, and virus titers were determined by plaque assays on QT6 cells.

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