HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation
<p>HPV16 E7 variants examined in this study. Schematic representation of amino acid (AA) sequences of HPV16 E7 single AA substitution mutants and their location in functional domains. Variants from cancer-free controls shown above and variants from pre-cancer samples below the functional domain. The numbers 1 to number 98 indicate the amino acid position of E7 variants. *: cervical intraepithelial neoplasia 2 (CIN2); the CXXC motif is shown in red (AA.58–61 and 91–94). Conserved regions 1, 2 and 3 are indicated (CR1, CR2, CR3).</p> "> Figure 2
<p>Western blot analyses of HPV16 E7 from HeLa (<b>A</b>) and 293T (<b>B</b>) cells transiently transfected with empty vector, wild-type and indicated mutant E7 constructs. The position of the E7, Tubulin and TBP (TATA binding protein) are indicated. N-P and C-P represent the nuclear and cytoplasm proteins, respectively. Statistical analysis of three independent experiments is shown in <a href="#app1-cancers-14-04895" class="html-app">Figure S2</a>.</p> "> Figure 3
<p>Morphology and anchorage-independent growth of NIH3T3 transfected with HPV16 E7 WT and variant types. (<b>A</b>). Morphology of NIH3T3 cells stably expressing empty vector, WT and the indicated variant types. Morphology of stable NIH3T3 cells (10x, 20x). (<b>B</b>). Quantitation of anchorage-independent cell growth of HPV16 E7 variants on soft agar and each variant was compared to WT that cell growth was adjusted to 100%. *, <span class="html-italic">p</span> < 0.05 for comparison with WT cells.</p> "> Figure 4
<p>Migration of stably transfected NIH3T3 cell lines in a wound healing (scratch) assay. Stable NIH3T3 cell clones were plated onto 24-well plates. Wounds in the monolayer were created with an automated tool, plates were washed to remove detached cells, and closure of the wound was followed for 24 h with hourly imaging in a BioTek Lionheart FX Automated Live Cell Imager. Images are shown in (<b>A</b>) and quantitation over time in (<b>B</b>). Results are expressed relative to WT E7 migration at 10 h. At 10 h: WT vs. Vector (<span class="html-italic">p</span> = 0.0022); WT-H9 R (<span class="html-italic">p</span> = 0.0079); WT-E33K (<span class="html-italic">p</span> = 0.0022); WT-D14E (<span class="html-italic">p</span> > 0.05); WT-P92L (<span class="html-italic">p</span> = 0.0080) WT-P92S (<span class="html-italic">p</span> = 0.0082). If we apply a highly conservative Bonferroni multiple testing correction of six tests of mutant versus WT, only vector and E33K remain significant.</p> ">
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Plasmid Construction and antiHPV16 E7 Antibodies
2.2. Mutant Construction
2.3. Cell Culture and Transfection
2.4. Western Blotting
2.5. Determination of Morphological Transformation
2.6. Anchorage-Independent Cell Growth
2.7. Wound Healing Assay
2.8. Statistical Analysis
3. Results
3.1. HPV16 E7 Mutagenesis and Protein Expression
3.2. Reduced Protein Expression Levels of HPV16 E7 Variants
3.3. HPV16 E7 Variants Not Associated with Cancer Have a More Normal Cell Morphology and Reduced Anchorage-Independent Growth in NIH3T3 Cells
3.4. HPV16 E7 Variants Display Decreased NIH3T3 Cell Migration
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Variants | Original Codon | Mutant Codon | Nucleotide Change | Control (No.) | Precancer (No.) | E7 * |
---|---|---|---|---|---|---|
Wild type | 1 | |||||
Control | ||||||
H9R | CAT | CGT | A > G | 1 | 0 | 0.8 |
D14E | GAT | GAA | T > A | 0 | 1 (CIN2 **) | 0.11 |
D21N | GAT | AAT | G > A | 1 | 0 | 0.92 |
N29H | AAT | CAT | A > C | 0 | 1 (CIN2 **) | 0.3 |
N29S | AAT | AGT | A > G | 2 | 0 | 0.018 |
E33K | GAG | AAG | G > A | 2 | 0 | 0.2 |
T56I | ACC | ATC | C > T | 1 | 0 | 0.55 |
D62N | GAC | AAC | G > A | 1 | 0 | 0.11 |
S63F | TCT | TTT | C > T | 1 | 0 | 0.1 |
S63P | TCT | CCT | T > C | 1 | 0 | 0.3 |
T64M | ACG | ATG | C > T | 1 | 0 | 0.11 |
E80K | GAA | AAA | G > A | 1 | 0 | 0.26 |
D81N | GAC | AAC | G > A | 1 | 0 | 0.42 |
P92L | CCC | CTC | C > T | 2 | 0 | 0.1 |
P92S | CCC | TCC | C > T | 2 | 0 | 0.68 |
Precancer | ||||||
P6L | CCT | CTT | C > T | 1 | 1 (CIN3 **) | 1.1 |
H51N | CAT | AAT | C > A | 6 | 3 (CIN3/AIS ***) | 0.46 |
R77S | CGT | AGT | C > A | 1 | 1 (AIS) | 0.99 |
Construct | Cervical Disease | HPV16 Clearance | Function Domain | Nuclear Protein | Transforming Activity (% Growth in Soft Agar) | Wound Healing |
---|---|---|---|---|---|---|
Vector | + | |||||
D81N | Control | CR3 | ++ | NA | NA | |
E80K | Control | CR3 | ++ | NA | NA | |
P92S | Control | CR3 | ++ | ++ | ++ | |
S63F | Control | CR3 | + | NA | NA | |
T56I | Control | CR3 | ++ | NA | NA | |
T64M | Control | CR3 | + | NA | NA | |
D21N | Control | yes | CR2 | +++ | +++ | +++ |
D62N | Control | CR3 | + | NA | NA | |
P92L | Control | yes | CR3 | + | ++ | ++ |
S63P | Control | yes | CR3 | + | NA | NA |
E33K | Control | yes | CR2 | + | ++ | + |
H9R | Control | CR1 | +++ | ++ | ++ | |
N29S | Control, CIN2 | yes (both) | CR2 | + | NA | NA |
N29H | CIN2 | CR2 | ++ | +++ | NA | |
D14E | CIN2 | CR1 | ++ | +++ | +++ | |
H51N * | CIN3/AIS | CR3 | ++ | NA | NA | |
P6L | CIN3 | CR1 | ++++ | NA | NA | |
R77S | AIS | CR3 | ++++ | ++++ | ++++ | |
Wild type | ++++ | ++++ | ++++ |
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Lou, H.; Boland, J.F.; Li, H.; Burk, R.; Yeager, M.; Anderson, S.K.; Wentzensen, N.; Schiffman, M.; Mirabello, L.; Dean, M. HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation. Cancers 2022, 14, 4895. https://doi.org/10.3390/cancers14194895
Lou H, Boland JF, Li H, Burk R, Yeager M, Anderson SK, Wentzensen N, Schiffman M, Mirabello L, Dean M. HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation. Cancers. 2022; 14(19):4895. https://doi.org/10.3390/cancers14194895
Chicago/Turabian StyleLou, Hong, Joseph F. Boland, Hongchuan Li, Robert Burk, Meredith Yeager, Stephen K. Anderson, Nicolas Wentzensen, Mark Schiffman, Lisa Mirabello, and Michael Dean. 2022. "HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation" Cancers 14, no. 19: 4895. https://doi.org/10.3390/cancers14194895