Replication kinetics of parental and chimeric viruses.
To define the host transcriptional response to the 1918 influenza virus and to determine the role of the NS1 gene in inducing this response, we made use of a panel of wild-type (WT) and chimeric viruses. These viruses included the eight-gene 1918 pandemic H1N1 virus, A/Texas/36/91 (a seasonal isolate of human H1N1 influenza virus), and two chimeric viruses in which the NS gene segment of the 1918 and A/Texas/36/91 viruses was interchanged to generate 1918:Tx/91(NS) (the 1918 virus with the A/Texas/36/91 NS1 gene) and Tx/91:1918(NS) (the A/Texas/36/91 virus with the 1918 NS1 gene).
To determine whether these influenza viruses had similar replication kinetics, we infected A549 lung epithelial cells at an MOI of 2 and harvested total RNA at 2, 6, and 24 h postinfection. Quantitative real-time PCR was then used to measure viral NS1 and HA RNA levels at each time point relative to mock-infected cells. We found that the 1918 virus produced approximately 1 to 2 log units more viral RNA than did the 1918:Tx/91(NS) virus at each time point (Fig. ). Both viruses showed robust replication patterns, with viral NS1 and HA RNA levels increasing consistently over time. Thus, although these viruses have been reported to replicate at similar levels in normal human bronchial epithelial cells, a primary multicellular culture system (39
), substituting the Tx/91 NS gene into the 1918 virus appeared to lower the amount of viral RNA produced in A549 cells. We next compared viral NS1 and NP protein levels for the 1918 and 1918:Tx/91(NS) viruses (Fig. ). By 24 h postinfection, viral protein expression was significant for each of the viruses, despite the differences in viral RNA levels observed between these viruses at 24 h.
FIG. 1. Viral NS1 (A and C) and HA (B and D) transcript levels at 2, 6, and 24 hours postinfection in A549 human lung epithelial cells as measured by quantitative real-time PCR. Average log10 ratio values were calculated by averaging threshold cycle (CT) values (more ...)
The Tx/91 and Tx/91:1918(NS) viruses also replicated efficiently in A549 cells, with viral RNA levels steadily increasing between 2 and 24 h. Substituting the 1918 NS gene into the Tx/91 virus did not have an effect on replication, as measured by plaque assay. At 2 h postinfection, the Tx/91 and Tx/91:1918(NS) viruses had plaque assay titers of 11.5 × 104 and 3.5 × 104 PFU/ml, respectively. At 24 h postinfection, the Tx/91 and Tx/91:1918(NS) viruses had plaque assay titers of 2.5 × 107 and 4.0 × 107 PFU/ml, respectively. Viral NS1 and HA RNA levels were nearly equivalent for both viruses at each time point (Fig. , respectively).
Global host transcriptional response to parental and chimeric viruses.
After confirming that each virus was able to establish a robust infection in A549 cells, we next analyzed global gene expression patterns in response to infection. For these experiments, A549 cells were infected in triplicate using an MOI of 2. Total RNA was then harvested at 2, 6, and 24 h postinfection, and Agilent 4,000 × 44,000 human oligonucleotide microarrays were used to compare cellular expression patterns of infected cells with those of mock-infected cells at each time point.
Global gene expression patterns induced by the 1918 and 1918:Tx/91(NS) influenza viruses were particularly distinct at the 6- and 24-h time points, with the 1918 virus inducing considerably more gene expression changes than the 1918:Tx/91(NS) virus did (Fig. ). The greater host transcriptional response to the 1918 virus may have been due in part to its higher level of viral RNA expression; however, at the 2-h time point, when the difference in viral RNA levels was the greatest between the 1918 and 1918:Tx/91(NS) viruses, the cellular gene expression profiles were quite similar. Conversely, at the 24-h time point, when the viral RNA levels for the two viruses were more similar, the differences in cellular gene expression patterns were more pronounced. Therefore, there did not appear to be a strong correlation between viral RNA levels and the number of differentially expressed genes. Rather, differences in the host response appear to have been driven by the NS1 gene expressed by the virus. Because NS1 protein expression was high for both the 1918 and 1918:Tx/91(NS) viruses at 24 h postinfection, we focused on this time point for many of our microarray analyses.
