Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation

Version 1 : Received: 5 November 2021 / Approved: 8 November 2021 / Online: 8 November 2021 (12:28:30 CET)

How to cite: Lou, H.; Boland, J.F.; Burk, R.; Yeager, M.; Wentzensen, N.; Schiffman, M.; Mirabello, L.; Dean, M. HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation. Preprints 2021, 2021110134 (doi: 10.20944/preprints202111.0134.v1). Lou, H.; Boland, J.F.; Burk, R.; Yeager, M.; Wentzensen, N.; Schiffman, M.; Mirabello, L.; Dean, M. HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation. Preprints 2021, 2021110134 (doi: 10.20944/preprints202111.0134.v1).

Abstract

The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from the controls showed a reduced level of steady-state E7 protein, with 7/13 variants having deficient protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-normal E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2); and the R77S (CIN3) had activity only slightly reduced from wildtype E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay as compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration further demonstrating the importance of fully active E7 in clinical cancer development.

Keywords

HPV16; E7 variants; cervical cancer; transformation; wound healing; western blotting

Subject

MEDICINE & PHARMACOLOGY, Oncology & Oncogenics

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