Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses

Abstract

Human influenza A viruses replicate in the upper respiratory tract at a temperature of about 33 degrees C, whereas avian viruses replicate in the intestinal tract at a temperature close to 41 degrees C. In the present study, we analyzed the influence of low temperature (33 degrees C) on RNA replication of avian and human viruses in cultured cells. The kinetics of replication of the NP segment were similar at 33 and 37 degrees C for the human A/Puerto-Rico/8/34 and A/Sydney/5/97 viruses, whereas replication was delayed at 33 degrees C compared to 37 degrees C for the avian A/FPV/Rostock/34 and A/Mallard/NY/6750/78 viruses. Making use of a genetic system for the in vivo reconstitution of functional ribonucleoproteins, we observed that the polymerase complexes derived from avian viruses but not human viruses exhibited cold sensitivity in mammalian cells, which was determined mostly by residue 627 of PB2. Our results suggest that a reduced ability of the polymerase complex of avian viruses to ensure replication of the viral genome at 33 degrees C could contribute to their inability to grow efficiently in humans.

FIG. 1

Comparison of virus titers and plaque size for human and avian influenza viruses at 37 and 33°C. MDCK cells were infected in duplicate with various dilutions of human PR8, SYD, or BAY or avian MAL, FPV, or PIN influenza virus. For each virus, MDCK cells which were infected with the same dilution (10⁻⁶ or 10⁻⁵) at either 37 or 33°C are shown. Titers were determined from dilutions that gave a minimum of 10 plaques.

FIG. 2

Kinetics of replication of the NP RNA segment from human and avian influenza viruses at 37 and 33°C. (A) MDCK cells were infected at an MOI of 20 PFU/cell with either PR8, SYD, MAL, FPV, or PIN virus or mock infected. (B) COS-1 cells were infected at an MOI of 10 PFU/cell with either PR8, SYD, BAY, MAL, FPV, or PIN virus or mock infected. Infected cells were incubated at either 37°C (solid symbols, solid lines) or 33°C (open symbols, dashed lines). The amount of NP vRNA was evaluated at 0, 1, 3, 5, 7, 9, 12, and 21 or 24 h p.i. The signal measured for a given virus at a given time was corrected by subtracting the signal measured at the same time point in mock-infected cells. For each virus, the corrected values were then expressed as a percentage of the value obtained at 21 or 24 h p.i. and at 37°C. The results are from one experiment representative of two independent experiments (A, PR8, MAL, and FPV) or of one experiment (A, SYD and PIN, and B).

FIG. 3

Transcription-replication of a virus-like CAT reporter RNA with homospecific polymerase complexes at 37 and 33°C in COS-1 cells. COS-1 cells were cotransfected in duplicate with pPolI-CAT-RT and four plasmids expressing the PB1, PB2, PA, and NP proteins derived from PR8, VIC, FPV, or MAL. Cell extracts were prepared and tested for CAT expression following 48 h of incubation at 37 or 33°C. For a given complex, the CAT levels measured at 33°C (grey bars) were compared to the CAT levels measured at 37°C (black bars). The results are expressed as concentration values and as the mean ± standard deviation (SD) of duplicate samples from one experiment representative of two independent experiments (A) or as percentages and as the mean ± SD of two independent experiments (B).

FIG. 4

Transcription-replication of a virus-like CAT reporter RNA with heterospecific polymerase complexes at 37 and 33°C in COS-1 cells. COS-1 cells were cotransfected with pPolI-CAT-RT and four plasmids encoding the PB1, PB2, PA, and NP proteins derived from PR8, FPV, or MAL viruses, including, in the case of FPV and MAL, either the wild-type PB2 (A and B) or mutant E627K-PB2 (B), as indicated. Cell extracts were prepared and tested for CAT expression following 48 h of incubation at 37 or 33°C. For a given combination of PB1, PB2, PA, and NP, the CAT levels measured at 33°C were compared to the CAT levels measured at 37°C (100%, solid line). The results are expressed as percentages and as the mean ± SD of duplicate samples from one experiment representative of two independent experiments (A) or as the mean ± SD of two independent experiments (B).

FIG. 5

Transcription-replication of a virus-like CAT reporter RNA with homospecific polymerase complexes at 37 and 33°C in QT6 cells and COS-1 cells. COS-1 cells (A) or QT6 cells (B) were infected for 1 h at an MOI of 5 PFU/cell with a recombinant vaccinia virus expressing the T7 RNA polymerase and then transfected with pT7-CAT-RT and four plasmids encoding the PB1, PB2, PA, and NP proteins derived from VIC or MAL viruses, including in the latter case either the wild-type MAL PB2 or the mutant MAL E627K-PB2, as indicated. Cell extracts were prepared and tested for CAT expression following 18 h of incubation at 37 or 33°C. For a given complex, the CAT levels measured at 33°C (grey bars) were compared to the CAT levels measured at 37°C (black bars). The mean ± SD values are from duplicate samples from one experiment representative of two independent experiments.

FIG. 6

Synthesis of cRNA and mRNA at 37 and 33°C with avian and human virus-derived polymerase complexes. COS-1 cells were cotransfected with pPolI-CAT-RT and the PB1, PB2, PA, and NP expression plasmids derived from PR8 (lanes 4 and 10) or from MAL, including in the latter case either the wild-type MAL PB2 (lanes 5 and 11) or the mutant MAL E627K-PB2 (lanes 6 and 12) expression plasmid. Controls included COS-1 cells transfected with pPolI-CAT-RT without protein expression plasmids (lanes 1 and 7), PR8 protein expression plasmids without pPolI-CAT-RT (lanes 2 and 8), and MAL protein expression plasmids without pPolI-CAT-RT (lanes 3 and 9). Following 48 h of incubation at 37°C (lanes 1 to 6) or 33°C (lanes 7 to 12), total RNAs were extracted, and 5 μg was analyzed by RNase protection assay as described in the text. The image obtained by scanning the gel with a STORM820 optical scanner is shown. The length expected for the undigested riboprobe was 190 nucleotides (nt) (lane C+). No signal was expected when 5 μg of yeast tRNA was analyzed (lane C−). The expected lengths for the riboprobe fragments protected by cRNA or mRNA were 175 and 159 nt, respectively. MW, molecular size markers.