FIG. 2. Global host transcriptional response to infection. Signature genes were defined as having greater than twofold differential expression compared with expression in mock-infected cells (P < 0.01). (A) Cells infected with the 1918 influenza virus (more ...)
Global gene expression patterns induced by the Tx/91 and Tx/91:1918(NS) viruses were more similar to one another (Fig. ), with the largest number of gene expression changes occurring at the 6- and 24-h time points. Because these viruses replicated to similar levels in A549 cells, differences in the host response again appear to have been driven by the NS1 gene expressed by the virus. Surprisingly, adding the 1918 NS1 gene to the Tx/91 virus had less of an impact on the host response than adding the Tx/91 NS1 gene to the 1918 virus did. This suggests that the NS1 gene of the 1918 virus may be maximally functional only in the context of the complete 1918 viral genome. While we cannot exclude the possibility that cooperativity between viral gene segments affected NS1 function and/or the global gene expression patterns shown in Fig. , it is known that viruses lacking a functional NS1 protein cannot replicate efficiently in IFN-competent systems (15
). Because we saw an increase in viral mRNA levels over time for all four viruses, appreciable levels of viral proteins expressed at 24 h for the 1918 and 1918:Tx/91(NS) viruses, and an increase in viral titer over time for the WT Tx/91 and Tx/91:1918(NS) viruses, we can assume that the NS1 proteins in all four viruses are functional within their larger constellations of viral gene segments.
Direct comparison of the host transcriptional response to parental and chimeric viruses.
To further analyze the gene expression data for differences in the host response that could be attributed to the NS1 gene, we performed one-way analysis of variance (ANOVA) on data generated from the 24-h time point of the 1918 and 1918:Tx/91(NS) experiments. Our ANOVA was generated based on three biological replicates from each group. [Note that these biological replicates were not pooled for analysis, whereas the WT Tx/91 and Tx/91:1918(NS) replicates were pooled for analysis. The latter set were pooled only after it was established, using qRT-PCR, that the replicates contained equivalent viral mRNA levels and had equivalent levels expression of various cellular genes.] From the resulting list of significant (ANOVA P ≤ 0.01) genes, we utilized in silico analysis (described in Materials and Methods) to further filter gene expression patterns induced by the 1918 and 1918:Tx/91(NS) viruses. We selected genes that were at least twofold different in their expression in this direct comparison and then used Ingenuity Pathways Analysis to group those genes according to their function. This allowed us to determine whether genes within a given functional category were expressed at a higher level in response to one virus compared to the other virus and to identify categories containing large numbers of differentially expressed genes (Fig. ). This approach is particularly useful for depicting general trends and highlighting differences in the host response to each virus, which was one of the primary purposes of our analyses. However, the results must be interpreted carefully since the direct comparison does not provide information about gene expression changes relative to mock-infected cells. Therefore, genes identified as being differentially expressed in direct comparisons were also analyzed to measure their expression values relative to mock-infected cells (see Fig. , , , and ) (also see Table S1 in the supplemental material).
FIG. 3. Functional categories of differentially expressed genes as generated using Ingenuity Pathways Analysis. (A) Direct comparison of the 1918 influenza virus versus 1918:Tx/91(NS). (B) Direct comparison of Tx/91 versus Tx/91:1918(NS). Genes from cells infected (more ...)
FIG. 4. Regulation of IFN-stimulated gene expression in A549 cells infected with the 1918 or 1918:Tx/9(NS) influenza virus at 24 h postinfection. IFN-stimulated genes suppressed by the 1918 virus are shown. (A) Network diagram showing a subset of IFN-stimulated (more ...)
FIG. 5. Regulation of immune response genes. (A) IPA network diagram generated using genes from the in silico comparison of the 1918 and 1918:Tx/91(NS) influenza viruses (P < 0.01). Genes that were expressed at a higher level in cells infected with the (more ...)
