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Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product
S M Stirdivant1, H E Huber, D R Patrick
1Department of Cancer Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Abstract:
The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.
Insights
Human papillomavirus E7 protein inhibits retinoblastoma protein (pRB) DNA binding, a key function for pRB
Area of Science:
- Molecular Biology
- Virology
- Oncology
Background:
- Human papillomavirus (HPV) E7 oncoprotein transforms cells.
- E7 protein binds to the retinoblastoma tumor suppressor protein (pRB).
- pRB normally binds DNA and inhibits cell growth.
Purpose of the Study:
- To investigate the mechanism by which HPV E7 protein affects pRB function.
- To determine if E7 binding to pRB inhibits pRB's DNA-binding activity.
- To identify the region of E7 responsible for inhibiting pRB DNA binding.
Main Methods:
- Used recombinant E7 protein and a 60-kDa version of pRB (pRB60) in DNA-binding assays.
- Employed mutant E7 proteins and specific E7 peptides to map functional regions.
- Assessed the effect of E7 on pRB60's ability to bind DNA-cellulose.
Main Results:
- Recombinant E7 protein significantly blocked pRB60 binding to DNA in a dose-dependent manner.
- A mutant E7 protein with reduced pRB binding showed decreased inhibition of pRB DNA binding.
- Specific E7 peptides could either restore pRB DNA binding or block it, suggesting a critical E7 region (residues 32-75).
Conclusions:
- HPV E7 protein directly inhibits pRB's DNA-binding activity.
- The E7 region between amino acid residues 32 and 75 is crucial for this inhibitory function.
- pRB's DNA-binding activity is likely essential for its tumor-suppressive function.