Virus-Host Protein-Protein Interactions between Human Papillomavirus 16 E6 A1 and D2/D3 Sub-Lineages: Variances and Similarities

Abstract

High-risk strains of human papillomavirus are causative agents for cervical and other mucosal cancers, with type 16 being the most frequent. Compared to the European Prototype (EP; A1), the Asian-American (AA; D2/D3) sub-lineage seems to have increased abilities to promote carcinogenesis. Here, we studied protein-protein interactions (PPIs) between host proteins and sub-lineages of the key transforming E6 protein. We transduced human keratinocyte with EP or AA E6 genes and co-immunoprecipitated E6 proteins along with interacting cellular proteins to detect virus-host binding partners. AAE6 and EPE6 may have unique PPIs with host cellular proteins, conferring gain or loss of function and resulting in varied abilities to promote carcinogenesis. Using liquid chromatography-mass spectrometry and stringent interactor selection criteria based on the number of peptides, we identified 25 candidates: 6 unique to AAE6 and EPE6, along with 13 E6 targets common to both. A novel approach based on pathway selection discovered 171 target proteins: 90 unique AAE6 and 61 unique EPE6 along with 20 common E6 targets. Interpretations were made using databases, such as UniProt, BioGRID, and Reactome. Detected E6 targets were differentially implicated in important hallmarks of cancer: deregulating Notch signaling, energetics and hypoxia, DNA replication and repair, and immune response.

Keywords: E6 oncoprotein; carcinogenesis; co-immunoprecipitation; human papillomavirus; interactome study; mass spectrometry; metabolic pathways; proteomics; sub-lineages.

Figure 1

To scale depiction of HPV16 E6 SNPs between AAE6 and EPE6. The yellow cones indicate an SNP that results in an amino acid change (missense mutation), whereas the pink cones indicate no amino acid changes at that particular site (nonsense mutation). SNPs resulting in amino acid changes from EPE6 to AAE6 (Q14H, H78Y, and L83V) are found at nucleotide (NT) positions G145T, C335T, and T350G. SNPs that do not result in any change in amino acids are found at NT positions: T286A, A289G, and G532A [28].

Figure 2

Venn diagrams representing AAE6 and EPE6 host cellular targets for both selection approaches: Peptide Method (A) and Protein-Pathway Method (B). EPE6-specific interactors are in yellow, AAE6-specific interactors are in blue and proteins common to both variants are in green. Protein names are abbreviated based on UniProt protein rather than gene nomenclature, enabling easy identification for searches and further information.

Figure 3

Summary of individual and tandem effects between AAE6 and EPE6 PPIs with host cellular proteins by which E6 may promote cell-transforming processes. Candidates from both approaches are shown for AAE6 only due to the lack of significant FDR values for EPE6 and AAE6/EPE6 combined using the Protein-Pathway Method (upright font = Peptide Method, italic font = Protein-Pathway Method). Candidates from the Peptide Method are shown for AAE6, EPE6, and AAE6/EPE6 (upright font). The first-described E6 binder (P53; Table S1) is most likely targeted through several mechanisms shared by both E6 variants (middle, in green). AAE6 and EPE6 proteins equally bound 3 proteins known to affect P53 inactivation: TRIPC, CHM4B, and NOG2. Collectively, these interactions could result in decreased P53 function far beyond its well-described E6- and E6AP-mediated degradation through the proteasome. Potential interactions unique to AAE6 (right, in blue) are mostly associated with Notch signaling, hypoxia, energetics, DNA base excision repair, and to some extent with the innate immune system. Notably, some AAE6-targeted molecules have multiple roles, e.g., MTOR is associated with hypoxia and metabolism, CDK8 with hypoxia and Notch signaling, and MSK2 with hypoxia and chromatin remodeling, further underlining this variant’s “lead” in the malignant process. AAE6′s association with MUTYH, IN80B, and MSK2 could promote its integration potential and the binding to PROK2 could promote both angiogenesis and hypoxia within infected cells. Being a chemokine-like molecule, PROK2 is implicated in the innate immune system normally attracting macrophages to the site of inflammation. In the hypoxic and consequently more acidic tumour environment, tumour-associated macrophages (TAMs) may develop from original site-filtrating macrophages adapting to the tumour microenvironment. Tumour growth is then promoted by the positive feedback loop between TAMs and epithelial cells via the expression of colony-stimulating factor and epithelial growth factor, respectively. EPE6′s binding to MX2 (left, in yellow) may limit this variant’s ability to integrate into the host genome effectively while it may be more successful in deregulating the host’s viral defence. MX2 also affects the P53 pathway, e.g., via EHMT2 and the expression of pro-apoptotic genes Bax and PUMA, further duplicating well-established anti-P53 E6 activities.