Integrative analyses reveal novel strategies in HPV11,-16 and -45 early infection

Abstract

The interaction between human papillomavirus (HPV) and host cells is not well understood. We investigate the early stage of HPV infections by global expression profiling in a cell model, in which HaCaT cells were transfected with HPV11, HPV16 or HPV45 genomes. We report the differential expression of genes not previously implicated in HPV biology, such as the PSG family and ANKRD1, and of genes implicated in the biology of other viruses, e.g. MX1, IFI44 and DDX60. Carcinogenesis-related genes, e.g. ABL2, MGLL and CYR61, were upregulated by high-risk HPV16 and -45. The integrative analysis revealed the suppression of DNA repair by HPV11 and -16, and downregulation of cytoskeleton genes by all HPV types. Various signalling pathways were affected by the HPVs: IL-2 by HPV11; JAK-STAT by HPV16; and TGF-β, NOTCH and tyrosine kinase signalling by HPV45. This study uncovered novel strategies employed by HPV to establish infection and promote uncontrolled growth.

Figure 1. HaCaT cells were transfected with full genomic DNA and grown under G418 selection for two weeks.

Cells were seeded for measurement of proliferation using the MTT assay,which was conducted without G418 selection. Cultures were harvested for proliferation measurement every 24 hours for a total of 96 hours. The cell cultures transfected with HPV genomes grew more slowly than the control (pSV2neo), and some cells died following the transfection.

Figure 2

Right panel: Venn diagram of differentially expressed genes (numbers of upregulated genes are shown in green and downregulated in red). Left panel: The heatmap of the expression of genes that were differentially expressed by at least two virus subtypes. The PSG family, ANKRD1 and IFIT2 were upregulated by all three HPV subtypes. ABL2, MGLL and CYR61, which have angiogenic and oncogenic potential, were upregulated by HPV16 and -45. IFI44 and DDX60 were upregulated by HPV11 and downregulated by HPV16 and -45. There were 16 genes that were significantly downregulated by at least two HPVs. These gene showed tendency to be downregulated across all 3 type, among them we find ANKRD11, AOAH, FOXN1 and the oncogene BCL11A. Lists of all the differentially expressed genes are available in Supplement 2. The upregulated genes are depicted in red, downregulated in blue, and not differentially expressed in white.

Figure 3. Heatmap showing the expression of differentially expressed genes upon transfection with HPV11, -16 or -45 genomes.

The genes were grouped into six clusters. Clusters 2, 5 and 6 were enriched with cell cycle and DNA repair genes from BRCA1 (clusters 2 and 5) and BRCA2 (cluster 6) networks. Cluster 4 was enriched with JUN transcription factor target genes and cluster 3 was enriched with interferon response genes. The genes of each of the six clusters are listed in Supplement 3. The upregulated genes are depicted in red, downregulated in blue, and not differentially expressed in white.

Figure 4. The networks represent the differentially expressed regions in the human protein-protein interaction (PPI) network upon infection with A) HPV11, B) HPV16 and C) HPV45.

The fold changes of differential expression are represented by colour of the nodes: upregulated – red, downregulated – green and not changed – white. The subcellular localisation is illustrated by shape of the nodes: ellipse – cytoplasm, triangle – extracellular, hexagon – nucleus, rectangle – plasma membrane and parallelogram – unknown.