Avian Influenza A Virus Polymerase Can Utilize Human ANP32 Proteins To Support cRNA but Not vRNA Synthesis

Figures

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  FIG 1. cRNA synthesis is inhibited in human cells lacking ANP32A/B. (A to C) Segment 4, vRNA (A), cRNA (B), or mRNA (C) accumulation over time in eHAP WT versus dKO cells following infection with PR8 (MOI = 3). The fold change was calculated versus input (0 hpi). n = 3 biological replicates, plotted as means ± the standard deviations (SD). Significance between WT and dKO cells was assessed at 2 hpi using an unpaired t test following log transformation. (D to F) Segment 6 vRNA (D), cRNA (E), or mRNA (F) accumulation over time in eHAP WT versus dKO cells after infection with PR8 (MOI = 3). The fold change was calculated versus mock-infected cells. n = 3 biological replicates, plotted as means ± the SD. Significance was assessed using multiple unpaired t tests following log transformation and corrected for multiple comparisons using the false discovery rate. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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  FIG 2. cRNA can be visualized using in situ replication assays. (A) Schematic illustrating influenza polymerase activity under the conditions of an ActD-treated replication assay. (B) Schematic illustrating influenza polymerase activity under the conditions of an ActD-treated cRNP stabilization assay. (C to F) Validation of ActD-treated replication and cRNP stabilization assays analyzed by RNA FISH. Assays were performed in HEK293T cells, following infection with PR8 (MOI = 3). Pretransfected PR8 polymerase mixes were: WT = PB2, PB1, PA, and NP (replication assay); D446Y = PB2, PB1 D446Y, PA, and NP (cRNP stabilization assay); −PB1 = PB2, PA, and NP (control). (C to E) RT-qPCR analysis of segment 6 vRNA (C), cRNA (D), and mRNA (E) accumulation at 6 hpi. Pretransfected polymerase mixes are indicated on the x axis. The dotted line indicates input levels of RNA (0 hpi). Fold change was calculated versus mock-infected cells. n = 3 biological replicates, plotted as means ± the SD. Significance compared to −PB1 was assessed using one-way ANOVA with Dunnett’s multiple-comparison test, following log transformation. (F) Accumulation of NA vRNA and +RNA at 3 hpi, analyzed by RNAscope with…

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  FIG 3. Human ANP32 proteins play a direct role in primary cRNA synthesis. ActD-treated cRNP stabilization assay in eHAP WT and dKO cells, following infection with PR8 (MOI = 3), 3 hpi. (A) Accumulation of segment 6 vRNA and +RNA in eHAP WT and dKO cells, analyzed using RNAscope. Magenta arrowheads highlight a subset of NA vRNA-stained puncta. Images are representative maximum-intensity projections. (B to D) Accumulation of segment 6 vRNA (B), cRNA (C), or mRNA (D) analyzed by RT-qPCR. The dotted line indicates the background RNA present in control samples transfected with a plasmid mix lacking PB1. The fold change was calculated versus mock-infected cells. n = 3 biological replicates, plotted as means ± the SD. Significance was assessed using an unpaired t test following log transformation. ns, not significant; ****, P < 0.0001.

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  FIG 4. ANP32 proteins are not required for primary transcription. (A) Schematic illustrating influenza polymerase activity under the conditions of a cRNP stabilization assay with CHX. (B to D) cRNP stabilization assay with CHX in eHAP WT, dKO, and tKO cells. Segment 4 vRNA (B), cRNA (C), or mRNA (D) accumulation following infection with PR8 (MOI = 3) is shown. The fold change was calculated versus the input (0 hpi). n = 3 biological replicates, plotted as means ± the SD. Significant differences between cell types was assessed using multiple unpaired t tests following log transformation, corrected for multiple comparisons using the false discovery rate. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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  FIG 5. Avian polymerase is not restricted in cRNA synthesis in mammalian cells. (A) Schematic outlining assay setup and expected polymerase combinations during replication. As indicated on the left, in the cRNP stabilization assay, only the first layer of activity (primary cRNA synthesis) will occur. Both layers of activity can occur in replication assays. Unknown polymerase activity is indicated by a dashed black arrow. (B and C) Simultaneous cRNP stabilization and replication assays with ActD, 6 hpi. Segment 6 vRNA (B) and cRNA (C) accumulation following infection with either 5092E or 5092K as indicated (MOI = 0.1) is shown. The dotted line indicates levels of vRNA/cRNA present in a control lacking PB2 in the transfection mix. The fold change was calculated versus input (0 hpi). n = 3 biological replicates, plotted as means ± the SD. Significance was assessed using one-way ANOVA with Dunnett’s multiple-comparison test, following log transformation. (D and E) Replication assays with ActD and chANP32A, 6 hpi. Segment 6 vRNA (D) and cRNA (E) accumulation following infection with either 5092E or 5092K as indicated (MOI = 0.1) is shown. The dotted line indicates the levels of vRNA/cRNA…

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  FIG 6. Summarizing schematic. (i) When human-adapted influenza viruses encoding a lysine at position 627 of the PB2 polymerase protein (PB2 627K) infect wild-type human cells in which only human ANP32 proteins are present, both cRNA and vRNA synthesis can occur. (ii) When influenza viruses (PB2 627K) infect human cells lacking ANP32 proteins, neither cRNA nor vRNA accumulate, demonstrating that ANP32 is required to support both cRNA and vRNA synthesis. (iii) When avian influenza viruses encoding a glutamic acid at position 627 of the PB2 polymerase protein (PB2 627E) infect wild-type human cells in which only human ANP32 proteins are present, cRNA synthesis but not vRNA synthesis can occur. Since scenario ii illustrates that ANP32 proteins are required for cRNA synthesis, this suggests that avian polymerase can functionally interact with human ANP32 proteins to support cRNA synthesis. However, avian polymerase cannot utilize human ANP32 proteins to support vRNA synthesis; thus, host restriction is imposed on this step of replication. (iv) In the presence of exogenously expressed avian ANP32A, avian influenza viruses (PB2 627E) can undergo both cRNA and vRNA synthesis, confirming…