FIG. 6. Regulation of lipid metabolism gene expression in A549 cells infected with the 1918 or 1918:Tx/91(NS) influenza virus at 24 h postinfection. (A) Network of lipid metabolism genes identified by the in silico comparison of the 1918 and 1918:Tx/91(NS) viruses (more ...)
Overall, this analysis revealed that both viruses induced gene expression changes in numerous functional categories. Genes associated with several of these categories, including cell signaling and immune response, tended to be more highly expressed in cells infected with the 1918 virus. Unexpectedly, genes associated with cell turnover (including the categories of cell cycle, cell death, and cell growth and proliferation) and with metabolic pathways (including the categories of amino acid, carbohydrate, and lipid metabolism) tended to be more highly expressed in cells infected with 1918:Tx/91(NS). Thus, even though the 1918 virus induced more gene expression changes overall than 1918:Tx/91 did (Fig. ), Tx/91 NS1 preferentially induced the expression of specific functional categories of genes. This may be due to structural differences in the two NS1 proteins, which may contribute to differential virus-host factor interactions during the course of infection.
Due to the pooling of biological replicates in the Tx/91 and Tx/91:1918(NS) experiments, we next went directly to the in silico analysis to compare the gene expression patterns induced by the Tx/91 and Tx/91:1918(NS) viruses. Consistent with the findings described above, we found that genes associated with cell turnover and metabolic pathways tended to be expressed at higher levels in cells infected with the Tx/91 virus (Fig. ). Since a similar trend was observed in cells infected with 1918:Tx/91(NS), this host response appears to be correlated with viral expression of the Tx/91 NS1 gene. In contrast, genes associated with the category of immune response also tended to be expressed at higher levels in cells infected with the Tx/91 virus. Therefore, the expression of such genes was elevated in response to both the parental 1918 and Tx/91 viruses compared with the response to the chimeric viruses. Again, the context of viral genes in which NS1 is present appears to impact the functioning of the NS1 protein and the virus-host interactions responsible for the induction of immune response genes.
Regulation of IFN-stimulated and immune function genes.
We next used IPA to generate interactive networks of the genes appearing in the functional categories shown in Fig. . The top-scoring network generated from the gene lists distinguishing the 1918 and 1918:Tx/91(NS) viruses was a network of IFN-stimulated genes (Fig. ). This is consistent with the fact that the 1918:Tx/91(NS) virus induced more than eight times the amount of beta interferon than the 1918 virus at 24 h postinfection, as measured by microarray. All of the genes in the network in Fig. were expressed at a higher level in cells infected with 1918:Tx/91(NS) than in cells infected with the 1918 virus at 24 h, highlighting a striking functional difference between the two viruses. Thus, while immune response genes in general were expressed at higher levels in response to infection with the parental viruses, the expression of IFN-stimulated genes in particular was repressed by the 1918 virus. This is consistent with our earlier report that the NS1 gene of the 1918 virus may act to antagonize IFN-stimulated gene expression (16
To extend this finding, we used qRT-PCR to measure the expression of a panel of 12 IFN-stimulated genes (Fig. ). The expression of all but one of these genes (OAS2) was significantly upregulated in cells infected with the 1918:Tx/91(NS) virus relative to mock-infected controls. In contrast, all but one of these genes (IFI44) was downregulated in cells infected with the 1918 virus compared with that observed in mock-infected cells, indicating that the 1918 NS1 protein suppressed even low-level, constitutive expression of these genes. We did not observe the same pattern of IFN-stimulated gene downregulation by the WT Tx/91 virus. Although the mechanism underlying the inhibition of IFN-stimulated gene expression by the 1918 NS1 protein cannot be directly ascertained by our analyses, amino acid sequences and structural differences between NS1 proteins and the consequent differences in virus-host factor interactions are likely to contribute to this effect. Because the viral