Functional consequences of directed mutations in human papillomavirus E6 proteins: abrogation of p53-mediated cell cycle arrest correlates with p53 binding and degradation in vitro.
Clinical and epidemiological studies have implicated the involvement of human papillomavirus (HPV) infection in cervical tumorigenesis. We have previously shown that expression of high-risk (HPV16) E6 can abrogate an important cell cycle checkpoint mediated by p53. Sublethal DNA damage causes p53 accumulation and G1 arrest in normal cells, but not in cells with mutant or absent p53, or in cells that express HPV16-E6. To investigate the functional consequences of low-risk (HPV11) E6 expression and to evaluate regions of E6 believed to mediate interaction with p53, we generated several E6 expression constructs, including HPV11-E6, and fourdifferent E6 mutants. HPV16E6 deltaD and HPV16E6 deltaB had short deletions of nucleotides encoding amino acids previously implicated in p53 degradation and binding, respectively. HPV16E6HL and HPV11E6LH had the putative p53 binding domain exchanged between the high- and the low-risk types. Unlike HPV16-E6, HPV11-E6 and the mutant E6 proteins were not able to bind or degrade p53 in in vitro assays. When expressed in RKO cells, HPV11-E6 or the mutant E6 proteins did not prevent p53 accumulation or interfere with p53-dependent WAF1/CIP1 mRNA expression, allowing p53-mediated G, cell cycle arrest after DNA damage. These findings demonstrate that low-risk and high-risk E6 proteins differ in their effects on p53-mediated cell cycle control and that rather subtle mutations of high-risk E6 can alter its ability to abrogate this important cellular response.
Slebos
R J
RJ
Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA.
Kessis
T D
TD
Chen
A W
AW
Han
S M
SM
Hedrick
L
L
Cho
K R
KR
D002453:Cell Cycle / Q000235:genetics; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D005260:Female; D015967:Gene Expression Regulation, Viral; D006801:Humans; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics* / Q000502:physiology; D030361:Papillomavirus Infections / Q000235:genetics / Q000473:pathology / Q000821:virology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000502:physiology; D014412:Tumor Virus Infections / Q000235:genetics / Q000473:pathology / Q000821:virology*; D002583:Uterine Cervical Neoplasms / Q000821:virology
D009856:Oncogene Proteins, Viral; D016159:Tumor Suppressor Protein p53