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.
Highlights
Human papillomaviruses (HPVs) are double-stranded DNA viruses that infect keratinocytes of skin and mucosal tissues [1]
Peptide Method: E6 Activities Relate to P53 Tumour Suppression, Hypoxia, Energetics, Chromosome Remodeling, and Innate Immunity For the Peptide Method, two stringent rules were followed: any EPE6- or AAE6-targeted protein must have a sum of at least 2 peptides in either trial, independent of treatment, OR any EPE6or AAE6-targeted protein must have at least 1 peptide in both trials, independent of treatment
After sorting the proteins with Excel, our peptide selection strategy left us with 25 candidates: 6 unique AAE6 and 6 unique EPE6-interacting proteins along with 13 common E6-interacting proteins between AAE6 and EPE6 (Figure 2A; Table S4)
Summary
Human papillomaviruses (HPVs) are double-stranded DNA viruses that infect keratinocytes of skin and mucosal tissues [1]. Intracellular oncoproteins (E6 and E7) play an important role in the immortalization and malignant transformation of HPV-infected cells [5] Even though both proteins are important for carcinogenesis, studying them independently allows for identification of pathways targeted by each oncoprotein. The deregulation of HIF-1α was caused by AAE6 s ability to augment mitogen-activated protein kinase/extracellular-related kinase (MAPK/ERK) signaling. We started filling this gap by investigating two common naturally occurring E6 variants of the A1 (EPE6) and D2/D3 (AAE6) sub-lineages, based on our hypothesis that the type of PPIs between E6 and host cell proteins affects the carcinogenic potential of the HPV16 variant. FFigiguurree11. .TTooscsacaleleddepepicitciotinonofoHf HPVP1V61E66ES6NSPNsPbsebtweteweneeAnAAEA6Ea6ndanEdPEE6P.ET6h. eTyhellyoewllocwonceosnineds iicnadteicaante SaNnPSNthPatthreastureltssuilntsain anmainmoinaociadcicdhacnhganeg(em(imssiesnsesnesme umtuatiaotnio)n, )w, whehreeraesatshtehepipniknkcoconnesesininddicicaatetennoo aammininooaaccididcchhaannggeessaattththaattppaarrtitcicuulalarrssitiete(n(noonnsseennsseemmuutatatitoionn).).SSNNPPssrreessuultltininggininaammininooaaccididcchhaannggeess ffrroommEEPPEE66ttooAAAAEE66((QQ1144HH,,HH7788YY,,aannddLL8833VV))aarreeffoouunnddaattnnuucclleeoottiiddee((NNTT))ppoossiittiioonnss GG114455TT,,CC333355TT,, aannddTT335500GG. .SSNNPPsstthhaattddoonnoottrreessuultltininaannyycchhaannggeeininaammininooaaccididssaarreefofouunnddaat tNNTTppoossitiitoionnss: :TT228866AA,, AA228899GG, ,aannddGG553322AA[2[288].]
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