polymerase complexes are identical in the 1918 and 1918:Tx/91(NS) viruses, it is unlikely that differences exist between the two viruses in their respective abilities to undergo cap snatching in the nucleus (excluding this as a probable mechanism for the differences in IFN-stimulated gene expression observed between them). In addition, a number of the genes repressed by the 1918 NS1 protein, including RIG-I (DDX58), PKR, and OAS1/OAS2, have been reported to interact directly (or through a dsRNA intermediate) with the NS1 protein during infection, and it is possible that in some cases NS1 may impose an additional block on the activities of these proteins at the posttranslational level (4
As noted, immune response genes in general were expressed at higher levels in cells infected by the 1918 virus than in cells infected by the Tx/91:1918(NS) virus, and these genes also formed a high-scoring IPA network (Fig. ). This network included genes associated with chemokine signaling (CCL2, CCL19, CCL21, CCR10, and LGALS3), cytokine signaling (FES and NFATC4), lymphocyte activation (CD4, LCK, PTPRCAP, SELL, SELPLG, SLAMF1, THY1, CD79A, and SPI1), and neutrophil activation (ELA2, MMP9, NCF1, and SELL). Importantly, during an in vivo infection, the increased expression of these genes may serve to attract infiltrating immune cells to the site of infection and to increase proinflammatory processes and subsequent lung pathology. Expression relative to mock-infected cells of the genes from Fig. is shown in Fig. . The fact that the 1918 virus paradoxically upregulated many chemokine genes but downregulated many IFN-stimulated genes is one of the intriguing findings of our study and challenges the traditional dogma that IFN is required for the cell to signal to immune infiltrates.
Regulation of lipid metabolism genes.
Perhaps most intriguingly, our examination of high-scoring networks generated from the gene lists distinguishing the 1918 and 1918:Tx/91(NS) viruses revealed an extensive, direct network of lipid metabolism genes that were expressed at higher levels in cells infected with the 1918:Tx/91(NS) virus than in cells infected with the 1918 virus (Fig. ). This network contained key activators of cell proliferation and differentiation, including PIK3R1, PLCE1, RARB, and SIRT1. Cell proliferation is typically associated with an increased de novo synthesis of fatty acids, phospholipids, and cholesterol to build cellular membrane components, and there was an increase in the expression of genes involved in these pathways.
To extend this finding, we used qRT-PCR to measure the expression of a panel of 11 lipid metabolism genes (Fig. ). The expression of all but one of these genes (RGS2) was significantly downregulated in cells infected with the 1918 virus relative to mock-infected controls. In contrast, the 1918:Tx/91(NS) virus induced the expression of some of these genes relative to mock-infected cells. In every case, the 1918 virus significantly suppressed the expression of these genes relative to the 1918:Tx/91(NS) virus. As some of the genes represented in Fig. function in proinflammatory (and potentially antiviral) pathways, it is possible that an early suppression of these genes contributes to the severe lethality associated with the 1918 virus.
Cells infected with 1918:Tx/91(NS) also had higher levels of expression of genes encoding proinflammatory lipid mediators (including BDKRB2, LTA4H, PLA2G4A, PTGS2, and VIM) and genes encoding different representatives of the ATP-binding cassette family of proteins (ABC transporters) (Fig. ). Certain members of this family, including ABCB1, are reported to stimulate dendritic cell activation (40
). Several CYP family members were also induced by the 1918:Tx/91(NS) virus, including CYP24A1, which is reported to induce monocyte and macrophage differentiation, and CYP3A4, which aids in the oxidative catabolism of lipids (7
). Thus, the 1918 NS1 protein may also play a role in inhibiting the transcription of certain lipid-based proinflammatory mediators that function as part of the host antiviral response. Consistent with this idea, the expression of many of the same genes related to lipid metabolism were also expressed at a higher level in cells infected with the Tx/91 virus than in cells infected with Tx/91:1918(NS) (Fig. ). These included CYP family genes, genes encoding proinflammatory mediators, and cholesterol biosynthesis genes.